Moraxellaceae O-linked oligosaccharyltransferases, glycosylated fragments, and uses thereof

Abstract

A method for producing glycoconjugates and compositions thereof, comprising covalently attaching an oligosaccharide or polysaccharide to an acceptor protein comprising (or consisting of) a TfpM-associated pilin-like protein or a glycosylated fragment thereof using TfpM oligosaccharyltransferase (OTase).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 414,185, filed October 7, 2022, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant R44AI131742 awarded by the National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in this invention.

[0003] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing in ST26 format (Name VAXNEWMO_229640_ST26_SeqList.xml, Size: 194,162 bytes, and Creation Date: October 5, 2023) attached to this application are incorporated herein by reference in their entirety. [Background technology]

[0004] Technical field to which the invention belongs The present disclosure relates to the field of in vivo protein glycosylation and conjugate vaccine development.

[0005] Protein glycosylation is the most common post-translational modification and is widespread in all three domains of life. In bacteria, protein glycosylation can be mediated directly by glycosyltransferases, which sequentially glycosylate acceptor proteins with individual monosaccharides, or by oligosaccharyltransferases (OTases), which en bloc transfer preassembled oligosaccharides to acceptor proteins. Bacterial oligosaccharyltransferases comprise a family of glycosyltransferases that are divided into two major categories: N-linked and O-linked (Harding, C.M., and Feldman, M.F. (2019) Glycobiology 29, 519-529; Nothaft, H., and Szymanski, C.M. (2010) Nature Reviews Microbiology 8, 765-778). N-linked oligosaccharyltransferases catalyze the covalent attachment of preassembled oligosaccharides to the side chains of asparagine residues in acceptor proteins, whereas O-linked oligosaccharyltransferases typically transfer glycans to the side chains of serine or threonine residues (Nothaft, H., and Szymanski, C. M. (2010) Nature Reviews Microbiology 8, 765-778). The amino acid motif on the substrate protein recognized by an oligosaccharyltransferase is called a sequon. Both N-linked and O-linked oligosaccharyltransferases are membrane-bound enzymes present in the inner membrane that glycosylate periplasmic proteins using glycans derived from lipid-linked precursors. The sugar substrates for these glycosylation reactions are usually derived from a highly conserved pathway that synthesizes lipid-linked oligosaccharides in the inner leaflet of the plasma membrane, which are then "flipped" to the outer leaflet in a manner similar to the Wzy-dependent pathway for O-antigen biosynthesis (Raetz, CRH, and Whitfield, C. (2002) Annual Review of Biochemistry 71, 635-700). The lipid carrier molecule in these reactions is typically undecaprenol pyrophosphate (Und-PP), and oligosaccharyltransferases catalyze the transfer of Und-PP-linked glycans to acceptor proteins.Many O-linked oligosaccharyltransferases have been shown to react promiscuously with polysaccharide substrates and transfer a variety of bacterial glycans to acceptor proteins (Faridmoayer, A., et al. (2008) Journal of Biological Chemistry 283, 34596-34604; DiGiandomenico, A., et al. (2002) Molecular Microbiology 46, 519-530). These bacterial glycans typically consist of repeating sets of 2–7 monosaccharides, known as repeat units, which are polymerized into polysaccharides in the periplasm before transfer (Harding, C.M., and Feldman, M.F. (2019) Glycobiology 29, 519-529). Aside from providing fundamental insights into bacterial glycosylation pathways, oligosaccharyltransferases have attracted considerable interest due to their ability to transfer various bacterial O-antigens and capsular polysaccharides onto specific residues of engineered carrier proteins in a process called bioconjugation or protein glycan coupling (Harding, C.M., and Feldman, M.F. (2019) Glycobiology 29, 519-529; Feldman, M.F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016; Kay, E.J., et al. (2016) Open Biology 6, 150243; Kay, E., et al. (2019) npj Vaccines 4, 16).The resulting bioconjugated glycoproteins are being utilized as glycoconjugate vaccines against a variety of bacterial pathogens, with several candidates currently in clinical trials (GSK 2022 Infectious Disease Pipeline (2022) available on the World Wide Web at: gsk.com / en-gb / research-and-development / our-pipeline / ?infectious-diseases; Johnson & Johnson Infectious Diseases and Vaccines, Global Public Health Pipeline (2022) available on the World Wide Web at: investor.jnj.com / pharmaceutical-pipeline-information).

[0006] Among other substrates, O-linked oligosaccharyltransferases most notably catalyze the glycosylation of type IV pilin-like proteins (Schaeffer, C., and Messner, P. (2017) FEMS Microbiology Reviews 41, 49-91). Type IV pili are primarily composed of protein subunits called major pilins, which interact noncovalently to form the fibrous structure of the pilin shaft (Giltner, Carmen, L., et al. (2012) Microbiology and Molecular Biology Reviews 76, 740-772). The roles of pilin glycosylation in bacterial lifestyles are numerous. Pilin glycosylation has been shown to aid bacterial phage defense by inhibiting binding to surface proteins of bacterial phage particles (Harvey, H., et al. (2018) Nature Microbiology 3, 47-52). In pathogenic bacteria such as Neisseria, Pseudomonas, and Burkholderia, pilin glycosylation regulates tissue adhesion and invasion (Marceau, M., et al. (1998) Molecular Microbiology 27, 705-715; Willcocks, SJ, et al. (2020) Future Microbiology 15, 241-257; Nguyen, LC, et al. (2012) Molecular Plant Pathology 13, 764-774), inhibits complement protein binding, and causes immune system evasion (Tan Rommel, et al. (2015) Infection and Immunity 83, 1339-1346), resulting in various effects that lead to increased virulence (Yakovlieva, L., et al. (2021) Frontiers in Microbiology 12). This highlights the importance of exploring bacterial glycosylation systems as novel antibacterial and antivirulence targets (Yakovlieva, L., et al. (2021) Frontiers in Microbiology 12).

[0007] Historically, O-linked oligosaccharyltransferases have been functionally classified based on several characteristics, including (i) the type of substrate protein they glycosylate, (ii) the type of glycan they can transfer (often defined by the monosaccharide or disaccharide at the reducing end (the sugar covalently linked to Und-PP)), (iii) the position of the glycosylated amino acid on the substrate protein, and (iv) the typical number of sugar monomers or repeating oligosaccharide units in the glycan transferred to the acceptor protein. Three major types of bacterial O-linked OTases have been identified so far: those from Neisseria (Faridmoayer, A., et al., (2007) Journal of Bacteriology 189, 8088-8098), those from Pseudomonas (Castric, P. (1995) Microbiology 141, 1247-1254; Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136; Harvey, H., et al. (2009) Journal of Bacteriology 191, 6513-6524; Qutyan, M., et al. (2010) Journal of Bacteriology 191, 6513-6524). Typical representatives from Bacillus subtilis (192, 5972-5981) and Acinetobacter (Harding, C.M., et al. (2015) Molecular Microbiology 96, 1023-1041; Harding, C.M., et al. (2019) Nature Communications 10, 891; Knoot, C.J., et al. (2021) Glycobiology 31, 1192-1203) have been characterized. TfpO (formerly known as PilO) is the first bacterial oligosaccharyltransferase discovered and exclusively catalyzes the glycosylation of a single protein, PilA, the major pilin subunit of type IV pili (Giltner, Carmen, L., et al. (2012) Microbiology and Molecular Biology Reviews 76, 740-772).TfpO was first characterized in P. aeruginosa strain 1244 (Castric, P. (1995) Microbiology 141, 1247-1254; Comer Jason, E., et al. (2002) Infection and Immunity 70, 2837-2845), but more recently, TfpO orthologues from other organisms have been characterized in medically relevant Acinetobacter species (Harding, C. M., et al. (2015) Molecular Microbiology 96, 1023-1041). The TfpO protein catalyzes the attachment of a single O-antigen repeat unit to the C-terminal serine residue of PilA (Comer Jason, E., et al. (2002) Infection and Immunity 70, 2837-2845). The PglL (also called PglO) protein is another class of bacterial O-linked oligosaccharyltransferases, considered the "general" oligosaccharyltransferase, which catalyzes the glycosylation of multiple periplasmic and membrane-associated proteins, including the major pilin subunit PilE of Neisseria type IV pili (Faridmoayer, A., et al. (2007) Journal of Bacteriology 189, 8088-8098; Vik, Å., et al. (2009) Proceedings of the National Academy of Sciences 106, 4447; Hayes, AJ, et al. (2021) Communications Biology 4, 1045). PglL orthologs naturally transfer glycans that have 2-N-acetyl sugars or hexose galactose at the reducing end of the glycan (Faridmoayer, A., et al. (2008) Journal of Biological Chemistry 283, 34596-34604), but do not naturally transfer glycans that have glucose as the reducing end sugar (Harding, CM, et al. (2019) Nature Communications 10, 891).The PglS orthologue is the third type of bacterial O-linked oligosaccharyltransferase and the most recently discovered of the three types (Harding, CM, et al. (2015) Molecular Microbiology 96, 1023-1041). PglS from Acinetobacter baylyi ADP1 (PglS). ADP1 ) is the best characterized and naturally catalyzes the glycosylation of a single pilin protein called ComP, which forms the type IV-like pili involved in natural competence (Harding, C.M., et al. (2015) Molecular Microbiology 96, 1023-1041; Porstendoerfer, D., et al. (2000) Journal of Bacteriology 182, 3673-3680). In particular, PglS ADP1 has the broadest substrate range of all characterized oligosaccharyltransferases, transferring virtually any glycan to ComP, including those bearing 2-N-acetyl sugars, galactose, or glucose at the reducing end (Harding, C.M., et al. (2019) Nature Communications 10, 891).

[0008] Despite differences in sequence and surface protein structure, there are several notable differences between the TfpO, PglL, and PglS proteins. First, TfpO glycosylates the C-terminal serine or threonine residues of substrate proteins (Comer Jason, E., et al. (2002) Infection and Immunity 70, 2837-2845), whereas PglL and PglS glycosylate sequons located within the amino acid sequence of receptor proteins (Harding, C. M., et al. (2019) Nature Communications 10, 891; Vik, A., et al. (2009) Proceedings of the National Academy of Sciences 106, 4447). Second, while the TfpO protein is limited to transferring only short oligosaccharides to acceptor proteins, the PglL and PglS proteins, when heterologously expressed in glycoengineered Escherichia coli (E. coli) systems, can transfer long-chain polysaccharides with multiple repeating units consisting of hundreds of monosaccharides (Faridmoayer, A., et al. (2007) Journal of Bacteriology 189, 8088-8098; Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136; Harding, C.M., et al. (2015) Molecular Microbiology 96, 1023-1041; Harding, C.M., et al. (2019) Nature Communications 10, 891; Comer Jason, E., et al. (2002) Infection and Immunity 70, 2837-2845). However, the reason for this difference in glycan substrate size and reducing end sugar preference is unclear, in part because the protein structure of any bacterial O-linked oligosaccharyltransferase is not available.

[0009] Therefore, there remains a need to identify new members of the bacterial O-linked OTase family. Comparison of known bacterial O-linked OTases with new representatives will facilitate the study of OTase structure-function relationships and may identify determinants of OTase-glycan specificity.

[0010] Summary of the Invention The present disclosure relates to glycoconjugates comprising an oligosaccharide or polysaccharide covalently attached to a receptor protein, wherein the receptor protein comprises or consists of a TfpM-associated pilin-like protein or a glycosylated fragment thereof, the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof, the TfpM-associated pilin-like protein or glycosylated fragment thereof comprising a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide covalently attached to the C-terminal serine or threonine. In certain embodiments, the receptor protein is a fusion protein comprising a TfpM-associated pilin-like protein or glycosylated fragment thereof translationally fused to a heterologous carrier protein, wherein the TfpM-associated pilin-like protein or glycosylated fragment thereof is the most C-terminal sequence of the receptor protein, such that the receptor fusion protein comprises a C-terminal serine or threonine residue and the oligosaccharide or polysaccharide is covalently attached to the C-terminal serine or threonine. In certain embodiments, the glycoconjugates are immunogenic.

[0011] The present disclosure also relates to glycosylated fragments of pilin-like proteins, comprising or consisting of isolated fragments of TfpM-associated pilin-like proteins, wherein the TfpM-associated pilin-like proteins or glycosylated fragments thereof are Pil. Mo (SEQ ID NO: 57), or Pil lacking the amino acids corresponding to residues 1 to 28 Mo (Pil MoΔ28, SEQ ID NO:58), or a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, optionally with a C-terminal threonine substituted with serine, and the TfpM-related pilin-like protein or glycosylated fragment of the pilin-like protein is Pil. DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment (e.g., C-terminal fragment) and / or variant thereof, wherein the C-terminal threonine is replaced with serine, and / or glycosylated fragment of a pilin-like protein is Pil. Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo In certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil_DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148). 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine. In certain embodiments, the glycosylated fragment of a pilin-like protein consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or variants thereof with 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine.

[0012] The present disclosure also relates to a fusion protein comprising a TfpM-associated pilin-like protein or glycosylated fragment thereof translationally fused to a heterologous carrier protein, wherein the pilin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue, wherein the pilin-like protein or glycosylated fragment is the most C-terminal sequence of the fusion protein, and wherein the fusion protein comprises a C-terminal serine or threonine residue. In certain embodiments, the fusion protein is glycosylated with an oligosaccharide or polysaccharide comprising a glucose at its reducing end covalently linked to the C-terminal serine or threonine.

[0013] The present disclosure relates to methods for producing glycoconjugates, comprising using a TfpM oligosaccharyltransferase (OTase) to covalently attach an oligosaccharide or polysaccharide to an acceptor protein comprising (or consisting of) a TfpM-associated pilin-like protein or a glycosylated fragment thereof, wherein the pilin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue, the acceptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently attached to the C-terminal serine or threonine residue of the acceptor protein. In certain embodiments, the method is a method for in vivo attachment of an oligosaccharide or polysaccharide to an acceptor protein. In certain embodiments, the TfpM OTase is a TfpM-associated pilin-like protein or glycosylated fragment thereof. Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), or TfpM 72-O-c (SEQ ID NO: 81).

[0014] The present disclosure provides isolated nucleic acids encoding glycosylated fragments and / or fusion proteins of the above-described pilin-like proteins, any of the embodiments thereof described anywhere herein, and host cells comprising the isolated nucleic acids.

[0015] The present disclosure provides a method for inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need of an immune response an effective amount of a glycoconjugate, conjugate vaccine, fusion protein, or any composition thereof disclosed anywhere herein.

[0016] The present disclosure provides a method of preventing or treating bacterial and / or infectious diseases in a subject comprising administering to a subject in need thereof a glycoconjugate, conjugate vaccine, fusion protein, or any composition thereof disclosed anywhere herein.

[0017] The present disclosure provides a method for producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising: (a) isolating a glycoconjugate or glycosylated fusion protein disclosed anywhere herein; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and / or carrier.

[0018] The present disclosure provides recombinant nucleic acid constructs comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence. In certain embodiments, the recombinant nucleic acid construct further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or a glycosylated fragment thereof, or a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof operably linked to a nucleotide sequence encoding the TfpM OTase. In certain embodiments, the recombinant nucleic acid construct further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or a glycosylated fragment thereof, or a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof 5′ to the nucleotide sequence encoding the TfpM OTase and operably linked to a nucleotide sequence. [Brief explanation of the drawings]

[0019] [Figure 1]A, B, C, and D show the characteristics of 13 TfpM orthologues from species in the Moraxellaceae family. A) Phylogenetic tree of 20 TfpM orthologues, including representative TfpO, PglL, and PglS from Pseudomonas, Neisseria, and Acinetobacter, respectively. Branch reliability is indicated in red. OTase / pilin pairs marked with a star were cloned and tested in bioconjugation experiments. B) Design of EPA-pilin fusion proteins and TfpM constructs. Colored arrows indicate genes. Gene expression was driven by an IPTG-inducible tac promoter with the lacO operator (tac1O). The rrnB T2 terminator is marked with a black hairpin structure. C) and D) Anti-EPA Western blots of whole-cell Escherichia coli (E. coli) extracts expressing different EPA pilin carrier open reading frames and the tfpM gene. Panel D is the same image as panel C, but at a higher exposure. The whole-cell extracts loaded in each lane were normalized by OD600. H286A indicates a Moraxella osloensis (M. osloensis) TfpM site-specific OTase active site mutant. "g0" indicates unglycosylated EPA pilin, and "gn" indicates a CPS8-glycosylated EPA pilin protein. Reference protein masses are marked in kDa on the left side of the Western blot. [Figure 2] A phylogenetic tree showing the relative distances of TfpM, PilO, PglL, and PglS orthologues is shown. The tree was generated using the phylogeny.fr server (available on the World Wide Web at phylogeny.fr / ) and uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively. [Figure 3]Phylogenetic tree showing the relative distances between TfpM-related pilin-like proteins, selected PilA proteins from Neisseria and Pseudomonas, and ComP from Acinetobacter soli CIP 110264. Red numbers indicate the reliability of the branches. The phylogeny was generated using the phylogeny.fr server (available on the World Wide Web at phylogeny.fr / ) and uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively. [Figure 4] Figure 1 shows a multiple sequence alignment of selected bacterial O-linked oligosaccharyltransferases. The alignment was generated using Clustal Omega with default settings, available on the World Wide Web at ebi.ac.uk / Tools / msa / clustalo / . N_menigitidis_MC58_PglL (SEQ ID NO: 105), A_baylyi_ADP1_PglS (SEQ ID NO: 106), P_aeruginosa_1244_TfPO (SEQ ID NO: 107), M_osloensis_1202_TfpM (SEQ ID NO: 56), A_nosocomialis_M2_TfpO (SEQ ID NO: 108). [Figure 5] Anti-EPA whole-cell Western blots examining the glycosylation status of the EPA-PilMoΔ28 fusion and the C-terminal Thr167 mutant of EPA-PilMoΔ28 are shown. All lanes were normalized to the same OD600. The masses of reference proteins are marked in kDa next to the Western blot. [Figure 6] A and B show targeted MS / MS analysis of the HexHexA-modified C-terminal EPA-PilMoΔ28 peptide 762FLPANCRGT770 (SEQ ID NO: 61). A) EThcD fragmentation allowed localization of the HexHexA glycosylation event to the terminal residue Thr770. B) HCD fragmentation allowed confirmation of the peptide sequence and the attachment of the disaccharide HexHexA via the Hex monosaccharide by observing multiple y ions bound exclusively to Hex residues. [Figure 7]Figures A, B, C, D, E, F, and G show that TfpMMo can transfer diverse bacterial glycans to the EPA-PilMoΔ28 fusion protein. Figure A shows the repeating unit structures of five bacterial glycans tested with TfpMMo. The bonds between sugar monomers are indicated by parentheses. Glycan abbreviations used: CPS8 is Streptococcus pneumoniae capsular polysaccharide type 8; GBSIII is Streptococcus group B capsular polysaccharide type III; LT2 is Salmonella enterica group B serotype LT2 O antigen; O16 is E. coli serotype O16 O antigen; O2a is Klebsiella pneumoniae serotype O2a O antigen. All sugars are in the pyranose form unless otherwise noted. Abbreviations used: Glc is glucose, Gal is galactose, Galf is galactofuranose, Rha is rhamnose, GlcNAc is N-acetylglucosamine, Abe is abequase, and NeuNAc is N-acetylneuraminic acid, sialic acid. B)-F) Anti-glycan Western blots using partially purified TfpMMo-derived bioconjugates. B) Anti-CPS8; C) Anti-O16; D) Anti-LT2; E) Anti-O2a; F) Anti-GBSIII; G) Anti-EPA. The + / - labels in panels B)-G) indicate whether samples were incubated with (+) or without (-) proteinase K prior to SDS-PAGE separation. Reference protein masses are marked in kDa next to the Western blots. [Figure 8]Panels A and B show that TfpMMo glycosylates an EPA-fused pilin mutant truncated by as little as three amino acids. Panel A shows the sequence of the EPA-fused PilMo fragment tested for bioconjugation with TfpMMo. Blue text marks the C-terminal residue of EPA (i.e., EDLK, SEQ ID NO: 132). The underlined residues indicate the glycine linker placed between the EPA and pilin sequences. Panel B shows an anti-EPA Western blot of whole-cell extracts expressing the truncated pilin mutant, CPS8, and TfpMMo. The calculated mass of EPA-PilMoΔ28 is 80.3 kDa, while the masses of the truncated mutants range from 67.1 to 69.0 kDa. All lanes were normalized to the same OD600. The masses of reference proteins are marked in kDa on the left side of the Western blot. "g0" indicates unglycosylated truncated EPA pilin, and "gn" indicates glycosylated EPA pilin protein. Non-glycosylated EPA-PilMoΔ28 migrates at around 75 kDa. Pil20 (SEQ ID NO: 60). Pil15[A] (SEQ ID NO: 171). Pil13[A] (SEQ ID NO: 173). GGGG plus Pil10[A] is Pil10L[A] (SEQ ID NO: 111). Pil10[A] (SEQ ID NO: 176). Pil7[A] (SEQ ID NO: 179). Pil6[A] (SEQ ID NO: 180). Pil5[A] (SEQ ID NO: 181). Pil4[A] (SEQ ID NO: 182). Pil3 "RGT" (SEQ ID NO: 117). EDLK plus Pil2 (SEQ ID NO: 118). EDLKGGGG plus Pil20 (SEQ ID NO: 122). EDLK plus Pil15[A] (SEQ ID NO: 123). EDLK plus Pil13[A] (SEQ ID NO: 124). EDLK plus Pil10L[A] (SEQ ID NO: 125). EDLK plus Pil10[A] (SEQ ID NO: 126). EDLK plus Pil7[A] (SEQ ID NO: 127). EDLK plus Pil6[A] (SEQ ID NO: 128). EDLK plus Pil5[A] (SEQ ID NO: 129). EDLK plus Pil4[A] (SEQ ID NO: 130). EDLK plus Pil3 (SEQ ID NO: 131). [Figure 9]Figure 1 shows a multiple sequence alignment of selected pilin proteins. The accession numbers of these proteins are listed in the text. The alignment was generated using Clustal Omega with default settings, available on the World Wide Web at ebi.ac.uk / Tools / msa / clustalo / . P_aeruginosa_1244_PilA (SEQ ID NO: 119). N_menigitidis_M2_PilA (SEQ ID NO: 120). A_junii_65_pilin (SEQ ID NO: 97). A_CIP102143_pilin (SEQ ID NO: 88). A_CIP102637_pilin (SEQ ID NO: 100). A_YZS-X1-1_pilin (SEQ ID NO: 98). A_soli_110264_ComP (SEQ ID NO: 121). A_YH01026_pilin (SEQ ID NO: 87). M_osloensis_1202_pilin (SEQ ID NO: 57). A_junii_TUM15069_pilin (sequence number 84). [Figure 10] Figures A, B, C, D, E, and F show that purified TfpMMo-derived GBSIII bioconjugates induce potent IgG immune responses in mice. A) Western blots of purified GBSIII-291 bioconjugates, anti-EPA channel, and anti-GBSIII. B) Anti-GBSIII. C) A merged image of A and B. D) Coomassie staining of purified GBSIII-291 bioconjugates. E) Full MS1 spectrum of purified GBSIII-291 bioconjugates. The theoretical mass of the 291 protein (EPA-Pil20) is 69,582.19 Da. The GBSIII-291 bioconjugates are observed in multiple states of increasing mass, separated by approximately 980 Da, corresponding to the calculated mass of the GBSIII glycan repeat unit. F) GBSIII-specific IgG kinetics over the course of immunization, measured by ELISA and converted to ng / mL IgG using a standard IgG curve. **P<0.01. [Figure 11]Panels A and B show the glycosylation of EPA constructs containing sequons from different O-linked oligosaccharyltransferase systems. A) Schematic of a plasmid-based operon expressing EPA with a PglS- or TfpM-specific sequon. The internal glycan tag ("iGT") is a 23-amino acid fragment from ComP110264 inserted between EPA Ala489-Arg490 and / or Glu548-Gly549. B) Anti-EPA Western blot of SDB1 periplasmic extracts expressing one of the four constructs and the Escherichia coli (E. coli) O16 O antigen. Loading per lane was normalized to OD600. "g0" indicates unglycosylated EPA carrier protein, and singly or doubly glycosylated EPA proteins are indicated. Reference protein masses are marked in kDa on the left side of the Western blot. DETAILED DESCRIPTION OF THE INVENTION

[0020] To the extent necessary to provide descriptive support, the subject matter and / or text of the appended claims is incorporated herein by reference in its entirety.

[0021] It will be understood by any reader of this specification that the exemplary aspects and embodiments described and claimed herein may suitably be practiced in the absence of any recited feature, element, or step that may or may not be specifically disclosed herein.

[0022] definition It should be noted that the terms "a" or "an" refer to one or more of that entity; for example, "a polysaccharide" is understood to refer to one or more polysaccharides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0023] Furthermore, as used herein, "and / or" is to be construed as specifically disclosing each of the specified features or components with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0024] Whenever an embodiment is described herein with the words "comprising" or "comprises," it is understood that other similar embodiments described with terms such as "consisting of," "consists of," "consisting essentially of," and / or "consists essentially of" are also provided.

[0025] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0026] Numeric ranges are inclusive of the numbers defining the range. Even if not expressly identified as "and any range therebetween," if a list of values ​​is given, e.g., 1, 2, 3, or 4, the disclosure is intended to specifically include any range between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc., unless otherwise stated.

[0027] The headings provided herein are for ease of reference only and do not limit the various aspects or aspects of the disclosure that can be had by reference to the entire specification.

[0028] As used herein, the term "non-naturally occurring" substance, composition, entity, and / or any combination of substances, compositions, entities, or grammatical variations thereof, is a qualified term that expressly excludes, but only excludes, forms of substance, composition, entity, and / or any combination of substances, compositions, entities that are well understood to be "naturally occurring" by those of ordinary skill in the art or that have been, or may at any time be, determined or interpreted as "naturally occurring" by a judge, administrative agency, or judicial body.

[0029] As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or other terms used to refer to a chain of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of or interchangeably with these terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be produced by any method, including chemical synthesis.

[0030] As used herein, "protein" can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but it can also refer to two or more polypeptides associated, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to form a multimeric protein.

[0031] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not found in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0032] As used herein, the term "non-naturally occurring" polypeptide, or grammatical variations thereof, is a qualified term that expressly excludes, but only excludes, polypeptide forms that are well understood by those of skill in the art to be "naturally occurring" or that have been, or may at any time be, determined or interpreted by a judge, administrative agency, or judicial body to be "naturally occurring."

[0033] Disclosed herein may be a particular binding molecule, or an antigen-binding fragment, variant, or derivative thereof. Except when specifically referring to a full-sized antibody, such as a naturally occurring antibody, the term "binding molecule" encompasses not only full-sized antibodies, but also antigen-binding fragments, variants, analogs, or derivatives of such antibodies (e.g., naturally occurring antibodies or immunoglobulin molecules, or artificial antibody molecules or fragments that bind to antigens in a manner similar to antibody molecules).

[0034] As used herein, the term "binding molecule" refers, in its broadest sense, to a molecule that specifically binds to an antigenic determinant. As further described herein, a binding molecule may contain one or more "binding domains." As used herein, a "binding domain" is a two- or three-dimensional polypeptide structure that can specifically bind to a given antigenic determinant or epitope. A non-limiting example of a binding molecule is an antibody or fragment thereof that contains a binding domain that specifically binds to an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody that contains a first binding domain that binds to a first epitope and a second binding domain that binds to a second epitope.

[0035] The terms "antibody" and "immunoglobulin" may be used interchangeably herein. An antibody (or fragment, variant, or derivative thereof) disclosed herein comprises at least a heavy chain variable domain and at least heavy and light chain variable domains. Basic immunoglobulin structure in vertebrate systems is relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0036] Binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab'), Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either the VL or VH domain, fragments produced by a Fab expression library, and the like. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Immunoglobulin or antibody molecules encompassed by the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0037] "Specifically binds" means that a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope. As used herein, the term "specificity" is used to modify the relative affinity with which a particular binding molecule binds to a particular epitope. For example, binding molecule "A" can be considered to have higher specificity for a particular epitope than binding molecule "B," or binding molecule "A" can be said to bind epitope "C" with higher specificity than to related epitope "D."

[0038] The term "polynucleotide" is intended to encompass both single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of a polynucleotide. Isolated polynucleotides or nucleic acids also include such molecules produced synthetically. Additionally, a polynucleotide or nucleic acid may be or include a regulatory element such as a promoter, ribosome binding site, or transcription terminator.

[0039] Two DNA segments (such as a polypeptide coding region and its associated promoter) can be considered "operably associated" or "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA segments does not interfere with the ability of the expression control sequences to direct expression of the gene product or to transcribe the DNA template. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. Two adjacent or overlapping coding sequences would be operably linked if both are capable of efficient transcription.

[0040] As used herein, a "non-naturally occurring" polynucleotide, or grammatical variations thereof, is a qualified definition that expressly excludes, but only excludes, forms of polynucleotides that are well understood by those of skill in the art to be "naturally occurring" or that have been, or may at any time be, determined or interpreted by a judge, administrative agency, or judicial body to be "naturally occurring."

[0041] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide may be RNA.

[0042] A "vector" (used interchangeably herein with "plasmid") is a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain nucleic acid sequences that enable it to replicate in the host cell, such as an origin of replication. A vector may encode and express proteins. A vector may also contain one or more selectable marker genes and other genetic elements known in the art.

[0043] A "transformed" cell, or "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, electroporation, lipofection, and introduction of naked DNA by particle gun acceleration. A transformed or host cell can be a bacterial cell or a eukaryotic cell.

[0044] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, e.g., a polypeptide. This process includes any expression of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. This includes, but is not limited to, transcription of a gene into messenger RNA (mRNA) and translation of such mRNA into polypeptide(s). When the final desired product is a biochemical, expression includes the production of that biochemical and its precursors. Expression of a gene produces a "gene product." As used herein, a gene product is either a nucleic acid, such as a messenger RNA, produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids that have undergone post-transcriptional modifications, e.g., polyadenylation, or polypeptides that have undergone post-translational modifications, e.g., methylation, glycosylation, addition of lipids, conjugation to other protein subunits, or proteolytic cleavage.

[0045] As used herein, the terms "treat," "treatment," or "treatment of" (e.g., the phrase "treating a subject") refer to reducing the likelihood of a disease condition, reducing the occurrence of a disease symptom, e.g., to the point where the subject survives longer or experiences less discomfort. For example, treatment can refer to the ability of a therapy administered to a subject to alleviate the symptoms, signs, or causes of a disease. Treatment can also refer to alleviating or reducing at least one clinical symptom and / or inhibiting or slowing the progression of a disease state and / or preventing or delaying the onset of a disease or condition.

[0046] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, farm animals, industrial animals, sport animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0047] The terms "pharmaceutical composition" or "therapeutic composition" refer to a formulation in which the biological activity of the active ingredient is effective and which contains no additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0048] As used herein, a "sequon" refers to a specific amino acid sequence of amino acid residues that is recognized and subsequently glycosylated by a specific oligosaccharyltransferase.

[0049] As used herein, a "glycoconjugate" refers to a polypeptide covalently attached to a carbohydrate moiety. It is understood that the carbohydrate moiety can be a monosaccharide, oligosaccharide, or polysaccharide. For purposes of this disclosure, a "glycoconjugate" is a specific type of "bioconjugate" referred to herein. In certain art-recognized embodiments, a glycoprotein is a glycoconjugate.

[0050] overview Conjugate vaccines, consisting of polysaccharides linked to proteins, are lifesaving prophylactics. Traditionally, conjugate vaccines are produced using chemical methods. However, in vivo bacterial conjugation has emerged as an alternative method of production. In vivo conjugation (bioconjugation) utilizes oligosaccharyltransferase (OTase) to attach polysaccharides to proteins.

[0051] Despite the introduction and implementation of pneumococcal conjugate vaccines over the past two decades, approximately 1.5 million people die from Streptococcus pneumoniae (S. pneumoniae) each year. This is due in part to the presence of over 90 serotypes of S. pneumoniae and the complex manufacturing process required to synthesize pneumococcal conjugate vaccines. These factors combine to hinder the global distribution and development of broader, more protective vaccines. To expedite development and reduce manufacturing costs, we disclose a platform for developing and manufacturing conjugate vaccines, such as pneumococcal conjugate vaccines, using in vivo conjugation. This streamlined process could complement existing manufacturing pipelines or completely avoid reliance on chemical conjugation methodologies, enabling the production of more comprehensive conjugate vaccines.

[0052] While traditional chemical conjugation vaccine synthesis is considered complex, costly, and laborious (Frasch, CE Vaccine 27, 6468-6470 (2009)), in vivo conjugation has made extensive progress as a viable biosynthetic alternative (Huttner, A. et al. Lancet Infect Dis 17, 528-537 (2017)). These advances are best highlighted by the success of GlycoVaxyn (now LimmaTech Biologics AG, an independent company with direct ties to GlaxoSmithKline), a clinical-stage biopharmaceutical company with multiple bioconjugate vaccines in various stages of clinical trials, one of which (Flexyn2a) has completed a Phase 2b challenge trial. While GlycoVaxyn is at the forefront of the in vivo conjugation revolution, the ability to efficiently glycosylate carrier / acceptor proteins with polysaccharides containing glucose (Glc) as the reducing end sugar has remained elusive and, as expected, has hindered the development of bioconjugate vaccines targeting multiple pathogens, including but not limited to Streptococcus pneumoniae, Streptococcus agalactiae, Klebsiella pneumoniae, and Enterococcus.

[0053] Protein glycosylation in bacteria is typically mediated by oligosaccharyltransferases, which transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Originally, O-linked oligosaccharyltransferases typically transfer a single repeating unit of an O-antigen or capsular polysaccharide to the side chain of a serine or threonine residue on an acceptor protein. Three major families of bacterial O-linked oligosaccharyltransferases (PglL, PglS, and TfpO) have been described across diverse genera. TfpO enzymes are limited to the transfer of short oligosaccharides both in their native context and when heterologously expressed in glycoengineered Escherichia coli (E. coli). In contrast, PglL and PglS enzymes can transfer long-chain polysaccharides when heterologously expressed.

[0054] Because bacteria in the genus Acinetobacter have been shown to possess genes encoding TfpO, PglL, and PglS orthologues, we were interested in searching for novel O-linked oligosaccharyltransferases within the Moraxellaceae family, to which Acinetobacter belongs. Because different types of O-linked oligosaccharyltransferases cannot be clearly distinguished based on primary amino acid sequence alone, final family assignment usually relies on functional analysis. Nevertheless, the size of TfpO proteins is typically approximately 420–460 amino acids, smaller than the PglS and PglL proteins, which are approximately 525–600 amino acids. Furthermore, the amino acid sequences of the pilin glycosylated by O-linked oligosaccharyltransferases also differ sufficiently among oligosaccharyltransferase families to help identify which family an oligosaccharyltransferase belongs to. In the case of PglS, only a single protein, the type IV pilin-like protein ComP, functions as its cognate receptor in its natural context (Harding, CM, et al. (2015) Molecular Microbiology 96, 1023-1041). The PglS sequon is flanked by two cysteine ​​residues and contains a conserved internal serine residue that is the glycosylation site (Harding, CM, et al. (2019) Nature Communications 10, 891; Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203). In contrast, the smallest TfpO sequon is located at the C-terminus of the type IV pilin protein PilA (Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136; Harvey, H., et al. (2009) Journal of Bacteriology 191, 6513-6524; Comer Jason, E., et al. (2002) Infection and Immunity 70, 2837-2845).

[0055] Herein, we disclose the discovery and functional characterization of a new family of bacterial O-linked oligosaccharyltransferases, termed TfpM, from bacteria of the Moraxellaceae family. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analysis demonstrates that TfpM proteins cluster in a distinct clade from known bacterial oligosaccharyltransferases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylate the C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We demonstrated that TfpM has broad substrate tolerance and can transfer a variety of polysaccharides containing reducing-end glucose, galactose, or 2-N-acetyl sugars. Furthermore, TfpM-derived bioconjugates were shown to be immunogenic and elicit serotype-specific polysaccharide IgG responses in mice. Thus, the diversity of TfpM glycan substrates and the identification of the minimal TfpM sequon make this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

[0056] Bioinformatics-based identification of a new class of OTases possessed by bacteria of the Moraxellaceae family. To identify genes encoding O-linked oligosaccharyltransferases, we first performed a basic local alignment search tool (BLAST) and PglS ADP1 The amino acid sequence (SEQ ID NO: 1) was used as a query to search the NCBI genome and whole-genome shotgun contig sequence databases. Sequence number 1_PglS ADP1 Amino acid sequence MNSIFKKIKNYTIVSGVFFLGSAFIIPNTSNLSSTLYKELIAVLGLLILLTVKSFDYKKILIPKNFYWFLFVIFIIFIQLIVGEIYFFQDFFFSISFLVILFLSFLLGFNERLNGDDLIVKKIAWIFIIVVQISFLI AINQKIEIVQNFFLFSSSYNGRSTANLGQPNQFSTLILITLFLLCYLREKNSLNNMVFNILSFCLIFANVMTQSRSAWISVILISLLYLLKFQKKIELRRVIFFNIVFWTLVYCVPLLFNLIFFQKNSYSTFDRLTM GSSRFEIWPQLLKAVFHKPFIGYGWGQTGVAQLETINKSSTKGEWFTYSHNLFLDLMLWNGFFIGLIISILILCFLIELYSSIKNKSDLFLFFCVVAFFVHCLLEYPFAYTYFLIPVGFLCGYISTQNIKNSISYFN LSKRKLTLFLGCCWLGYVAFWVEVLDISKKNEIYARQFLFSNHVKFYNIENYILDGFSKQLDFQYLDYCELKDKYQLLDFKKVAYRYPNASIVYKYYSISAEMKMDQKSANQIIRAYSVIKNQKIIKPKLKFCSIEY

[0057] Shorten the hit list and PglS ADP1 To reduce the chance of identifying a very similar orthologue of PglS, the search was ADP1 The list was further narrowed to candidates with less than 50% amino acid sequence identity to TfpO. Several of the top hits from this narrowed list were proteins much more similar in size to the TfpO protein, but the upstream cognate pilin protein contained both the ComP disulfide-flanked sequon and a C-terminal PilA-like sequon. The first identified pilin-oligosaccharyltransferase pair was encoded by two Acinetobacter species: A. parvus DSM 16617 and A. townerii ZZC-3 (Table 1). [Table 1-1]

Table 1-2

Table 1-3

Table 1-4

[0058] The two oligosaccharyltransferases from these strains were closely related, sharing >96% sequence identity. Intrigued by these findings, we investigated further and identified other strains of the Moraxellaceae family that contained genes encoding similar putative oligosaccharyltransferase / pilin pairs. While numerous genes similar to those found in A. parvus DSM 16617 and A. townerii ZZC-3 were found, most of the related pilin proteins encoded upstream of the oligosaccharyltransferases lacked ComP-like sequons. The accession numbers and protein sizes of 20 of these putative oligosaccharyltransferase-pilin-like protein pairs are shown in Table 1. Because the inventors did not observe any homologs in species outside the Moraxellaceae family, they named these different oligosaccharyltransferases TfpM proteins ("M" stands for Moraxellaceae) to distinguish them from other known enzymes. Because the size of the TfpM proteins is similar to that of the known TfpO proteins, it was initially speculated that these genes might encode variants of the TfpO-PilA pair found in Acinetobacter and Pseudomonas (Harding, CM, et al. (2015) Molecular Microbiology 96, 1023-1041). However, multiple sequence alignment of the 20 TfpM proteins with the known PglS, PglL, and TfpO proteins demonstrated that the former share less than 26% sequence identity with typical oligosaccharyltransferases. Analysis of the phylogenetic tree generated by the multiple alignment showed that the TfpM protein clustered in a distinct clade from the TfpO, PglS, and PglL proteins (Figures 1A and 2). In contrast, the pilin gene located immediately upstream of tfpM did not cluster in a discrete clade (Figure 3) and showed high overall identity with the PilA protein, i.e., 37% to 60%.Based on the coding sequences, most of the related pilin proteins belong to the type IV major pilin family, except for the pilin proteins of Acinetobacter sp. CIP102143 and Acinetobacter sp. CIP102637, which lack the characteristic type III signal sequence at their N-terminus (Giltner, Carmen, L., et al. (2012) Microbiology and Molecular Biology Reviews 76, 740-772).

[0059] The TfpM orthologue uses the capsular polysaccharide of Streptococcus pneumoniae serotype 8 to glycosylate an engineered pilin fusion protein. Although similar in size to TfpO, TfpM differs sufficiently in amino acid sequence to warrant further investigation. We were particularly interested in determining whether TfpM can transfer only short oligosaccharides to acceptor proteins like TfpO. Of the 20 TfpM oligosaccharyltransferases listed in Table 1, we selected 13 representatives from different clades and tested their glycosylation activity in glycoengineered Escherichia coli (E. coli) strains (Harding, C.M., and Feldman, M.F. (2019) Glycobiology 29, 519-529; Feldman, M.F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016). Previously, we developed a chimeric receptor protein strategy that fuses different sized soluble fragments of ComP (the natural substrate of PglS) with the exotoxin A protein (EPA) from Pseudomonas aeruginosa (Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203). All type IV pilin-like proteins contain a conserved N-terminal pilin signal sequence and membrane anchor domain, which are not required for glycosylation but are essential for pilin stability. The fusion protein approach allows for the removal of the conserved N-terminal pilin signal sequence and membrane anchor domain, resulting in the formation of a chimeric receptor protein that binds PglS. ADP1This approach was used to determine the smallest sequon that can be recognized and efficiently glycosylated by EPA (Harding, C.M., et al. (2019) Nature Communications 10, 891; Knoot, C.J., et al. (2021) Glycobiology 31, 1192-1203). We adopted this approach and designed 13 synthetic double-stranded DNA blocks encoding N-terminal truncated fragments of the upstream pilin gene and the downstream tfpM gene. In most strains carrying tfpM, the protein-coding region of the upstream pilin gene and the start codon of tfpM overlap by a single nucleotide. This gene structure was left intact in the expression construct. The synthetic DNA blocks were designed so that, when cloned into the EPA expression vector using Gibson assembly, the pilin-coding region was placed in frame with the C-terminus of EPA, creating a gene fusion with the immediately downstream tfpM gene that would be translated as a single protein (Figure 1B). The sizes of the truncated pilin fragments ranged from 113 to 140 amino acids. A purification tag was not added to the C-terminus of the EPA-pilin fusion because a previous study using TfpO from P. aeruginosa 1244 reported that adding an additional C-terminal residue after the serine residue prevented glycosylation (Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136). Expression of the EPA pilin and TfpM proteins was driven from an IPTG-inducible tac promoter on the pEXT20 plasmid (Dykxhoorn, DM, et al. (1996) Gene 177, 133-136). The fusion proteins were secreted into the periplasm using the DsbA signal sequence at the N-terminus of EPA. The oligos and primers used for assembly are listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0060] Using this design, we evaluated the ability of 13 TfpM proteins to transfer the Streptococcus pneumoniae capsular polysaccharide 8 (CPS8) glycan to the corresponding pilin domain of an EPA-pilin fusion. The CPS8 repeating unit is a tetrasaccharide with a glucose residue at the reducing end. Notably, PglS is the only known oligosaccharyltransferase capable of naturally transferring this glycan to an acceptor protein (Harding, CM, et al. (2019) Nature Communications 10, 891). The 13 EPA-pilin fusion / TfpM expression vectors were individually transformed into the Escherichia coli SDB1 strain, which expresses the CPS8 glycan (Feldman, MF, et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016), and protein glycosylation was evaluated. The predicted masses of the unglycosylated fusion proteins ranged from 78.3 to 80.5 kDa. Some TfpM proteins glycosylated the corresponding EPA-pilin fusions, and glycosylation was indicated by a high molecular weight ladder (g) above the unglycosylated band (g). n) (Figure 1C). Each high-mass band represents a glycan containing one additional CPS8 repeat unit attached to the EPA pilin protein. Glycosylation was readily observed in the seven TfpM orthologues examined: Acinetobacter sp. YZSX-1-1, Acinetobacter sp. CIP102637, Acinetobacter sp. YH01026, Acinetobacter junii 65, Acinetobacter sp. CIP102143, Acinetobacter sp. TUM15069, and Moraxella osloensis 1202 (Figure 1C). Some of these extracts required higher exposure of the Western blot to observe the glycosylation pattern (Figure 1D). As a control, we cloned the conserved histidine (His) previously shown to be required for the activity of wzy_C domain-containing enzymes. 286 We generated mutants of the Moraxella osloensis (M. osloensis) TfpM protein gene with a single residue change (Figure 4). The wzy_C family pfam04932 is an "O-antigen ligase" domain present in membrane-bound enzymes that catalyze the covalent transfer of lipid-linked oligosaccharides (liposaccharides) to lipid A or protein substrates (Ruan, X., et al. (2012) Glycobiology 22, 288-299; Musumeci, MA, et al. (2014) Glycobiology 24, 39-50).

[0061] No glycosylation was observed in SDB1 cell extracts expressing this H286A mutant together with the EPA-pilin fusion protein and CPS8 glycans (Fig. 1C), indicating that glycosylation is due to the activity of the tfpM gene product and not His. 286 We show that α- and β-actin are required for the catalytic activity and / or stability of this oligosaccharyltransferase.

[0062] TfpMMo is an O-linked oligosaccharyltransferase that glycosylates the C-terminal threonine of pilin substrates. Of the 13 TfpM-pilin pairs tested in previous experiments, the pair from Moraxella osloensis 1202 and Acinetobacter sp. YH01026 showed the most efficient transfer of glycans of various sizes. Due to the slightly higher apparent stability of the pilin from M. osloensis FDAARGOS_1202 (hereafter referred to as 1202), we selected the oligosaccharyltransferase from this organism as a representative for further characterization and compared the enzyme with TfpM. Mo For clarity, the complete native M. osloensis 1202 pilin protein is referred to in the text as PilMo (SEQ ID NO: 57), and the N-terminally truncated fusion domain is referred to as Pil Mo Δ28 (SEQ ID NO: 58). Mo Using Pil Mo We identified the glycosylation site at Δ28 and therefore determined whether the enzyme acts like the TfpO protein, glycosylating the C-terminal amino acid of the cognate pilin receptor, or like the PglL or PglS proteins, glycosylating an internal residue. TfpO proteins typically transfer short oligosaccharides containing 3–6 sugars to the side chain of a C-terminal serine residue of the corresponding pilin. All but one of the cognate pilin proteins located immediately upstream of the tfpM open reading frame terminates with a C-terminal threonine residue, specifically, the one from Psychrobacter sp. 72-OC. Based on this observation and the similarity in size between the TfpM and TfpO proteins, we hypothesized that the TfpM enzyme also transfers glycans to C-terminal residues and engineered pilin point mutants to test this. The C-terminal pilin threonine (Thr) 167 Two mutants of Pil were generated, in which this residue was converted to serine or alanine, and glycosylation by CPS8 was examined. MoWhole-cell Escherichia coli (E. coli) extracts of strains expressing the Δ28 mutant were probed using anti-EPA antibodies in Western blots. As shown in Figure 5, TfpM Mo EPA-Pil Mo Δ28 T167S was also glycosylated, but no glycosylation was observed with the alanine mutant. 167 or Ser 167 The hydroxyl groups on the side chains of the residues are Mo These results indicate that the glycan binding site is likely to be the glycan binding site of β-actin.

[0063] TfpM Mo To confirm the site of pilin glycosylation by CPS8, we analyzed CPS8-glycosylated EPA-Pil Mo Δ28 was partially purified and separated by SDS-PAGE analysis, and the separated glycoproteins were stained with Coomassie. Mo The gel slice corresponding to Δ28 was excised, digested with LysC, and glycopeptides were analyzed. Mo Open search-based analysis of the Δ28 peptide (Chick, JM, et al. (2015) Nature Biotechnology 33, 743-749; Polasky, DA, et al. (2020) Nature Methods 17, 1125-1132) revealed a hexose (Hex)-hexuronic acid (HexA) modification consistent with an incomplete monomer (HexHexAHex2) of the CPS8 glycan. 762 FLPANCRGT 770The peptide was identified. High-energy C-trap dissociation (HCD) analysis confirmed that this disaccharide was linked via a hexose residue, and targeted electron transfer / high-energy collision dissociation (EThcD) analysis confirmed the attachment of HexHexA to the C-terminal threonine residue (Figures 6A and 6B). This MS analysis did not identify the CPS8 tetrasaccharide multimer, which is not surprising, given the extreme difficulty of detecting elongated glycoconjugates using peptide-centric LC-MS methods without the use of specialized chemical additives such as supercharging agents (Lin, C.-W., et al. (2016) Analytical Chemistry 88, 8484-8494). Nevertheless, the identification of a disaccharide consistent with the partially completed CPS8 tetrasaccharide still confirms the identity of the high-molecular-weight ladder as a polymerized CPS8 tetrasaccharide.

[0064] TfpM Mo transfers polysaccharides containing glucose, galactose, or 2-N-acetyl monosaccharides at the reducing end. Next, we transformed TfpM in a glycoengineered E. coli background. Mo We aimed to explore the range of polysaccharide substrates that TfpM can transfer. We selected polysaccharides containing different reducing end sugars, different disaccharide sugar linkages near the reducing end, and / or polymers consisting of linear or branched repeating units. In addition to pneumococcal CPS8, four polysaccharides were identified that were involved in TfpM transfer: E. coli O16 antigen, Salmonella enterica LT2 O antigen, Klebsiella pneumoniae O2a antigen, and type III group B Streptococcus capsular polysaccharide. MoThe structures of the five repeating units tested are shown in Figure 7A (Liu, B., et al. (2020) FEMS Microbiology Reviews 44, 655-683; Curd, H., et al. (1998) Journal of Bacteriology 180, 1002-1007; Whitfield, C., et al. (1992) Journal of Bacteriology 174, 4913-4919; Pinto, V., and Berti, F. (2014) Journal of Pharmaceutical and Biomedical Analysis 98, 9-15; Geno, KA, et al. (2015) Clinical Microbiology Reviews 28, 871-899). Each of the five polysaccharides was synthesized by EPA-Pil in Escherichia coli (E. coli) SDB1 cells. Mo Δ28 and TfpM Mo The TfpM glycoproteins were coexpressed with α- and β-glucanase (SDB1), induced, and grown for subsequent glycoprotein purification. Periplasmic extracts of SDB1 cells were partially purified using anion exchange chromatography to remove any contaminating undecaprenol pyrophosphate-linked polysaccharides that could complicate Western blot interpretation. To verify that the glycan-specific antibody signals observed on Western blots were indeed derived from glycosylated proteins and not from contaminating lipid-linked polysaccharides from whole-cell lysates, the purified glycoproteins were divided into two equal fractions, one half of which was digested with proteinase K before SDS-PAGE separation and Western blot analysis. Because all antibodies used in this experiment were derived from rabbits, Western blots were probed with antisera specific for each polysaccharide and, separately, with an anti-EPA antibody. As shown in Figure 7, TfpM glycoproteins were specifically expressed in the TfpM glycoproteins. Mo All five different polysaccharides were identified as EPA-Pil MoDigestion with proteinase K eliminated both the anti-glycan signals (Figures 7B, 7C, 7D, 7E, and 7F) and the anti-EPA signals (Figure 7G) in Western blots, confirming that the anti-glycan signals were derived from protein-bound polysaccharides rather than contaminating lipid-bound polysaccharides.

[0065] TfpM Mo Disconnect Pil Mo The Δ28 mutant can be glycosylated. All previous experiments have used PilA, which is 139 amino acids in length. Mo An N-terminal truncation mutant of TfpM was used. Mo To gain insight into the minimal features required for C-terminal pilin glycosylation by Pil Mo A series of further truncated mutants of Δ28 were generated and tested for their ability to be glycosylated. We first synthesized Pil fused to the C-terminus of EPA via a flexible four-residue glycine linker. Mo 20 amino acid fragments of Pil 20 We named this fragment "DSL" (Figure 8A). This 20 amino acid fragment was selected because it contains a disulfide loop ("DSL") region that is conserved in many type IV pilins (Figure 9) (Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136; Harvey, H., et al. (2009) Journal of Bacteriology 191, 6513-6524). It is noteworthy that this DSL corresponds to a distinct motif from the disulfide-bonded sequon present in the ComP protein (Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203). Based on sequence alignment with P. aeruginosa 1244 PilA, the DSL of M. osloensis pilin is located at residue Cys. 148 and Cys 164 Therefore, the inventors have determined that the sequence downstream of the glycine linker is Cys148 It was designed to start with EPA-Pil 20 The plasmid containing the construct encoding Pil and TfpM was named pVNM297. 20 Glycosylation experiments of Pil Mo TfpM with CPS8 at levels similar to Δ28 Mo It was revealed that the DSL region can be glycosylated by α-Cys (Figure 7B). To test whether the DSL region is required for glycosylation, several short mutants lacking this feature were generated. In these small constructs, the inventors also removed Cys to prevent the formation of non-native disulfide bonds upon oxidation in the periplasm. 164 was mutated to alanine (Harvey, H., et al. (2009) Journal of Bacteriology 191, 6513-6524). Mo Mutants were tested, one of the 10-amino acid versions containing an amino acid linker (e.g., a GGGG linker) and one without. These constructs were each 15 , Pil 13 , Pil 10L , Pil 10 It was named TfpM (Figure 8A). Mo Pil 20 and Pil Mo All four of these mutants were able to glycosylate, albeit at lower levels than Δ28 (Fig. 5B). 10 and Pil 10L Both are similarly glycosylated, and the presence of an upstream glycine linker is Mo This linker was omitted from all subsequent constructs. 13 was significantly less glycosylated than the other proteins.

[0066] Sequence alignment revealed that each pilin protein glycosylated by TfpM OTase contains a conserved "PAN / ECRG" (SEQ ID NO: 200) motif near the C-terminus, immediately upstream of the penultimate threonine residue (Figure 9). The presence of this feature in all glycosylated pilins suggests that this motif is a TfpM Mo The inventors have investigated whether Pil is required for glycosylation by PIL, which consists of a similar motif ("PANARGT" (SEQ ID NO: 179) modified with a cysteine ​​mutated to an alanine (bold residue)). Mo A 7-amino acid mutant of "PANARGT" (SEQ ID NO: 179) (designated Pil7) was fused to EPA and glycosylation was assessed (Figure 8A). Thus, in certain embodiments, a glycosylation motif of the invention can contain an N, E, or A at the fourth position from the C-terminus (e.g., corresponding to the N / E position in SEQ ID NO: 200 and the second A position in SEQ ID NO: 179). We also constructed stepwise single amino acid truncations of this "PANARGT" (SEQ ID NO: 179) sequence from 7 amino acids to 2 amino acids and assessed the ability of TfpM to glycosylate these fragments with CPS8. Results showed that all mutants except Pil2 were able to glycosylate Pil 10 TfpM at similar levels Mo The overall glycosylation was confirmed by Pil 20 and Pil Mo The glycosylated Pil2 was barely detectable, but some traces of ladder were visible on increasing exposure of the Western blot. This is due to the presence of TfpM. Mo These results demonstrate that TfpM can glycosylate this mutant, but to a much lower level than the construct containing the single additional amino acid. Mo When fused to the C-terminus of a heterologous EPA protein, the 3-amino acid Pil Mo It was concluded that the fragment could be recognized and glycosylated.

[0067] Immunogenicity of TfpMMo-derived GBSIII bioconjugates. EPA-Pil 20 Given that the construct was efficiently glycosylated by TfpM, we next synthesized EPA-Pil glycosylated with type III capsular polysaccharide from group B Streptococcus (GBSIII). 20 The protein was evaluated for its immunogenic potential in a mouse vaccination model. To aid in protein purification for these experiments, EPA-Pil was used. 20 A plasmid expressing an N-terminal 6x-His tagged variant of the carrier protein (pVNM291) was constructed. The His tag was added immediately downstream of the predicted N-terminal DsbA signal sequence cleavage site of EPA. pVNM291 was transfected into SDB1 cells expressing GBSIII glycans, and the resulting bioconjugate was purified by nickel-immobilized metal affinity chromatography (IMAC), followed by anion exchange and size exclusion chromatography on an FPLC. Western blot and Coomassie staining of the GBSIII-291 bioconjugate separated by SDS-PAGE demonstrated the EPA-Pil 20 High molecular weight glycosylation of the protein and GBSIII glycans was confirmed (Figures 10A, 10B, 10C, and 10D). 20 Intact protein MS of the GBSIII-GBSIII ("GBSIII-291") conjugate supported a 20% glycan:protein ratio (Figure 10E). Each dose was formulated to contain 1 μg of GBSIII polysaccharide. As a control for these experiments, non-glycosylated pVNM291-derived carrier protein ("291") was purified from SDB1 cells without the glycan plasmid and administered at the same protein concentration as the GBSIII bioconjugate.

[0068] TfpM MoTo test the immunogenicity of the GBSIII-291 bioconjugate produced in this study, we immunized 5-week-old female CD-1 mice. Mice received either placebo (non-glycosylated 291 carrier protein) or the GBSIII-291 bioconjugate at 2-week intervals, starting with a priming dose followed by two booster doses. All vaccines were formulated using Alhydrogel® 2% as an adjuvant at a 1:9 ratio. Serum was collected before each immunization and 2 weeks after the final booster dose. To measure the levels of GBSIII-specific antibodies elicited, we used enzyme-linked immunosorbent assay (ELISA). All mice immunized with bioconjugate GBSIII-291 expressed high levels of anti-GBSIII IgG antibodies, although one mouse with a low anti-GBSIII IgG response was able to be boosted during the immunization process (Figure 10F). As expected, GBSIII-specific IgG titers were increased in GBSIII bioconjugate-vaccinated mice compared to mock-vaccinated mice (291 only, Figure 10F). Taken together, these data suggest that TfpM Mo These results suggest that bioconjugates capable of eliciting polysaccharide-specific IgG responses can be generated.

[0069] TfpM Mo and PglS ADP1 (PglL ComP ) glycosylate a single protein designed to contain a sequon unique to each oligosaccharyltransferase. Finally, we wished to determine whether a protein designed to contain sequons from two different OTase systems would be glycosylated by both OTases at each site. To this end, we constructed an EPA fusion protein containing a sequon associated with TfpM and a sequon associated with PglS. To this end, we used the Ala residue as previously described. 489 and Arg 490The PglS sequon (CTGVTQIASGASAATTNVASAQC) (SEQ ID NO: 59) inserted between the C-terminal Pil 20 An EPA fusion protein containing the sequon (CGGTGTTVAAKFLPANCRGT) (SEQ ID NO: 60, identical to Pil DSL) was designed (Figure 11A). As previously described, this construct was designed so that the open reading frame of the gene encoding the EPA fusion overlapped the start codon of tfpM by one nucleotide. The open reading frame encoding pglS from Acinetobacter baylyi ADP1 was cloned 100 bp downstream of the tfpM open reading frame stop codon. This vector (pVNM337) was introduced into E. coli expressing the E. coli O16 antigen, and glycosylation was examined by Western blotting. To compare with proteins containing only a single sequon, we individually introduced the following constructs into E. coli SDB1 expressing the O16 antigen: (i) TfpM-associated Pil 20 EPA (pVNM297) containing only the sequon, or (ii) residue Ala 489 and Arg 490 EPA (pVNM167) containing the PglS sequon inserted between residues Ala and β. 489 and Arg 490 Between and residue Glu 548 and Gly 549 An EPA construct containing a sequon integrated between ComP or Pil was also introduced (pVNM245) (EPA_PglS sequon 2X). A diagram of these structures is shown in Figure 11A. As shown in Figure 11B, MoWestern blot analysis of EPA constructs containing only one of the sequons showed a glycosylation profile at approximately 100 kDa, suggesting monoglycosylation. EPA constructs containing two PglS sequons showed a predominantly monoglycosylated profile around 100 kDa, but also a diglycosylated population migrating around 150 kDa. Mo and PglS ADP1 Western blot analysis of EPA fusions containing sequons from displayed both mono- and diglycosylated populations, similar to those seen in construct pVNM245. From these results, it was concluded that receptor proteins can be glycosylated by two different OTase classes in one expression system.

[0070] Complex carbohydrates The present disclosure provides glycoconjugates comprising an oligosaccharide or polysaccharide covalently bound to a receptor protein. In certain embodiments, the receptor protein comprises or consists of a TfpM-associated pilin-like protein or glycosylated fragment thereof of the present disclosure. In certain embodiments, the oligosaccharide or polysaccharide is covalently bound to the pilin-like protein or glycosylated fragment thereof. Also, in certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment thereof comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently bound to the C-terminal serine or threonine. Furthermore, in certain embodiments, the receptor protein is a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof translationally fused / conjugated to a heterologous amino acid sequence (e.g., a carrier protein), where the TfpM-associated pilin-like protein or a glycosylated fragment thereof is the most C-terminal sequence of the receptor protein, such that the receptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently attached to the C-terminal serine or threonine. Specific examples of carrier proteins include, but are not limited to, Pseudomonas aeruginosa exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, or any fragment thereof. In certain embodiments, the TfpM-associated pilin-like protein or a glycosylated fragment thereof is translationally fused / conjugated to the heterologous amino acid sequence / carrier protein via an amino acid linker. In certain embodiments, the oligosaccharide or polysaccharide comprises a glucose at its reducing end. In certain embodiments, the glycoconjugates are immunogenic.

[0071] In certain embodiments of the glycoconjugates of the present disclosure, the receptor protein comprises or consists of a full-length TfpM-associated pilin-like protein. In certain embodiments, the receptor protein comprises or consists of a glycosylated fragment of a TfpM-associated pilin-like protein that is shorter than the full-length TfpM-associated pilin-like protein. In certain embodiments, the glycosylated fragment of a TfpM-associated pilin-like protein comprises a C-terminal serine or threonine residue. In certain embodiments, the glycosylated fragment of a pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin. In certain embodiments, the length of the glycosylated fragment of a pilin-like protein is between 3 and 138 amino acids, between 10 and 138 amino acids, between 20 and 138 amino acids, between 50 and 138 amino acids, between 100 and 138 amino acids, or between 116 and 138 amino acids. In certain embodiments, the length of a glycosylated fragment of a pilin-like protein is 3 to 139 amino acids, 10 to 139 amino acids, 20 to 139 amino acids, 50 to 139 amino acids, 100 to 139 amino acids, or 116 to 139 amino acids. In certain embodiments, the length of a glycosylated fragment is 3 to 140 amino acids, 10 to 140 amino acids, 20 to 140 amino acids, 50 to 140 amino acids, 100 to 140 amino acids, or 116 to 140 amino acids. In certain embodiments, the length of a glycosylated fragment is 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 3 to 21 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids. In certain embodiments, the glycosylated fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.In certain embodiments, the length of the glycosylated fragment is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0072] In certain embodiments of the glycoconjugates of the present disclosure, the TfpM-associated pilin-like protein or glycosylated fragment thereof is Pil Mo (SEQ ID NO: 57), or Pil lacking the amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or a polypeptide comprising 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, e.g., where the C-terminal threonine is replaced with serine. In certain embodiments of the glycoconjugates of the present disclosure, the TfpM-associated pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment of a pilin-like protein is selected from the group consisting of Pil DSM16617(SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment (e.g., C-terminal fragment) and / or variant thereof, wherein the C-terminal threonine is replaced with serine. In certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of an amino acid sequence selected from the group consisting of Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148). Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or variants thereof having one, two, three, four, or five amino acid substitutions and maintaining a C-terminal serine. In any of the above cases, the glycosylated fragment of the pyrin-like protein comprises at least the last three amino acids from the pyrin C-terminus (RGT) or at least the last three amino acids from the pyrin C-terminus (RGS) except that the C-terminal threonine is replaced with serine. Furthermore, in certain embodiments, the glycosylated fragment of the pyrin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin.

[0073] In certain embodiments of glycoconjugates of the present disclosure, the acceptor protein can be glycosylated at two or more different positions. In certain embodiments, the acceptor protein can be glycosylated by at least two different OTase classes in a single expression system. For example, in certain embodiments, the acceptor protein is a fusion protein that, in addition to a glycosylation fragment of a TfpM-related pilin-like protein located at its C-terminus, further comprises an additional glycosylation sequence (e.g., a glycosylation fragment) of an OTase other than the TfpM oligosaccharyltransferase (OTase). For example, the other OTase can be PglB, PglL, or PglS. In certain embodiments, the additional glycosylation sequence is an internal sequence of the fusion protein (i.e., not the majority of the C- or N-terminal sequence). In certain embodiments, the additional glycosylation sequence is an internal sequence within the sequence of the carrier protein (e.g., Figure 11A). In certain embodiments, the additional glycosylation sequence is also covalently attached to an oligosaccharide or polysaccharide. In certain embodiments, the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, three or more, five or more, ten or more, fifteen or more, twenty or more additional glycosylation sequences. In certain embodiments, the fusion protein comprises more than two, three or more, five or more, ten or more, fifteen or more, twenty or more, or twenty or more additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical. In certain embodiments, at least one additional glycosylation sequence is different from the others. In certain embodiments, at least three, at least four, or at least five of the additional glycosylation sequences are all different from one another. In certain embodiments, none of the additional glycosylation sequences are the same. In certain embodiments, the receptor protein is a fusion protein, and the fusion protein further comprises an internal glycosylation fragment of ComP in addition to a glycosylation fragment of a TfpM-related pilin-like protein located at its C-terminus. In certain embodiments, the ComP glycosylation fragment is present within the sequence of the carrier protein. In certain embodiments, the ComP glycosylated fragment is also covalently linked to an oligosaccharide or polysaccharide.Also, in certain embodiments, the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least amino acids ASA at positions 11-13. In certain embodiments, the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more ComP glycosylation fragments. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty-five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments are different from one another. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments are all different from one another. In certain embodiments, none of the ComP glycosylation fragments are the same.

[0074] In certain embodiments of the glycoconjugates of the present disclosure, the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least 3 repeating units of the oligosaccharide or polysaccharide structure. In certain embodiments of the glycoconjugates of the present disclosure, the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least 10 monosaccharides.

[0075] In certain embodiments of the glycoconjugates of the present disclosure, the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus (e.g., S. pneumoniae or S. agalactiae), and the polysaccharide is a capsular polysaccharide, such as type Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX.

[0076] In certain embodiments of the glycoconjugates of the present disclosure, the oligosaccharide or polysaccharide is produced by Klebsiella (e.g., K. pneumoniae), and the polysaccharide is a capsular polysaccharide or an O-antigen polysaccharide.

[0077] In certain embodiments of the glycoconjugates of the present disclosure, the oligosaccharide or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is an O-antigen polysaccharide, hi certain embodiments, the bacterium is S. enterica and the S. enterica polysaccharide is a group B O-antigen.

[0078] In certain embodiments of the glycoconjugates of the present disclosure, the glycoconjugates are produced in vivo, such as in bacterial cells. In certain embodiments, the bacterium is Escherichia coli. In certain embodiments, the bacterium is from the genus Klebsiella. In certain embodiments, the bacterial species is Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca. In certain embodiments, the glycoconjugates are produced in a cell-free system.

[0079] In certain embodiments of the glycoconjugates of the present disclosure, the bioconjugates are conjugate vaccines that induce an immune response when administered to a subject. In certain embodiments, the immune response induces long-term memory (memory B cells and T cells) and is an antibody response, optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, the antibody response is an IgG response, e.g., an IgG1 response. Also, in certain embodiments, the conjugate vaccine generates immune memory in a subject administered the vaccine.

[0080] Glycosylated fragment The present disclosure provides glycosylated fragments of pilin-like proteins that comprise or consist of isolated fragments of the TfpM-associated pilin-like proteins of the present disclosure. In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment of the pilin-like protein is Pil. Mo (SEQ ID NO: 57), or Pil lacking the amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or a polypeptide comprising 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the C-terminal threonine is replaced by serine. In certain embodiments of the glycoconjugates of the present disclosure, the TfpM-related pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment of a pilin-like protein is selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment (e.g., C-terminal fragment) and / or variant thereof, wherein the C-terminal threonine is replaced with serine. In certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of an amino acid sequence selected from the group consisting of Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148). Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10(SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or variants thereof having one, two, three, four, or five amino acid substitutions and maintaining a C-terminal serine. In any of the above cases, the glycosylated fragment of the pyrin-like protein comprises at least the last three amino acids from the pyrin C-terminus (RGT) or at least the last three amino acids from the pyrin C-terminus (RGS) except that the C-terminal threonine is replaced with serine. Furthermore, in certain embodiments, the glycosylated fragment of the pyrin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A](SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin.

[0081] In certain embodiments of glycosylated fragments of the invention, the length of an isolated fragment of a TfpM-associated pilin-like protein of the disclosure is between 3 and 138 amino acids, between 10 and 138 amino acids, between 20 and 138 amino acids, between 50 and 138 amino acids, between 100 and 138 amino acids, or between 116 and 138 amino acids. In certain embodiments, the length of a glycosylated fragment is between 3 and 139 amino acids, between 10 and 139 amino acids, between 20 and 139 amino acids, between 50 and 139 amino acids, between 100 and 139 amino acids, or between 116 and 139 amino acids. In certain embodiments, the length of a glycosylated fragment is between 3 and 140 amino acids, between 10 and 140 amino acids, between 20 and 140 amino acids, between 50 and 140 amino acids, between 100 and 140 amino acids, or between 116 and 140 amino acids. In certain embodiments, the length of the glycosylated fragment is between 3 and 22 amino acids, between 10 and 22 amino acids, between 11 and 22 amino acids, between 5 and 21 amino acids, between 10 and 21 amino acids, or between 11 and 21 amino acids. In certain embodiments, the glycosylated fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylated fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In certain embodiments, the glycosylated fragment of a TfpM-associated pilin-like protein comprises a C-terminal serine or threonine residue. In certain embodiments, the glycosylated fragment of a pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin.

[0082] Fusion proteins Provided herein are fusion proteins comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof translationally fused / conjugated to a heterologous carrier protein, such as, but not limited to, Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, or any fragment thereof. In certain embodiments, the TfpM-associated pilin-like protein or a glycosylated fragment thereof is translationally fused / conjugated to the heterologous carrier protein via an amino acid linker. In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue. In certain embodiments, the pilin-like protein or glycosylated fragment is the most C-terminal sequence of the fusion protein. Also, in certain embodiments, the fusion protein comprises a C-terminal serine or threonine residue. In certain embodiments, the fusion protein is glycosylated with an oligosaccharide or polysaccharide covalently attached to the C-terminal serine or threonine. Furthermore, in certain embodiments, the fusion protein is glycosylated with an oligosaccharide or polysaccharide that contains glucose at its reducing end and is covalently linked to the C-terminal serine or threonine.In certain embodiments, the glycosylated fusion protein induces an immune response.In certain embodiments, the glycosylated fusion protein is a conjugate vaccine.

[0083] In certain embodiments of fusion proteins of the present disclosure, the fusion protein comprises a full-length TfpM-associated pilin-like protein. In certain embodiments, the fusion protein comprises or consists of a glycosylated fragment of a TfpM-associated pilin-like protein that is shorter than the full-length TfpM-associated pilin-like protein. In certain embodiments, the glycosylated fragment of a TfpM-associated pilin-like protein comprises a C-terminal serine or threonine residue. In certain embodiments, the glycosylated fragment of a pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin. In certain embodiments, the length of the glycosylated fragment of a pilin-like protein is 3 to 138 amino acids, 10 to 138 amino acids, 20 to 138 amino acids, 50 to 138 amino acids, 100 to 138 amino acids, or 116 to 138 amino acids. In certain embodiments, the length of the glycosylated fragment is 3 to 139 amino acids, 10 to 139 amino acids, 20 to 139 amino acids, 50 to 139 amino acids, 100 to 139 amino acids, or 116 to 139 amino acids. In certain embodiments, the length of the glycosylated fragment is 3 to 140 amino acids, 10 to 140 amino acids, 20 to 140 amino acids, 50 to 140 amino acids, 100 to 140 amino acids, or 116 to 140 amino acids. In certain embodiments, the length of the glycosylated fragment is 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids. In certain embodiments, the glycosylated fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.In certain embodiments, the length of the glycosylated fragment is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0084] In certain embodiments of the fusion proteins of the present disclosure, the glycosylated fragment of a TfpM-associated pilin-like protein or pilin-like protein is Pil Mo (SEQ ID NO: 57), or Pil lacking the amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or a polypeptide comprising 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the C-terminal threonine is replaced by serine. DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment of a pilin-like protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment (e.g., C-terminal fragment) and / or variant thereof, wherein the C-terminal threonine is replaced with serine. Also, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of an amino acid sequence selected from the group consisting of PilMo pilin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148). Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or variants thereof having one, two, three, four, or five amino acid substitutions and maintaining a C-terminal serine. In any of the above cases, the glycosylated fragment of the pyrin-like protein comprises at least the last three amino acids from the pyrin C-terminus (RGT) or at least the last three amino acids from the pyrin C-terminus (RGS) except that the C-terminal threonine is replaced with serine. Furthermore, in certain embodiments, the glycosylated fragment of the pyrin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin.

[0085] In certain embodiments of the fusion proteins of the present disclosure, the receptor protein can be glycosylated at two or more different positions. In certain embodiments, the fusion protein can be glycosylated by at least two different OTase classes in a single expression system. For example, in certain embodiments, the fusion protein further comprises, in addition to the glycosylation fragment of a TfpM-related pilin-like protein located at the C-terminus, a glycosylation sequence (e.g., a glycosylation fragment) of an OTase other than the TfpM oligosaccharyltransferase (OTase). For example, the other OTase can be PglB, PglL, or PglS. In certain embodiments, the additional glycosylation sequence is an internal sequence of the fusion protein (i.e., not the C- or N-terminal majority sequence). In certain embodiments, the additional glycosylation sequence is an internal sequence within the sequence of the carrier protein (e.g., Figure 11A). In certain embodiments, the additional glycosylation sequence is also covalently attached to an oligosaccharide or polysaccharide. In certain embodiments, the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, three or more, five or more, ten or more, fifteen or more, twenty or more additional glycosylation sequences. In certain embodiments, the fusion protein comprises more than two, three or more, five or more, ten or more, fifteen or more, twenty or more, or twenty or more additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical. In certain embodiments, at least one additional glycosylation sequence is different from the others. In certain embodiments, at least three, at least four, or at least five of the additional glycosylation sequences are all different from one another. In certain embodiments, none of the additional glycosylation sequences are the same. In certain embodiments, the fusion protein further comprises an internal glycosylation fragment of ComP in addition to a glycosylation fragment of a TfpM-related pilin-like protein located at its C-terminus. In certain embodiments, the ComP glycosylation fragment is also covalently linked to an oligosaccharide or polysaccharide. Also, in certain embodiments, the ComP glycosylated fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least amino acids ASA at positions 11-13.In certain embodiments, the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more ComP glycosylation fragments. In certain embodiments, the fusion protein does not comprise more than two, three or more, five or more, ten or more, fifteen or more, twenty or more, or twenty-five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments are different from one another. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments are all different from one another. In certain embodiments, none of the ComP glycosylation fragments are the same.

[0086] In certain embodiments of the fusion proteins of the present disclosure, the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least 3 repeating units of the oligosaccharide or polysaccharide structure. In certain embodiments of the fusion proteins of the present disclosure, the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least 10 monosaccharides.

[0087] In certain embodiments of the fusion proteins of the present disclosure, the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus (e.g., S. pneumoniae or S. agalactiae), and the polysaccharide is a capsular polysaccharide, such as Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX.

[0088] In certain embodiments of the fusion proteins of the present disclosure, the oligosaccharide or polysaccharide is produced by Klebsiella (e.g., K. pneumoniae), and the polysaccharide is a capsular polysaccharide or an O-antigen polysaccharide.

[0089] In certain embodiments of the fusion proteins of the present disclosure, the oligosaccharide or polysaccharide is produced by a bacterium of the genus Salmonella and the polysaccharide is an O-antigen polysaccharide, hi certain embodiments, the bacterium is Salmonella enterica and the S. enterica polysaccharide is a group B O-antigen.

[0090] In certain embodiments of the fusion proteins of the present disclosure, the glycosylated fusion protein is produced in vivo, such as in a bacterial cell. In certain embodiments, the bacterium is Escherichia coli. In certain embodiments, the bacterium is from the Klebsiella genus. In certain embodiments, the bacterial species is Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca.

[0091] In certain embodiments of the fusion proteins of the present disclosure, the fusion protein is a vaccine that elicits an immune response when administered to a subject. In certain embodiments, the immune response elicits long-term memory (memory B cells and T cells) and is an antibody response, optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, the antibody response is an IgG response, e.g., an IgG1 response. Also, in certain embodiments, the fusion protein generates immune memory in a subject to which the fusion protein is administered.

[0092] Methods for producing complex carbohydrates Provided herein are methods for producing glycoconjugates. In certain embodiments, the methods are performed in vivo. In certain aspects, the glycoconjugates are produced in a cell-free system. An example of the use of a cell-free system utilizing an OTase other than TfpM is described in WO 2013 / 067523 A1, which is incorporated herein by reference. In certain embodiments, the methods comprise using a TfpM oligosaccharyltransferase (OTase) of the present disclosure to covalently attach (conjugate) an oligosaccharide or polysaccharide to an acceptor protein comprising or consisting of a TfpM-associated pilin-like protein or a glycosylated fragment thereof. In certain embodiments, the pilin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue, the acceptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently attached to the C-terminal serine or threonine residue of the acceptor protein. In certain embodiments, the oligosaccharide or polysaccharide comprises glucose at its reducing end. In certain embodiments, the receptor protein is a fusion protein of the present disclosure, as described in detail elsewhere herein, and in certain embodiments, the glycoconjugate is immunogenic.

[0093] In certain embodiments of the disclosed glycoconjugate production methods, or other compositions or methods disclosed herein, the TfpM OTase contains a wzy_C superfamily domain, an O-antigen ligase domain as defined by the National Council of Science (NCBI) conserved protein domain family cl04850, and / or the TfpM OTase contains a wzy_C domain, an O-antigen ligase domain as defined by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI, EMBL-EBI) protein families (pfam) conserved protein domain family motif pfam04932, where pfam04932 is a protein domain family within the cl04850 superfamily protein domain. In certain embodiments, the TfpM OTase is a TfpM Mo (SEQ ID NO: 56), TfpM DSM16617(SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2(SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), or TfpM 72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase is TfpM Mo (SEQ ID NO: 56).

[0094] In certain embodiments of the disclosed methods for producing glycoconjugates, the acceptor protein comprises or consists of a full-length TfpM-associated pilin-like protein. In certain embodiments, the acceptor protein comprises or consists of a glycosylated fragment of a TfpM-associated pilin-like protein that is shorter than the full-length TfpM-associated pilin-like protein. In certain embodiments, the glycosylated fragment of a TfpM-associated pilin-like protein comprises a C-terminal serine or threonine residue. In certain embodiments, the glycosylated fragment of a pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin. In certain embodiments, the length of the glycosylated fragment of a pilin-like protein is between 3 and 138 amino acids, between 10 and 138 amino acids, between 20 and 138 amino acids, between 50 and 138 amino acids, between 100 and 138 amino acids, or between 116 and 138 amino acids. In certain embodiments, the length of a glycosylated fragment of a pilin-like protein is 3 to 139 amino acids, 10 to 139 amino acids, 20 to 139 amino acids, 50 to 139 amino acids, 100 to 139 amino acids, or 116 to 139 amino acids. In certain embodiments, the length of a glycosylated fragment is 3 to 140 amino acids, 10 to 140 amino acids, 20 to 140 amino acids, 50 to 140 amino acids, 100 to 140 amino acids, or 116 to 140 amino acids. In certain embodiments, the length of a glycosylated fragment is 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids. In certain embodiments, the glycosylated fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.In certain embodiments, the length of the glycosylated fragment is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0095] In certain embodiments of the disclosed methods of producing glycoconjugates, the TfpM-associated pilin-like protein or glycosylated fragment of a pilin-like protein is Pil Mo (SEQ ID NO: 57), or Pil lacking the amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or a polypeptide comprising 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the C-terminal threonine is replaced by serine. DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment of a pilin-like protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment (e.g., C-terminal fragment) and / or variant thereof, wherein the C-terminal threonine is replaced with serine. Also, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of an amino acid sequence selected from the group consisting of PilMo pilin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148). Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or variants thereof having one, two, three, four, or five amino acid substitutions and maintaining a C-terminal serine. In any of the above cases, the glycosylated fragment of the pyrin-like protein comprises at least the last three amino acids from the pyrin C-terminus (RGT) or at least the last three amino acids from the pyrin C-terminus (RGS) except that the C-terminal threonine is replaced with serine. Furthermore, in certain embodiments, the glycosylated fragment of the pyrin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin.

[0096] In certain embodiments of the disclosed methods for producing glycoconjugates, the acceptor protein is a fusion protein and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof. In certain embodiments, the TfpM-related pilin-like protein or glycosylated fragment thereof is translationally fused / conjugated to the heterologous carrier protein via an amino acid linker.

[0097] In certain embodiments of the disclosed methods for producing glycoconjugates, the acceptor protein is a fusion protein, and the method comprises glycosylating the acceptor protein at two or more different positions. In certain embodiments, the method comprises glycosylating the acceptor protein using at least two different OTase classes in a single expression system. In certain embodiments, the fusion protein comprises two or more glycosylation sequences (e.g., glycosylation fragments) associated with at least two different OTases. Representative examples of OTases that can be used in combination include PglB, PglL, PglS, TfpO, and TfpM. One of skill in the art will recognize that OTases cannot be used together if both OTases require a glycosylation sequence (sequon) at the same position, e.g., both N-terminus or both C-terminus. For example, TfpO and TfpM typically cannot be used together if both require a sequon at the C-terminus. For example, in certain non-limiting exemplary embodiments, the receptor protein comprises a glycosylation fragment of a TfpM-related pilin-like protein located at its C-terminus, in addition to an additional glycosylation sequence of an OTase other than the TfpM oligosaccharyltransferase (OTase). In certain embodiments, the other OTase is PglB, PglL, and / or PglS. In certain embodiments, one or more glycosylation sequences are internal sequences of the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, one or more glycosylation sequences are internal sequences within the sequence of the carrier protein (e.g., FIG. 11A). In certain embodiments, the additional glycosylation sequence is internal sequences of the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, the additional glycosylation sequence is internal sequences within the sequence of the carrier protein (e.g., FIG. 11A). In certain embodiments, at least two different glycosylation sequences of two different OTase systems are covalently attached to an oligosaccharide or polysaccharide. In certain embodiments, the glycosylation fragment of the TfpM-related pilin-like protein and the additional glycosylation sequence located at the C-terminus of the fusion protein are covalently linked to an oligosaccharide or polysaccharide.In certain embodiments, the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, three or more, five or more, ten or more, fifteen or more, twenty or more additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical. In certain embodiments, at least one additional glycosylation sequence is different from the others. In certain embodiments, at least three, at least four, or at least five of the additional glycosylation sequences are all different from one another. In certain embodiments, none of the additional glycosylation sequences are the same. For example, in certain embodiments, the method comprises, in addition to glycosylating a C-terminally located glycosylated fragment of the TfpM-related pilin-like protein, further glycosylation of an internal glycosylation fragment of ComP using PglS OTase. In certain embodiments, the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least amino acids ASA at positions 11-13. In certain embodiments, the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more ComP glycosylation fragments. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty-five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments are different from one another. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments are all different from one another. In certain embodiments, none of the ComP glycosylation fragments are the same.

[0098] In certain embodiments of the disclosed methods for producing glycoconjugates, (e.g., conjugation) occurs in vivo within a host cell. In certain embodiments, the host cell is a bacterial cell. In certain embodiments, conjugation occurs in Escherichia coli. In certain embodiments, conjugation occurs in a bacterium from the genus Klebsiella. In certain embodiments, the bacterial species is Klebsiella pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0099] In certain embodiments of the disclosed methods for producing glycoconjugates, the methods comprise culturing a host cell comprising (a) a gene cluster encoding proteins necessary to synthesize oligosaccharides or polysaccharides, (b) a TfpM OTase, and (3) a receptor protein.

[0100] In certain embodiments of the methods of producing glycoconjugates of the present disclosure, the methods produce conjugate vaccines.

[0101] Additional Embodiments Provided herein are host cells comprising (a) a gene cluster encoding proteins necessary for synthesizing oligosaccharides or polysaccharides, (b) a TfpM OTase of the present disclosure, and (3) a receptor protein comprising a TfpM-related pilin-like protein of the present disclosure, or a glycosylated fragment thereof. In certain embodiments, the receptor protein is a fusion protein. In certain embodiments, the host cell comprises a nucleic acid encoding the TfpM OTase. In certain embodiments, the host cell comprises a nucleic acid encoding the receptor protein. Also, in certain embodiments, the TfpM OTase and the receptor protein are encoded by the same nucleic acid.

[0102] Provided herein are isolated nucleic acids encoding glycosylated fragments and / or fusion proteins of the pilin-like proteins of the present disclosure. In certain embodiments, the nucleic acid is a vector. Also provided are host cells comprising the isolated nucleic acids of the present disclosure. In certain embodiments, the host cell is a bacterial cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is from the genus Klebsiella. Also, in certain embodiments, the host cell is Klebsiella pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0103] Provided herein are compositions comprising a conjugate vaccine or fusion protein of the present disclosure and an adjuvant and / or carrier. In certain embodiments, the composition is a pharmaceutical or therapeutic composition suitable for administration to a subject / patient.

[0104] Provided herein are methods for inducing a host immune response against a bacterial pathogen, comprising administering to a subject in need thereof an effective amount of a conjugate vaccine, fusion protein, or composition comprising the conjugate vaccine or fusion protein of the present disclosure and an adjuvant and / or carrier. Treatment with a pharmaceutical composition comprising an immunogenic composition can be administered separately or in combination with other treatments, as needed. An amount sufficient to achieve this is defined as an "effective amount," "effective dose," or "unit dose." Amounts effective for this purpose will vary depending, for example, on the composition of the glycoconjugate, the method of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician. In some aspects, a primary antigen administration is followed by booster administrations over a period of time. In certain embodiments, the immune response is an antibody response. In certain embodiments, the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, a cellular response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system crosstalk, and a combination of two or more of the immune responses. In certain embodiments, the immune response is selected from the group consisting of an innate response, a humoral response, an antibody response, a T cell response, and a combination of two or more of an immune response.

[0105] Provided herein are methods of preventing or treating bacterial and / or infectious diseases in a subject, the methods comprising administering to a subject in need thereof an effective amount of a conjugate vaccine, fusion protein, or a composition comprising a conjugate vaccine or fusion protein of the present disclosure and an adjuvant and / or carrier. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a companion animal. In certain embodiments, the subject is a livestock animal. In certain embodiments, the infection is a local or systemic infection of the skin, soft tissue, blood, or organ, or is autoimmune. In certain embodiments, the disease is pneumonia. In certain embodiments, the infection is a systemic and / or blood infection. In certain embodiments, the conjugate vaccine, fusion protein, or composition is administered by intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

[0106] Provided herein are methods for producing a pneumococcal conjugate vaccine against pneumococcal infection, comprising: (a) isolating a glycoconjugate or glycosylated fusion protein of the present disclosure; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and / or carrier.

[0107] Provided herein are glycoconjugates, glycosylated fusion proteins, or conjugate vaccines of the present disclosure, or any composition thereof, for use in inducing a host immune response against a bacterial pathogen and / or for preventing or treating bacterial disease and / or infection in a subject.

[0108] Provided herein are recombinant nucleic acid constructs comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence. In certain embodiments, the TfpM OTase is Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase is TfpM Mo (SEQ ID NO: 56). In certain embodiments, the heterologous transcriptional regulatory sequence is a promoter sequence. In certain embodiments, the recombinant nucleic acid construct further comprises a fusion protein of the present disclosure comprising a nucleotide sequence encoding a TfpM-associated pilin-like protein or a glycosylated fragment thereof of the present disclosure, or a TfpM-associated pilin-like protein or a glycosylated fragment thereof operably linked to a nucleotide sequence encoding a TfpM OTase. In certain embodiments, the recombinant nucleic acid construct further comprises a fusion protein of the present disclosure comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof 5' to the nucleotide sequence encoding the TfpM OTase and operably linked to the nucleotide sequence. In certain embodiments, the fusion protein of the construct also comprises the glycosylation sequence of an OTase other than TfpM, such as PglB, PglL, or PglS (e.g., ComP or a glycosylated fragment thereof). In certain embodiments, the coding sequence for a TfpM-associated pilin-like protein or a glycosylated fragment thereof, or a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof, is within 2, 5, 10, 20, 30, 40, or 50 nucleotides of the sequence encoding the TfpM OTase. In certain embodiments, the coding sequence for a TfpM-associated pilin-like protein or a glycosylated fragment thereof, or a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof overlaps with an operably linked nucleotide sequence encoding the TfpM OTase.

[0109] In certain embodiments, the TfpM-associated pyrin-like protein comprises or consists of a full-length TfpM-associated pyrin-like protein. In certain embodiments, the TfpM-associated pyrin-like protein comprises or consists of a glycosylated fragment of a TfpM-associated pyrin-like protein that is shorter than the full-length TfpM-associated pyrin-like protein. In certain embodiments, the glycosylated fragment of a TfpM-associated pyrin-like protein comprises a C-terminal serine or threonine residue. In certain embodiments, the glycosylated fragment of a pyrin-like protein comprises at least the last three amino acids from the C-terminus of the pyrin. In certain embodiments, the length of the glycosylated fragment of a pyrin-like protein is 3 to 138 amino acids, 10 to 138 amino acids, 20 to 138 amino acids, 50 to 138 amino acids, 100 to 138 amino acids, or 116 to 138 amino acids. In certain embodiments, the length of a glycosylated fragment of a pilin-like protein is 3 to 139 amino acids, 10 to 139 amino acids, 20 to 139 amino acids, 50 to 139 amino acids, 100 to 139 amino acids, or 116 to 139 amino acids. In certain embodiments, the length of a glycosylated fragment is 3 to 140 amino acids, 10 to 140 amino acids, 20 to 140 amino acids, 50 to 140 amino acids, 100 to 140 amino acids, or 116 to 140 amino acids. In certain embodiments, the length of a glycosylated fragment is 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids. In certain embodiments, the glycosylated fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.In certain embodiments, the length of the glycosylated fragment is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0110] In certain embodiments, the glycosylated fragment of a pilin-like protein is Pil Mo (SEQ ID NO: 57), or Pil lacking the amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or a polypeptide comprising 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the C-terminal threonine is replaced by serine. DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like protein or glycosylated fragment of a pilin-like protein is selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9(SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment (e.g., C-terminal fragment) and / or variant thereof, wherein the C-terminal threonine is replaced with serine. Also, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of an amino acid sequence selected from the group consisting of PilMo pilin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148). Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11(SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or variants thereof having one, two, three, four, or five amino acid substitutions and maintaining a C-terminal serine. In any of the above cases, the glycosylated fragment of the pyrin-like protein comprises at least the last three amino acids from the pyrin C-terminus (RGT) or at least the last three amino acids from the pyrin C-terminus (RGS) except that the C-terminal threonine is replaced with serine. Furthermore, in certain embodiments, the glycosylated fragment of the pyrin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A](SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin. Furthermore, in certain embodiments, the glycosylated fragment of a pilin-like protein comprises or consists of Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or variants thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining a C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of pilin.

[0111] In certain embodiments, the fusion protein is a fusion protein of the present disclosure. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding an additional OTase operably linked to the TpfM OTase, as described elsewhere herein. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding an additional OTase 3' of the TpfM OTase, operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding an additional OTase 5' of the TpfM OTase, operably linked to the TpfM OTase. In certain embodiments, the coding sequence for the additional OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 3' operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding the PglS OTase 3' of the TpfM OTase, operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding the PglS OTase 5' of the TpfM OTase, operably linked to the TpfM OTase. In certain embodiments, the coding sequence for PglS OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TpfM OTase.

[0112] Further provided herein are vectors comprising the recombinant nucleic acid constructs. Also provided herein are host cells comprising the recombinant nucleic acid constructs or vectors. In certain embodiments, the host cell is a bacterial cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is from the genus Klebsiella. Also, in certain embodiments, the host cell is Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca. Provided herein are methods for producing TfpM OTase, including culturing a host cell, wherein the vector is an expression vector, and recovering TfpM OTase. [Example]

[0113] Example 1. Cloning and Plasmid Assembly All primers and oligos used in this study are listed in Table 2. The concentrations of antibiotics used in liquid culture and LB agar plates were as follows: ampicillin (Amp), 100 μg / mL; kanamycin (Kan), 20 μg / mL; tetracycline (Tet), 10 μg / mL; and spectinomycin (Sp), 50 μg / mL. To clone the tfpM pilin OTase gene, HiFi gblocks (Integrated DNA Technologies, IDT) designed with terminal 25-base pair overlaps for Gibson assembly using PCR-linearized plasmids were ordered. The plasmid backbone of these fragments was amplified from the pEXT20 plasmid (Dykxhoorn, DM, et al. (1996) Gene 177, 133-136), which encodes the P. aeruginosa EPA gene under the control of the tac promoter (pVNM57) (Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203). The EPA gene lacks residue E553, resulting in inactivation of the toxin. The linearized plasmid was individually mixed with each of the synthesized tfpM gBlocks and assembled using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs, NEB). After assembly, the plasmid was transformed into Escherichia coli (E. coli) stellate cells (Takara Bio) by heat shock, grown at 37°C for 1 hour, and plated on LB agar plates with Amp. Individual colonies were picked and cultured in LB medium with the appropriate antibiotics, and plasmids were isolated using the GeneJet Plasmid Miniprep Kit (Thermo Fisher). All plasmids were sequence-verified by the Sanger method (Genewiz). M. osloensis 1202 EPA-PilΔ28 fusion and TfpM Mo The plasmid expressing Pil was named pVNM227. MoTo generate site-directed mutants, we designed overlapping PCR primers to introduce the necessary codon changes into the pilin gene and amplify each fragment from the pVNM227 plasmid. The resulting PCR products were digested with DpnI (NEB) for 30 minutes at 37°C and gel-purified from an agarose gel using a Pure-Link Gel Extraction Kit (Thermo Fisher Scientific). To insert the truncated pilin gene region, complementary oligos with terminal 25-bp overlaps homologous to the pVNM227 PCR product were ordered. The oligos were resuspended in purified water, mixed, and annealed by heating to 98°C for 5 minutes in a thermocycler, followed by slow cooling to 4°C at 0.1°C / min. The annealed oligos were diluted 1:5 with water and assembled with PCR-linearized pVNM227 using the NEBuilder HiFi DNA Assembly Kit (NEB). The resulting DNA was transformed into Stellar cells, and the plasmid was isolated and verified as described above. EPA-Pil 20 The plasmid containing the construct encoding TfpM was named pVNM297. 20 The mutant was generated by linearizing pVNM297 using PCR and Gibson assembly of this fragment with complementary annealed oligos containing the 6xHis coding region and terminal homology regions to obtain pVNM291. pVNM167 was constructed using the previously described EPA iGTccThe plasmid was generated by digesting with SalI (Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203). The purified SalI fragment was Gibson assembled with the pglS gene with its native 100-bp 5' UTR amplified from Acinetobacter baylyi ADP1 gDNA. pVNM245 was generated by a separate PCR reaction from the pVNM167 template to amplify a product with overhangs for Gibson assembly: (i) a vector backbone carrying pgl and EPA with one iGT, (ii) a second iGT for integration between E548 and G549, and (iii) the C-terminus of EPA downstream of iGT. Plasmid pVNM337 was created by amplifying tfpM from pVNM291 using primers EPA 3′F1 and pglS-tfpM R1 and cloning the product into PCR-linearized pVNM167 amplified with pglS 5′F1 and EPA 3′R1. Phylogeny trees of TfpM and pilin proteins were generated using the phylogeny.fr server (located on the World Wide Web at phylogeny.fr / ), which uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively.

[0114] Example 2. Glycan Expression and Cloning of Klebsiella pneumoniae O2a Glycan Genes Streptococcus pneumoniae (S. pneumoniae) CPS8 glycans were expressed on the plasmid pB8 (Tet R ) (Kay, EJ, et al. (2016) Open Biology 6, 150243), the Salmonella enterica LT2 glycan was cloned into the plasmid pPR1347 (Kan R ) (Neal, BL, et al. (1993) Journal of Bacteriology 175, 7115-7118), and the E. coli O16 wbbL gene was carried on the plasmid pMF19 (Sp R) (Feldman, MF, et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016), and GBSIII glycans were expressed from pBBR1MCS2 derivatives (Duke, JA, et al. (2021) ACS Infectious Diseases 7, 3111-3123). Bioconjugation with Klebsiella pneumoniae O2a O antigen has not been reported previously. To clone the genes encoding the machinery required for O2a glycan synthesis, PCR was used to amplify the wzm, wzt, wbbM, glf, wbbN, and wbbO genes (Clarke, BR, et al. (2018) Journal of Biological Chemistry 293, 4666-4679) (44) from K. pneumoniae strain NTUH K2044 genomic DNA. K. pneumoniae was grown overnight in LB medium to saturation, and genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega). The plasmid backbone of the O2a cluster was plasmid pBBR1MCS2 (Kan R ) (Kovach, ME, et al. (1995) Gene 166, 175-176). Primers for these reactions are listed in Table 2, and PCR products from these reactions were assembled using Gibson assembly with the NEBuilder HiFi DNA Assembly Kit (NEB). Star cells were transformed and plasmids were isolated and verified as described in the previous section.

[0115] Example 3. Bioconjugation and Western Blot The E. coli strains used in bioconjugation experiments were either SDB1 or CLM24 (Feldman, MF, et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016). SDB1 is a W3110 E. coli derivative mutated in the genes encoding the glycosyltransferase WecA, which initiates the synthesis of endogenous E. coli O16 antigens, and WaaL, the enzyme that transfers Und-PP-linked glycans to lipid A core sugars to generate LPS. CLM24 is a W3110 derivative deleted only for waaL. Removal of these genes prevents crosstalk between the heterologous bioconjugation system and the endogenous E. coli glycosylation pathway. To prepare Escherichia coli (E. coli) strains for bioconjugation, we electroporated the plasmid using competent cells prepared as previously described (Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203), followed by growth in SOB medium at 37°C. Cells were plated on LB agar plates with the appropriate antibiotics. The next day, 8–10 colonies were picked and inoculated into LB or TB medium with antibiotics and grown overnight at 30°C with shaking. The next morning, the inoculum was inoculated into either 30 mL of medium in a 125 mL Erlenmeyer flask or 1 L of medium in a 2 L flask, and the starting optical density at 600 nm (OD ) was measured. 600 The OD was 0.05. 600 The cultures were grown with shaking at 175 RPM until the OD reached 0.4-0.6, at which point the cultures were induced with 1 mM IPTG. All bioconjugation experiments were performed at 30°C unless otherwise noted. After overnight induction, the OD was reached after a total of 20-24 hours of growth. 600 was measured and 0.5 OD units of cells were pelleted for analysis.

[0116] The cell pellet was suspended in 100 μl of 1X Laemmli Buffer (Biorad) and boiled for 10 minutes at 100°C. The boiled samples were briefly centrifuged at 10,000 rcf, and equal volumes were collected at the same OD per lane. 600 The data were normalized to 0.01 and loaded onto a 7.5% Mini-Protean TGX gel (Biorad) for SDS-PAGE separation. Proteins were transferred to a nitrocellulose membrane using a semi-dry electrode system and blocked with Intercept Blocking Buffer (Li-Cor) for 1 hour. The membrane was then incubated with primary antibodies in 1:1 blocking and TBST for 45 minutes. Commercially available rabbit anti-EPA and mouse anti-6xHis antibodies (Millipore-Sigma) were used for protein detection. Rabbit glycan antibodies for CPS8, GBSIII, and O16 were purchased from SSI Diagnostica. Klebsiella pneumoniae rabbit O2a antibody was a kind gift from Professor Chris Whitfield (University of Guelph) (Clarke, BR, et al. (2018) Journal of Biological Chemistry 293, 4666-4679). Salmonella group B rabbit antibody was purchased from BD. After the primary incubation, the membrane was washed three times with TBST buffer for a total of 15 minutes. It was then incubated with the secondary antibodies IRDye 680RD goat anti-mouse and / or IRDye 800CW goat anti-rabbit (Li-Cor) in a 1:1 ratio of blocking buffer to TBST for 30 minutes. After a final 15-minute wash with TBST, the membrane was imaged using a Li-Cor Odyssey CLx.

[0117] Example 4. Recombinant Moraxella osloensis (M. osloensis) Pil Mo Δ28 Lys-C digestion In-gel digestion was performed according to the protocol of Shevchenko et al. (Shevchenko, A., et al. (2006) Nat Protoc 1, 2856-2860) with minor modifications. Gel-separated glycosylated EPA-PilΔ28 was excised and destained twice for 10 minutes at room temperature with shaking at 750 RPM in destaining solution (50 mM NH4HCO3, 50% ethanol). The destained bands were dehydrated for 10 minutes in 100% ethanol, dried in a vacuum centrifuge for 10 minutes, and then rehydrated in 10 mM DTT in 50 mM NH4HCO3. Reduction was performed for 60 minutes at 56°C. The gel bands were then dehydrated twice for 10 minutes in 100% ethanol to remove any remaining reducing buffer. The reduced samples were sequentially alkylated with 55 mM iodoacetamide in 50 mM NH4HCO3 for 45 minutes at room temperature in the dark. The alkylated samples were then washed four times for 10 minutes with 50 mM NH4HCO3, followed by 100% ethanol, followed by 50 mM NH4HCO3, followed by 100% ethanol, and then dried by vacuum centrifugation. The dried alkylated samples were rehydrated with 20 ng / μl Lys-C endoprotease (Wako Chemicals) in 40 mM NH4HCO3 for 1 hour at 4°C. Excess Lys-C was removed, and the gel pieces were covered with NH4HCO3 and incubated overnight at 37°C. Peptides were purified by C 18 The mixture was concentrated and desalted using a stage tip (Ishihama, Y., et al. (2006) J Proteome Res 5, 988-994; Rappsilber, J., et al. (2007) Nat Protoc 2, 1896-1906), dried, eluted with buffer B (0.5% acetic acid, 80% acetonitrile (ACN)), and stored at -20°C before LC-MS analysis.

[0118] Example 5. Analysis of recombinant Moraxella osloensis (M. osloensis) PilMoΔ28 using reversed-phase LC-MS C 18 The concentrated digest is then added to Buffer A. *(0.1% TFA, 2% ACN) and resuspended in PepMap100 C 18 20mmx75μm Trap and PepMap C 18 Separation was performed using a two-column chromatography setup containing a 500 mm x 75 μm analytical column (Thermo Fisher Scientific). Samples were concentrated onto the trap column for 5 min at 5 μl / min using 0.1% formic acid (FA), then passed through the analytical column using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific) with varying concentrations of Buffer A (2% DMSO, 0.1% FA) and Buffer B (78% ACN, 2% DMSO, 0.1% FA) and then injected at 300 nl / min into an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer equipped with a FAIMS Pro interface (Thermo Fisher Scientific). A 140-minute analytical run was used for potential glycopeptide identification, and a 60-minute run was used for targeted analysis. During a specific analytical run, the buffer composition was changed from 3% Buffer B to 28% Buffer B over 120 minutes, from 28% Buffer B to 40% Buffer B over 9 minutes, from 40% Buffer B to 100% Buffer B over 3 minutes, then held at 100% Buffer B for 2 minutes, then lowered to 3% Buffer B over 2 minutes, and held at 3% Buffer B for 8 minutes. The Lumos™ mass spectrometer was operated in a stepwise FAIMS data-dependent mode with three different FAIMS CVs (-25, -45, and -65) as previously described (Ahmad Izaham, AR, et al. (2021) J Proteome Res 20, 599-612), acquiring a single Orbitrap MS scan (60k resolution) every 1.5 seconds and an Orbitrap HCD scan (maximum fill time 120 ms, AGC 2x10) at each of the three FAIMS CVs. 5 The Orbitrap MS-MS scan resolution was switched between 30k and NCE (25, 30, 45). MoFor targeted characterization of glycopeptides, the buffer composition was changed from 3% Buffer B to 15% Buffer B over 30 min, from 15% Buffer B to 30% Buffer B over 10 min, and from 30% Buffer B to 80% Buffer B over 5 min. The composition was then held at 100% Buffer B for 5 min, then lowered to 3% Buffer B over 1 min, and further held at 3% Buffer B for 9 min. Analysis was performed using HCD (maximum fill time 250 ms, AGC 2.5x10 at 30k resolution for Orbitrap MS-MS scans). 5 , 15, 30, 35 NCE) and EThcD (maximum fill time 250 ms, Orbitrap MS-MS scan resolution 30k, AGC 2.5x10 5 , and HexHexA-modified glycopeptides 762 FLPANCRGT 770 Parallel reaction monitoring using calibrated charge-dependent ETD parameters for the +2 charge state of (687.2972 m / z) (ETD reaction time controlled using Rose, C.M., et al. (2015) J Am Soc Mass Spectrom 26, 1848-1857) was performed on a FAIMS CV-45.

[0119] Example 6. Pil Mo Open search of Δ28 and annotation of HexHexA modified C-terminal peptides Pil Mo Identification of glycosylation events was achieved using open database searches as previously described (Lewis, JM, et al. (2021) J Vis Exp,). Briefly, data files were processed using MSfragger 3.4 in FragPipe (version 17.1) (Polasky, DA, et al. (2020) Nat Methods 17, 1125-1132; Kong, AT, et al. (2017) Nat Methods 14, 513-520) and analyzed using Moraxella osloensis Pil. MoThe sequence (NCBI accession number: WP_156627541.1) was searched. The search was performed using "Lys-C" enzyme specificity, with carbamidomethylation of cysteine ​​as the fixed modification and oxidation of methionine as the variable modification, allowing for up to two missing cleavages. A mass tolerance range of 0 to 2000 Da, termed delta mass, was allowed to allow for identification of potential glycosylation events. The C-terminal peptide 762 FLPANCRGT 770 The delta mass observed in (SEQ ID NO: 61) was manually inspected to identify potential glycosylation events. HexHexA modified glycopeptide 762 FLPANCRGT 770 Parallel reaction monitoring results corresponding to (SEQ ID NO: 62) were manually extracted using Freestyle Viewer (1.7 SP1, Thermo Fisher Scientific), and MS / MS data were annotated using the interactive peptide spectrum annotator (Brademan, DR, et al. (2019) Mol Cell Proteomics 18, S193-S201) (available on the World Wide Web at interactivepeptidespectralannotator.com / PeptideAnnotator.html). Spectral annotation allowed modification of the terminal T residue with HexHexA (338.0849 Da) and Hex (162.0528 Da). The obtained MS data and search results have been deposited in the PRIDE ProteomeXchange Consortium repository (Perez-Riverol, Y., et al. (2019) Nucleic Acids Res 47, D442-D450; Perez-Riverol, Y., et al. (2015) Proteomics 15, 930-949) and can be accessed under the identifier PXD033468.

[0120] Example 7. Bioconjugate Protein Purification Cells for protein purification were grown in 1 L of TB medium, and bioconjugates were isolated using an osmotic shock protocol. After overnight growth and induction, cells were pelleted by centrifugation and washed with 0.9% NaCl. The washed cell pellet was suspended in 200 mM Tris-HCl (pH 8.5), 100 mM EDTA, and 25% sucrose and incubated with rolling at 4°C for 30 minutes. Cells were pelleted by centrifugation at 4,700 rcf for 30 minutes, and the resulting pellet was suspended in 20 mM Tris-HCl (pH 8.5) and incubated with rolling at 4°C for 45 minutes. The suspension was centrifuged at 18,000 rcf for 30 minutes. The supernatant containing the periplasmic fraction was concentrated and loaded directly onto an FPLC anion exchange column, or, in the case of the His-tagged EPA-PilΔ28 bioconjugate, purified using nickel-IMAC as previously described (Knoot, CJ, et al. (2021) Glycobiology 31, 1192-1203). The periplasmic extract or IMAC eluate was concentrated, buffer-exchanged into 20 mM Tris-HCl (pH 8.0), filtered through a 0.2 μm PES filter, and then loaded onto an Aekta pure FPLC instrument (Cytiva) equipped with a SOURCE 15Q 4.6 / 100 PE anion exchange column (Cytiva). The bioconjugates were eluted at 2 mL / min using a step gradient of buffer A (20 mM Tris, pH 8) and buffer B (20 mM Tris, pH 8, 1 M NaCl), increasing in 5% increments from 0% B to 25% B over 10 column volumes. The bioconjugates for immunization were further purified using a Superdex 200 Increase 10 / 300GL column. The concentrated bioconjugates collected from the anion exchange column were loaded onto a pre-equilibrated Superdex 200 column in PBS buffer and eluted at a flow rate of 0.75 mL / min. Fractions containing the purified bioconjugates were pooled, concentrated, and stored frozen at -80°C.Protein concentrations for immunization and Western blotting were measured using the Pierce BCA Protein Assay kit (Thermo Fisher). The polysaccharide-to-protein ratio for calculating vaccine doses was determined using the method described by Duke et al. (Duke, JA, et al. (2021) ACS Infectious Diseases 7, 3111-3123).

[0121] Example 8. Immunization of mice All mouse immunizations were conducted in accordance with ethical guidelines for animal experimentation and research. Experiments were conducted at Washington University School of Medicine in St. Louis in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. Five-week-old female CD-1 inbred mice (Charles River Laboratories) were subcutaneously injected with 100 μL of the vaccine formulation on days 0, 14, and 28. Vaccine groups were 291 alone (5 μg protein) and GBSIII-291 (5 μg protein, 1 μg polysaccharide). Mouse sera were collected on days 0, 14, 28, and 42. All vaccines were formulated with Alhydrogel® 2% aluminum hydroxide gel (InvivoGen) in a 1:9 ratio (50 μL vaccine and 5.5 μL alum dissolved in 44.5 μL 1x sterile phosphate-buffered saline).

[0122] Example 9. Enzyme-linked immunosorbent assay (ELISA) IgG kinetic titers were measured using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates (TRP Immunomaxi plates) were coated overnight in triplicate with glycoengineered Escherichia coli (E. coli) expressing GBSIII capsular polysaccharide in sodium carbonate buffer (approximately 10 CFU / 100 mL). The coated E. coli strains were grown as described above, incubated overnight to induce GBSIII expression, washed, diluted, and used to coat the plates. The wells were blocked with 1% BSA in PBS and washed with 0.05% PBS-Tween (PBST), with all subsequent washes identical. Mouse serum was diluted 1:100, added to the wells, and incubated at room temperature for 1 hour before washing. Total IgG titers were detected by adding HRP-conjugated anti-mouse IgG (GE Lifesciences, 1:5000 dilution) to the wells for 1 hour at room temperature. After washing, the plate was developed using 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Biolegend) and stopped with 2 N H2SO4. Optical density was measured at 450 nm using a microplate reader (Bio-Tek). To generate a standard curve for data fitting, total IgG product was determined using IgG standards. Standard wells were coated with IgG in sodium carbonate buffer and then treated identically to the sample wells. All wells were normalized to blank wells, which were treated identically to all sample wells except for the primary mouse serum. Significance was determined using the Mann-Whitney nonparametric test, with P < 0.05. *****

[0123] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0124] Particular embodiments of the present disclosure may be defined in any of the following numbered paragraphs.

[0125] 1. A glycoconjugate comprising an oligosaccharide or polysaccharide covalently bound to a receptor protein, the receptor protein comprises or consists of a TfpM-associated pilin-like protein or a glycosylated fragment thereof, and the oligosaccharide or polysaccharide is covalently bound to the TfpM-associated pilin-like protein or a glycosylated fragment thereof; the TfpM-related pilin-like protein or glycosylated fragment thereof comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently linked to the C-terminal serine or threonine; Optionally, the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin-like protein; Optionally, the receptor protein is a fusion protein comprising the TfpM-associated pilin-like protein or glycosylated fragment thereof translationally fused to a heterologous carrier protein, wherein the TfpM-associated pilin-like protein or glycosylated fragment thereof is the most C-terminal sequence of the receptor protein, such that the receptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently linked to the C-terminal serine or threonine; Optionally, the oligosaccharide or polysaccharide comprises glucose at its reducing end; and / or Optionally, said glycoconjugate is immunogenic.

[0126] 2. The length of the glycosylated fragment of the TfpM-related pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 2. The glycoconjugate of paragraph 1, wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

[0127] 3. (a) The TfpM-associated pilin-like protein or a glycosylated fragment thereof is (i) Pil Mo(SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) The TfpM-associated pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100), (c) The TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last 3 amino acids from the C-terminus of the pilin; and / or (d) The glycosylated fragment of the above pilin-like protein is Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148), Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10(SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine; and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin; and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S(SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

[0128] 4. The glycoconjugate of any one of paragraphs 1 to 3, wherein the receptor protein is a fusion protein comprising a heterologous carrier protein, and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof.

[0129] 5. The receptor protein is a fusion protein, which, in addition to the glycosylation fragment of the TfpM-related pilin-like protein located at its C-terminus, further comprises an additional glycosylation sequence of an oligosaccharyltransferase (OTase) other than the TfpM OTase; Optionally, the additional glycosylation sequence is an internal glycosylation fragment of ComP, and further optionally, the glycosylation fragment of ComP comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59), or a fragment thereof comprising at least amino acids ASA at positions 11-13; 5. The glycoconjugate of any one of paragraphs 1 to 4, wherein optionally said additional glycosylation sequence is also covalently attached to the oligosaccharide or polysaccharide.

[0130] 6. The fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more additional glycosylation sequences; Optionally, the fusion protein does not comprise more than 2, more than 3, more than 5, more than 10, more than 15, more than 20, or more than 25 additional glycosylation sequences; Optionally, said additional glycosylation sequences are identical; Optionally, said additional glycosylation sequences are different from each other, and / or Optionally, at least three, at least four, or at least five of said additional glycosylation sequences are all different from one another; and / or Optionally, the glycoconjugate of paragraph 5, wherein none of said additional glycosylation sequences are the same.

[0131] 7. The glycoconjugate according to any one of paragraphs 1 to 6, wherein the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least three repeating units of the oligosaccharide or polysaccharide structure and / or a size of at least ten monosaccharides.

[0132] 8. (i) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, and the polysaccharide is a capsular polysaccharide; optionally, Streptococcus pneumoniae (S. pneumoniae) or Streptococcus agalactiae (S. agalactiae), and Optionally, the S. agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; (ii) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella, and the polysaccharide is a capsular polysaccharide or an O-antigen polysaccharide; Optionally, the bacterium is Klebsiella pneumoniae (K. pneumoniae), or (iii) the oligosaccharide or polysaccharide is produced by bacteria of the genus Salmonella, and the polysaccharide is an O-antigen polysaccharide; 8. The glycoconjugate of any one of paragraphs 1 to 7, optionally wherein the bacterium is Salmonella (S. enterica) and the Salmonella (S. enterica) polysaccharide is a group B O antigen.

[0133] 9. The above complex carbohydrates are Optionally, within a bacterial cell, Optionally, in Escherichia coli, Optionally produced in bacteria of the genus Klebsiella, and / or 9. The glycoconjugate of any one of paragraphs 1 to 8, wherein optionally the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0134] 10. The bioconjugate is a conjugate vaccine that induces an immune response when administered to a subject; Optionally, the immune response induces long-term memory (memory B cells and T cells) and is an antibody response, optionally a serotype-specific antibody response; Optionally, the antibody response is an IgG or IgM response; Optionally, the antibody response is an IgG response, optionally an IgG1 response; and / or 10. The glycoconjugate of any one of paragraphs 1 to 9, optionally wherein the conjugate vaccine generates immunological memory in a subject administered the vaccine.

[0135] 11. A glycosylated fragment of a pilin-like protein comprising or consisting of an isolated fragment of a TfpM-associated pilin-like protein, (a) The TfpM-associated pilin-like protein or a glycosylated fragment thereof is (i) Pil Mo (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1 to 28 Mo (Pil MoΔ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) The TfpM-associated pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100), (c) The TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71(SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last 3 amino acids from the C-terminus of the pilin; and / or (d) The glycosylated fragment of the above pilin-like protein is Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148), Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10(SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine; and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin; and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S(SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

[0136] 12. The length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 12. A glycosylated fragment of a pilin-like protein according to paragraph 11, wherein said glycosylated fragment of a TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

[0137] 13. A fusion protein comprising a TfpM-related pilin-like protein or a glycosylated fragment thereof translationally fused to a heterologous carrier protein, the TfpM-related pilin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue; the TfpM-related pilin-like protein or glycosylated fragment is the most C-terminal sequence of the fusion protein; and the fusion protein comprises a C-terminal serine or threonine residue; Optionally, the fusion protein is glycosylated with an oligosaccharide or polysaccharide covalently attached to the C-terminal serine or threonine; Optionally, the fusion protein is glycosylated with an oligosaccharide or polysaccharide comprising a glucose at its reducing end covalently linked to the C-terminal serine or threonine.

[0138] 14. The length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 14. The fusion protein of paragraph 13, wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

[0139] 15. (a) The TfpM-associated pilin-like protein or a glycosylated fragment thereof is (i) Pil Mo (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) The TfpM-associated pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100), (c) The TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last 3 amino acids from the C-terminus of the pilin; and / or (d) The glycosylated fragment of the above pilin-like protein is Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148), Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine; and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin; and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A](SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A](SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A] (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

[0140] 16. The fusion protein of any one of paragraphs 13 to 15, wherein the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof.

[0141] 17. The fusion protein further comprises, in addition to the glycosylation fragment of the TfpM-related pilin-like protein located at its C-terminus, an additional glycosylation sequence of an oligosaccharyltransferase (OTase) other than the TfpM OTase; Optionally, the additional glycosylation sequence is an internal glycosylation fragment of ComP, and further optionally, the glycosylation fragment of ComP comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59), or a fragment thereof comprising at least amino acids ASA at positions 11-13; 17. The fusion protein of any one of paragraphs 13 to 16, wherein optionally said additional glycosylation sequence is also covalently attached to the oligosaccharide or polysaccharide.

[0142] 18. The fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more additional glycosylation sequences; Optionally, the fusion protein does not comprise more than 2, more than 3, more than 5, more than 10, more than 15, more than 20, or more than 25 additional glycosylation sequences; Optionally, said additional glycosylation sequences are identical; Optionally, said additional glycosylation sequences are different from each other, and / or Optionally, at least three, at least four, or at least five of said additional glycosylation sequences are all different from one another; and / or Optionally, the fusion protein of paragraph 17, wherein none of the additional glycosylation sequences are the same.

[0143] 19. The fusion protein according to any one of paragraphs 13 to 18, wherein the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least three repeating units of the oligosaccharide or polysaccharide structure and / or a size of at least ten monosaccharides.

[0144] 20. (i) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, and the polysaccharide is a capsular polysaccharide; optionally, Streptococcus pneumoniae (S. pneumoniae) or Streptococcus agalactiae (S. agalactiae), and Optionally, the S. agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; (ii) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella, and the polysaccharide is a capsular polysaccharide or an O-antigen polysaccharide; Optionally, the bacterium is Klebsiella pneumoniae (K. pneumoniae), or (iii) the oligosaccharide or polysaccharide is produced by bacteria of the genus Salmonella, and the polysaccharide is an O-antigen polysaccharide; 20. The fusion protein of any one of paragraphs 13 to 19, optionally wherein the bacterium is Salmonella (S. enterica) and the Salmonella (S. enterica) polysaccharide is a group B O antigen.

[0145] 21. The above complex carbohydrates are Optionally, within a bacterial cell, Optionally, in Escherichia coli, Optionally produced in bacteria of the genus Klebsiella, and / or 21. The fusion protein of any one of paragraphs 13 to 20, optionally wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0146] 22. The fusion protein is a vaccine that induces an immune response when administered to a subject, Optionally, the immune response induces long-term memory (memory B cells and T cells) and is an antibody response, optionally a serotype-specific antibody response; Optionally, the antibody response is an IgG or IgM response; Optionally, the antibody response is an IgG response, optionally an IgG1 response; and / or 22. The fusion protein of any one of paragraphs 13 to 21, optionally wherein the fusion protein generates immune memory in a subject administered the vaccine.

[0147] 23. A method for producing glycoconjugates, the method comprising covalently attaching an oligosaccharide or polysaccharide to an acceptor protein comprising or consisting of a TfpM-associated pilin-like protein or a glycosylated fragment thereof using a TfpM oligosaccharyltransferase (OTase); the pyrin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue, the receptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently bound to the C-terminal serine or threonine residue of the receptor protein; Optionally, the oligosaccharide or polysaccharide comprises glucose at its reducing end; Optionally, the receptor protein is a fusion protein according to any one of paragraphs 13 to 22; Optionally, the method is a method for in vivo conjugation of an oligosaccharide or polysaccharide to an acceptor protein; and / or Optionally, the glycoconjugate is immunogenic.

[0148] 24. The above TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81), comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to Optionally, the TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), or TfpM 72-O-c (SEQ ID NO: 81), Optionally, the TfpM OTase is TfpM Mo 24. The method of paragraph 23, wherein the nucleic acid sequence is (SEQ ID NO: 56).

[0149] 25. The length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 25. The method of paragraph 23 or 24, wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

[0150] 26.(a) The TfpM-associated pilin-like protein or a glycosylated fragment thereof is (i) Pil Mo(SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) The TfpM-associated pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100), (c) The TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last 3 amino acids from the C-terminus of the pilin; and / or (d) The glycosylated fragment of the above pilin-like protein is Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148), Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10(SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine; and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin; and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A] (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S(SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A] (SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A](SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

[0151] 27. The method of any one of paragraphs 23 to 26, wherein the receptor protein is a fusion protein comprising a heterologous carrier protein, and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof.

[0152] 28. The receptor protein is a fusion protein comprising, in addition to the glycosylation fragment of the TfpM-related pilin-like protein located at its C-terminus, an additional glycosylation sequence of an oligosaccharyltransferase (OTase) other than the TfpM OTase, and the method further comprises covalently attaching an oligosaccharide or polysaccharide to the additional glycosylation sequence using an OTase other than the TfpM OTase; Optionally, the receptor protein is a fusion protein comprising a ComP glycosylation fragment, and the method further comprises covalently attaching an oligosaccharide or polysaccharide to the ComP glycosylation fragment using PglS OTase; 28. The method of any one of paragraphs 23 to 27, optionally wherein the ComP glycosylated fragment is an internal glycosylated fragment of ComP, and further optionally wherein the ComP glycosylated fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising amino acids ASA at least positions 11 to 13.

[0153] 29. The method of any one of paragraphs 23 to 28, wherein the binding occurs within a host cell.

[0154] 30. The host cell is a bacterial cell; Optionally, in Escherichia coli; Optionally, in a bacterium from the genus Klebsiella, or 30. The method of paragraph 29, optionally wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0155] 31. The method of paragraph 29 or 30, comprising culturing a host cell containing (a) a gene cluster encoding the proteins necessary to synthesize the oligosaccharide or polysaccharide, (b) TfpM OTase, and (3) the receptor protein.

[0156] 32. The method of any one of paragraphs 23 to 31, wherein the method produces a conjugate vaccine.

[0157] 33. A host cell comprising (a) a gene cluster encoding the above proteins necessary to synthesize the above oligosaccharide or polysaccharide, (b) a TfpM OTase, and (3) a receptor protein comprising a TfpM-related pilin-like protein or a glycosylated fragment thereof.

[0158] 34. The host cell of paragraph 33, wherein the receptor protein is a fusion protein.

[0159] 35. The host cell comprises a nucleic acid encoding the TfpM OTase, and / or the host cell contains a nucleic acid encoding the receptor protein; Optionally, the host cell of paragraph 33 or 34, wherein the TfpM OTase and the receptor protein are encoded by the same nucleic acid.

[0160] 36. An isolated nucleic acid encoding a glycosylated fragment of a pilin-like protein according to paragraph 11 or 12 and / or a fusion protein according to any one of paragraphs 13 to 22.

[0161] 37. The isolated nucleic acid of paragraph 36, wherein the nucleic acid is a vector.

[0162] 38. A host cell comprising the isolated nucleic acid of paragraph 36 or 37, Optionally, the host cell is a bacterial cell, and further comprising: Optionally, the host cell is Escherichia coli; Optionally, the host cell is from the genus Klebsiella; or Optionally, the host cell is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca host cell.

[0163] 39. A composition comprising the conjugate vaccine of paragraph 10 or the fusion protein of paragraph 22, and an adjuvant and / or carrier.

[0164] 40. A method for inducing a host immune response against a bacterial pathogen, said method comprising administering to a subject in need thereof an effective amount of the conjugate vaccine described in paragraph 10, the fusion protein described in paragraph 22, or the composition described in paragraph 39.

[0165] 41. The immune response is an antibody response; the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, a cellular response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system crosstalk, and a combination of two or more of the foregoing immune responses; and / or 41. The method of paragraph 40, wherein the immune response is selected from the group consisting of an innate response, a humoral response, an antibody response, a T cell response, and a combination of two or more of the foregoing immune responses.

[0166] 42. A method for preventing or treating a bacterial disease and / or infection in a subject, comprising administering to a subject in need thereof a conjugate vaccine according to paragraph 10, a fusion protein according to paragraph 22, or a composition according to paragraph 39; Optionally, the subject is a human.

[0167] 43. The infection is a local or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune; the disease is pneumonia, and / or 43. The method of paragraph 42, wherein the infection is a systemic infection and / or a blood infection.

[0168] 44. The method of any one of paragraphs 40 to 43, wherein the conjugate vaccine, the fusion protein, or the composition is administered by intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

[0169] 45. A method for producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising: (a) isolating a glycoconjugate according to any one of paragraphs 1 to 10 or a glycosylated fusion protein according to any one of paragraphs 13 to 22; (b) combining said isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and / or carrier.

[0170] 46. ​​A glycoconjugate, glycosylated fusion protein, or conjugate vaccine according to any one of the preceding claims for use in inducing a host immune response against a bacterial pathogen and / or for preventing or treating bacterial disease and / or infection in a subject.

[0171] 47. A recombinant nucleic acid construct comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one species transcriptional regulatory sequence.

[0172] 48. The above TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81), comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to Optionally, the TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40(SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpM YZS-X (SEQ ID NO: 76), TfpM CIP102637 (SEQ ID NO: 77), TfpM T-3-2 (SEQ ID NO: 78), TfpM BI730 (SEQ ID NO: 79), TfpM A3K91 (SEQ ID NO: 80), and / or TfpM 72-O-c (SEQ ID NO: 81), Optionally, the TfpM OTase is TfpM Mo 48. The recombinant nucleic acid construct of paragraph 47, wherein the construct is (SEQ ID NO: 56).

[0173] 49. The recombinant nucleic acid construct according to paragraph 47 or 48, wherein the heterologous transcriptional regulatory sequence is a promoter sequence.

[0174] 50. The method further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or a glycosylated fragment thereof, or a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof operably linked to the above-mentioned nucleotide sequence encoding the TfpM OTase, Optionally, the coding sequence for the TfpM-associated pilin-like protein or glycosylated fragment thereof, or a fusion protein comprising the TfpM-associated pilin-like protein or glycosylated fragment thereof, is within 2, 5, 10, 20, 30, 40, or 50 nucleotides of the sequence encoding the TfpM OTase; 50. The recombinant nucleic acid construct of any one of paragraphs 47 to 49, wherein optionally, the coding sequence for the TfpM-associated pilin-like protein or glycosylated fragment thereof, or a fusion protein comprising the TfpM-associated pilin-like protein or glycosylated fragment thereof, overlaps with an operably linked nucleotide sequence encoding TfpM OTase.

[0175] 51. The length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 51. The recombinant nucleic acid construct of paragraph 50, wherein said glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

[0176] 52.(a) The TfpM-associated pilin-like protein or a glycosylated fragment thereof is (i) Pil Mo (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1 to 28 Mo (Pil Mo Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) The TfpM-associated pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65(SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), and Pil CIP102637 (SEQ ID NO: 100), (c) The TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ ID NO: 98), Pil T-3-2 (SEQ ID NO: 99), Pil CIP102637 (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last 3 amino acids from the C-terminus of the pilin; and / or (d) The glycosylated fragment of the above pilin-like protein is Pil Mo Pilrin disulfide loop region (Pil 20 Also known as Pil Mo _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of pilin or a variant thereof, wherein the C-terminal threonine is replaced with serine (SEQ ID NO: 148), Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine; and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin; and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20[A] (SEQ ID NO: 166), Pil 19[A] (SEQ ID NO: 167), Pil 18[A] (SEQ ID NO: 168), Pil 17[A] (SEQ ID NO: 169), Pil 16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil 11[A] (SEQ ID NO: 175), Pil 10[A] (SEQ ID NO: 176), Pil 9[A] (SEQ ID NO: 177), Pil 8[A] (SEQ ID NO: 178), Pil 7[A] (SEQ ID NO: 179), Pil 6[A] (SEQ ID NO: 180), Pil 5[A] (SEQ ID NO: 181), or Pil 4[A](SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil 7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and optionally, the glycosylated fragment of the pilin-like protein comprises Pil 20S[A] (SEQ ID NO: 183), Pil 19S[A] (SEQ ID NO: 184), Pil 18S[A] (SEQ ID NO: 185), Pil 17S[A] (SEQ ID NO: 186), Pil 16S[A] (SEQ ID NO: 187), Pil 15S[A] (SEQ ID NO: 188), Pil 14S[A] (SEQ ID NO: 189), Pil 13S[A] (SEQ ID NO: 190), Pil 12S[A](SEQ ID NO: 191), Pil 11S[A] (SEQ ID NO: 192), Pil 10S[A] (SEQ ID NO: 193), Pil 9S[A] (SEQ ID NO: 194), Pil 8S[A] (SEQ ID NO: 195), Pil 7S[A] (SEQ ID NO: 196), Pil 6S[A] (SEQ ID NO: 197), Pil 5S[A] (SEQ ID NO: 198), or Pil 4S[A] (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

[0177] 53. A recombinant nucleic acid construct according to any one of paragraphs 50 to 52, wherein said fusion protein is a fusion protein according to any one of paragraphs 13 to 22.

[0178] 54. The method further comprises a nucleotide sequence encoding a PglS OTase operably linked to the TpfM OTase; 54. The recombinant nucleic acid construct of any one of paragraphs 50 to 53, optionally wherein the coding sequence for the PglS OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TfpM OTase.

[0179] 55. A vector comprising a recombinant nucleic acid construct according to any one of paragraphs 47 to 54.

[0180] 56. A host cell comprising a recombinant nucleic acid construct according to any one of paragraphs 47 to 54 or a vector according to paragraph 55, Optionally, the host cell is a bacterial cell, and further comprising: Optionally, the host cell is Escherichia coli; Optionally, the host cell is from the genus Klebsiella; or Optionally, the host cell is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca host cell.

[0181] 57. A method for producing TfpM OTase, comprising culturing a host cell according to paragraph 56, wherein the vector according to paragraph 54 is an expression vector, and recovering said TfpM OTase.

[0182] 58. A glycoconjugate comprising an oligosaccharide or polysaccharide covalently bound to a receptor protein, A glycoconjugate comprising means by which said receptor protein is covalently bound to said oligosaccharide or polysaccharide by TfpM OTase.

[0183] 59. The complex carbohydrate according to paragraph 58, wherein the complex carbohydrate is a complex carbohydrate according to any one of paragraphs 1 to 10.

[0184] 60. A fusion protein comprising a means for covalently binding an oligosaccharide or polysaccharide by TfpM OTase, said means being translationally fused to a heterologous carrier protein; The means is located in the most C-terminal sequence of the fusion protein.

[0185] 61. The fusion protein according to paragraph 60, wherein said fusion protein is a fusion protein according to any one of paragraphs 13 to 22.

[0186] 62. Use of a glycoconjugate according to paragraph 58 or 59, or a fusion protein according to paragraph 60 or 61, in any of the above methods.

[0187] Additional arrays >TfpM_Mo (SEQ ID NO: 56) MFNLIKHFKNKELFVALYFLLFIVGFSIYSFNFYNELRVLEVFLLIGLGLYGLIHKKNYFSKYEFCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFFNLNYNEF LSKLLIWLSFSIFLMLPIAITEFILSGVYPNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCIFNHAARF KLLFTYVATWLAYFSILFFSSHSASIVGSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWN LKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASTVSQVFGYIAYISIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_DSM16617 (SEQ ID NO: 63) MLNFNKHLKNKELFIALYFLTFILGLSIHISSNFYNESRVLETFLLLSLVFNKNLHLKKIEYIFIIFIIF HLLFLRNSQFIIFEILLYYLLYKAFLIINYNELLAKFIIWISFSIFSILPIEIVKYIHNSIYSNWYPTPWNIRVYNSYFLVLSVFAVWFYLKQEKFKIIYLFFLFLAFLSILLDGGRSATLAYSVFIACVCIFNRAVRFKLLFTYAATWLTYFSILFFSSHSASNVRSIVRESTSG RYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFEHAASFHPSTYEASRVSQVFGYIAYISIALAFIYLFFYTSALSESLASTPPRFWEYGYQLF >TfpM_ZZC3 (SEQ ID NO: 64) MLNFNKYLKNKELFIALYFLSFILGLSIHISSNFYNESRVLETFLLLSLVFNKNLHLKKIEYIFIIFIIF HLFFLRNSQFIIFEILLYYLLYKAFLVINYNELLAKFIIWISFSIFSMLPIEIVKYIHNSIYSNWYPTPW NIRVYNSYFFVLSIFAIWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCIFNRAARFKLLFTYAATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAH PHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIAYISIALAFIYLFFYTSALSESLPSTPRFWEYGYQLF >TfpM_TUM15069 (SEQ ID NO: 65) MFNLIKHFKNKELFVALYFFLFILGVSINGFNFYNELRVVEVFLLIFLGFYGLINKKNYFSKYEVCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEF LSKFVIWLSFLIFLMLPIAITEFIASGVYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLVFLFLAFLSILLDGGRSATLAYSVFIACVCIFNHAARF KLLFTYVATWLAYFSILFFSSHSASIVGSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWN LKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFEHAASFHPSTYEASRVSQVFGYIAYISIALAFIYLFFYTSALSESLASTPPRFWEYGYQLF >TfpM_AI7 (SEQ ID NO: 66) MFKYIKHFKNKELFIALYFLLFIVGFTIYSFNFYNELRAIEFFLLIFFGFHSLINKNYVSKYEFCFLIFI FLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKFVIWLSFLIFLMLPIAITEFILSGVYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCMFNRAVRFKLLFTYAATWLVYFSILFISSHSASGVRSIVRESTSGRYELWVNAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFEHAASFHPSTYEASRVSQVFGYIAYILIALAFIYLFSYTSALSESLPSTPPRFWEYGYQLF >TfpM_VE-C3 (Accession No. 67) MNFLVHFKNKELFVALYFLLFTVGFSIYSFNFYNELRALEFFLLIGLGLYGLIHKKNYFSKYEFCFLIFTLLGFLFWSNYYYIFFELILFYLLYKAFLNLNYNEFLSKLLIWLSFSIFLMLPIAITEFILSGVYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCMFNRAARFKLLFTSAATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDHYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIAYISIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_YH01026 (Accession No. 68) MFNLIKHFKNKELFIALYFLLFIVGFSIYSFNFYNELRVLEVFIFIGLVLFGLIHKKNYFSKYEFCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEF ISKLVIWLSFSIFLMLPIAITEFMLSGLYQNWYPMPWNIRVYNSYFLVLSIFAVWFYFKQEKFINIYLVFLFLAFLSILLDGGRSATLAYSVFIACVCIFNRAARF KLLFIYVATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGGFLILVICNILKRIKWN LKENYFVISAFIAVSIDLSFSGIHIYPITQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIAYILIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_CIP102143 (SEQ ID NO: 69) MFNLIKHLKNKELFVALYFLLFIVGFSIYSSNFYNELRALEFFLLIGLGLYVLIHKKNYFSKYELCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEF LSKLLIWLSFSIFLMLPIAITEFIISGVYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCIFNHAARF KLLFTYAVTWLAYFSILFFSSHSTSIVGSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWN LKENYFVISAFIAVSIDLSFSGIHIYPITQVALLWLFVFLLKNPEFQHATSFHPPIYEASKVSQVFEFMVYILIALAFIYLFFYTSALSESLASTPPRFWEYGYQLF >TfpM_AI40 (SEQ ID NO: 70) MFSVVSHLKNKELFIALYFLLFISGISIYSYNFYNELRVSEVFLLIGLGLCGLTHKKNYFSKYEFYFFIFILLGFLFWSNYNYILYEFILFYLLYQAFLNLNYSEF LSKFVIWLSFSIFLMFPIAVAEFLVSGVYQNWYPMPWNIRIYNSYFLVFSIFSVWFYLKEEKFKNIYLFFLFLAFLSILLDGGRSATLAYSIFIACICMFNRTARL KILFTYMATWLTYFSILFFSSNSASGVRSIARESTSGRYELWVSAFQCWLQNPLFGCGFYQLDLYSGIIPAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWN LKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHSSTYEASRVSQVFGYIVCILIALAFIYLFFYTSALSESLPSTPRFWEYGYQLF >TfpM_F78 (SEQ ID NO: 71) MSNLIKYLKNKELFIALYFFIFILGISIQLSFNFYNEARVLEILLLLCLGLYSFINNENLFFKKELVFLFFISFGFFYWFNFQIVFYEILLFYLLYKAFFFLKYNAIVSKLIVFSSFFIFIFLPMSLWEYLTTGKYQNWYPLPWNIRIYNSYFLIFSIFAIWFFLKEKYKSIYLAFIFLAFLSILLDGGRSAALAYTVFIGLLSIFNRLARL KLIFIYSLTWLAYFLIIYFSSQSGSSLRYIARDSTSGRYDLWLNAFQCWLQSPILGCGFYQLDKYSNLSAHPHNLFIQILTETGLIGLSFLLYIIFIILRNISWKF KENYFVISALIAVFIDLSFSGIHIYPITQVALLWLFVFLLKNPEFQHATYFSPSACEASKKSQFLEFIVYILIALAFIYLFFNTSALSESLPSTPRFWEYGYQIF >TfpM_S71 (SEQ ID NO: 72) MSNLIRHLKNKELFLALYFLTFILGVSLGVSYNFYNEARVLEVLLLLGFGFYSVFSKEIFFSKKEYLFLVVFISYLFFLKNSQFIIFDILLFYLLYKSFFILNYNL VVSKIIVLSSFLIFMTFPLSLLGYWGDGVYRNWYPMPWNIRVYNSYFLILLIFSTWLLMRGNRYTWVYLLFTYLSLLSILLDGGRSALLAYSTFFIIVIIFNKKVR LKLIFIYIISWLSFLLIVFSAGIASDGISIARVTTSRRSDLWMHALQCWIESPIFGCGFYQLGAYENLSAHPHNLFIQILTETGVMGFSFLALIIFGVLRNISWNI KENYFVIAAFFAIGVDLSFSGIHIYPITQVGLLWLFVFLLKNPEFRHAKYFSDILVQNPKSVWVVNFIIYLIITCAFIYLFVNTSALSESLAVTPPRFWEYGYQLF >TfpM_ANC4282 (SEQ ID NO: 73) MLNLIKNKELFIALYFLIFNLGFSVHISSNFYNEARLLEIFLLLSLGIFSGFVKNIVFHKIEYIFLLFFI FSIFFLKNQPFIFFEILLFYLLFKAFFALNYNSKISKAIILLSFLIFLMFPVSILHYLNSGLYQNWYPMPWNIRIYNSYFLIFSIFAIWFYLKEDKYKNIYLIFIFLAFLSILLDGGRSATLAYTIFIVIVCIFNRLERF KLLLIYCSTWLAYFSIVYFSSQSASTLRSITRESTSGRYELWLNAFQCWLENPILGCGFYQLDKYPSLSAHPHNLFVQILTETGLIGFIFLSFIIFKVVKNISWNF KQNYFVLAALFSVAIELSFSGIHIYPVTQVALLWLFVFLLKNPEFSHASYFNYLKIKNSKIDKLIQLFIYLFILIIFIYLFINTSVLSENLPSTPRFWEYGYQLF >TfpM_CIP102519 (SEQ ID NO: 74) MFNLIKHIKNKELFLALYFLLFSVGFSIYSFNFYNELRALEFFLLIGLGLYGLIHKKNYFSKYEVCFLIFTFLGFSFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKLVIWLSFSIFLMLPIAITEFMLNGIYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQDKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCMFNRAARFKLLFTYAVTWLVYFSILFFSSHSDSNVSSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPITQVALLWLFVFLLKNPEFEHAASFYPSTYEASRVSQVFGYIAYILIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_junii_65(Accession No. 75) MNFLVHFKNKELFVALYFLLFSVGFSIYSFNFYNELRVLEIFLFIGLGLNSLTHKKNYFSKYEFCFLIFTLLGFLFWSNYSYIFFELILFYLLYKAFLNLNYNEFLSKLVIWLSFSIFLMLPIAIKEFIASGVYQNWYPMPWNIRVYNSYFFILSIFAIWFYSKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCMFNRAARFKLLFTYAVTWLAYFSILFFSSHSTSIVGSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLEQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIVYILIALAFIYLFSYTSALSESLPSTPPRFWEYGYQLF >TpfM_YZS-X(Accession No. 76) MFNLIKHFKNKELFIALYFLLFIAGFSIDSNFYNELRIFEVFLLIGLGFYGLTHKKKYFSKYEFCFFIFILLGFLCWSNYSYILYEFILFYLLYKAFFDLNYNEF LSKLIIWLSFSIFLMFPIAVAEFLVSGVYQNWYPMPWNIRIYNSYFLVFSIFSVWFYLKEEKFKNIYLFFLFLAFLSILLDGGRSANLAYSIFIACICMFNRTARL KILFTYIATWLTYFSILFFSSNSASGVRSIARESTSGRYELWVSAFQCWLQNPLFGCGFYQLDLYSGIIPAHPHNLFIQILTETGLIGFGFLILVIWNILKRIKWN FKENYFVIPALIAVSIDLSFSGIHIYPITQIALLWLFVFLLKNPEFQYAASFNQSTHKGSKKSKNFELIVYILIALAFIYLFFHTSALSESLPSTPRFWEYGYRLF >TfpM_CIP102637 (SEQ ID NO: 77) MSNLIKHLKNKELLVALYFFLFILGVSINGFNFYNELRTLEVFLLIGLGFYSLIQKKSYFSKYEFCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEF LSKLVIWLSFSIFLMLPIAITEFMLSGVYQNWYPMPWNIRVYNSYFLVLSIFAIWFYLKQEKFNNIYLIFLFLAFLSILLDGGRSTALAYSVFIACVCMFNRAVRF KLLFTYAATWLTYFSILFFSSHSTSSVRSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWN LKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQRAASFHPSTYEASRVSQVFGYIAYILIALAFIYLFFYTSALSESLPSTPRFWEYGYQLF >TfpM_T-3-2 (SEQ ID NO: 78) MMKAIKLLAHKESFIGLYFLLFIMGVTVGSGYGIYNESRIAEIALLLGLGAHACFNKYYIVTKVEYLFFVFIIIGSFFWSNSFFIIIDLLLVYLLYKSFFFLEYRP LLTKIIVLASFLIFLLLPVAIWDYITSGIYTSNWYLLRLNIRIYNSYFLIMSIFAVWLYLTEKNYKKLYLSFIFLAFLSILMDGGRSATLAYTAFVIIICIFRRPVS WQLGFAYSMSWLTYLTINHLASLNAVETLGLGIARATTSQRYDIWMNAVQCWVQNPIWGCGFYQLDSTRNLASHPHNLFLQVLSETGLIGFGFLLAIIFSILKNISW NLNKDYFVIAALLAVVIETLLSGIHIYPITQIALLWLFIFLLKNPIFPHTLYFNSLIASFASNSYLSITVYLILTIFFLYFFINTSALIDPELLTRPRFLENGYNIF >TfpM_BI730 (SEQ ID NO: 79) MSTIVKRLKNKELFIALYFLLFILGFTIGITPKFYNEFRILQVTLLLNFGLHNIIHKHGYISRAELLFFVYIGIASLFWQNYEFIVIDLLLAYLLYKTFFLLKYNEL ATKVIVFFSLLIFPLLPLSVFDYISTGTYYPIWHPMPWNIRIYDSYLLIVSIFAVWFYITETKYKKIYLLFLLLAFFSVLLNGGRSATLAYTVFIAVIVVFNRIVRWQ ILATYAIAWLAYISISYLAISNLSMASPIGLQIARTTTSLRYDLWMNAIECWIQSPLVGCGFYQLYRYENLGAHPHNLLLQILTETGLIGFGFLLAIVVTILKHIDWQ LKRSYFVIAALLAIGVDTSLSGTHIYPITQMALLWLLVFLLKNPVFQHAAYFNRVPYITSVTDMVVSIVVYFSLTVIFIYLFLNTSVLFDSLMLATPPRFWEYGYQLF >TfpM_A3K91 (SEQ ID NO: 80) MYKKQLLNNQQMTVSIIKALANKESFVALYSLIFIVGVTANSGYGIYNESRIFEVALLLGLGAHACFNKYYIVTKVEYLFFVFIIIGSFFWSNSFFIIIDLLLVYLLYKSFFFLEYRPLLTKIIVLASFLIFLLLPVAIWDYITSGIYTSNWYLLRLNIRIYNSYFLIMSIFAVWLYLTEKNYKKLYLSFIFLAFLSILMDGGRSATLAYTAFVIIICIFRRPVSWQLGFAYSMSWLTYLTINHLASLNAVETLDLGIARATTSQRYDIWMNAVQCWVQNPIWGCGFYQLDSTRNLAAHPHNLFLQVLSETGLIGFWFLLAIIFFILKNISWNLNKDYFVIASLLAVVIETLLSGIHIYPITQIALLWLFVFLLKSSNFSHAIYFHSSIPSFVVNRYFSVTMFLVLVILFIYFFINTSALLDNEIMTRPRFLENGYNIF >TfpM_72 - O - c (Accession No. 81) MYNKNPLSKQHKLQTFIKSVRLLFNKELSIGLYFLLFILGMNTGSGHDSYNEFRVFQVTLLLVIGVSTWCYRRLFITKLELLFFAFIAFGSFFWQQPIFVLNDVLLVYLLYKSFYLLNYQPLLSKLIVLSSLLIFLLLPVALWNYIDTGKYSPIWYPLPWNMRVYDSYFLIISVFAVWFYLTEKQYRFLYLLFLFLAFLAVLLDGGRSVTLAYTVFIAIISLFHRRARWRLVLMYAMSWLTYIIVTYTANTSVTSLRIARDTRSDRYDLWINAFQCWSQHPLFGCGFYQLDKYPNIAAHPHNLFIQVLTETGLIGFGFLAFIIFKVAKNINWDLKQNYFVIAALLAISIDMSLSGVHIYPVTQIALLWLFVFLLKNPEFSHAHHFNKVVQQKKAIDNILPLIIYLSLTIWFIYLFTNTSSFLPGTPLTPPRFWVYGYQLW >Pil_DSM16617 (Accession No. 82) MNTMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTVRARVSEGITTASAMKATVSENILNAGALVAGTPSTAGSACVGVTEISGGTGNVASATCGASKAGQIIVTMGATTAKSVPVYLTPSYTASAVTWSCSTTAGNEKYVPSECRKVGT >Pil_ZZC3-9 (Accession No. 83) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTVRARVSEGITTASAMKATVSENILNAGALVAGAPSTAGSACVGVTEISGGTGNVASATCGASKPGQIIVTMGATTAKSVPVYLTPSYTASAVTWSCSTTTGNEKYVPSECRKVGT >Pil_TUM15069 (Accession No. 84) MNTMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGASPMDATALAGNKNVASITSSTAGVITIAYKPAVVPNGSNNLTLTPVGADGTTALDLSAAASAGSQVNWRCGGTGTTVAAKFLPANCRGT >Pil_AI7 (Accession No. 85) MNAQKGFTLIELMIVVAIIGILAAVAIPAYQDYTTRAKVSEVITAGAACKTSVAEYYQSTGSLPLNTEQAGCSSNATPMVKSLAVASGIITVTASDALAAKFSTSTQNTYVLEPTATTAAAPLTWSCTGSTIEGKYLPAECRGT >Pil_VE-C3 (Accession No. 86) MKSMQKGFTLIELMIVIAIIGILAAIAIPAYTDYTARAKITEAVGALASAKTSVSEYYTSMGKMPADAAAAGINTAPAGSYVDNVAYAKTSDTVSTVTATIKNVNSTADTKKFKLTGTGSVAGVTWACATVDLDQKYLPANCRST >Pil_YH01026 (Accession No. 87) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMECNNAATCALIGASPMDATALGGNKNVTSITSSEAGVITIAYKPAVVPAGANNLLLTPVGADGTTALNLSAASAGSQVNWQCGGTNGTTVAAKFLPANCRGT >Pil_Mo (SEQ ID NO: 57) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGASPMDATALAGNKNVASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGADGTTALNLSAASAGSQVNWRCGGTGTTVAAKFLPANCRGT >Pil_CIP102143 (sequence number 88) MIVVAIIGILAAVAIPAYQDYTTRAKVSEVITAGAACKTSVAEYYQSTGSLPVNTEQAGCSSNATPMVKSLAVDKGVITVTASDSLAAKFSTSTKNTYVLRPTATSAAAPLTWSCTGSTIEGKYLPAECRGT >Pil_AI40 (SEQ ID NO: 89) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTTRAKMSEVVNFAAAAKSAVSECAISTGDLDECDSNQKAGLAPAADLTSTYVESVTVGADGLITLAIQGTNVTALDNGSLTMEPTLDPVAGVTWVCKISSNTLNKYVPANCRAT >Pil_F78 (SEQ ID NO: 90) MNTVQKGFTLIELMIVIAIIGILAAIAIPAYTDYTVRARVSEAMTTASAMKATVSENIMNAGGTSIVATNKNCAGIPAFTATKNVATAACTDKTGVILVTTTEAAKSVPLTLTPTYSGGNVSWRCSTTSSFDKYVPSECRST >Pil_S71 (SEQ ID NO: 91) MNAQKGFTLIELMIVVAIIGILAAVAIPAYQDYTTRAKVSEVITAGAACKTSVAEFYQSSGELPGTLEQAGCSSNATPMVASLDVGADGVITVTASTDLAAKFSDSAKNTYVLAPTATTAAAPLTWSCTGSTIEGKYLPAECRGT >Pil_ANC4282 (SEQ ID NO: 92) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTTRAKVTEVMNYAAAAKSAVSECLSSTGVTTSCDTNEAGLEAATSLTSPYVTSVTVGTGGSITAVVKGTNATSGNVALDGASLVLTPALSNAGVAWTCKISNVALNKFVPQTCRST >Pil_72-Oc (SEQ ID NO: 93) MNAQKGFTLIELMIVIAIIGILAAIAIPAYQNYIAKSQAAEAFTLMGGAKTTINSNLQNNSCTNTDDDTQNTVNGKYGVLTIGGAVAQDSNPTAATGCTMSYLFKGTGVSSQLADLVIEAGLNNNGTLAIDDTATTVDDELLPKSFAS >Pil_BI730 (SEQ ID NO: 94) MNAQKGFTLIELMIVVAIIGILAAIAIPQYQNYIAKSQVSRAMGETSSVKTAVETCLNEGKGAAGACPLGITTSNIQATTVAGSAPKADGTNAAELTETTAIVATFGTGAAKTLQETGKNSVTWTRDATGSWKCNSTVQAKYAPAGCSAT >Pil_A3K91 (SEQ ID NO: 95) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQNYIAKSQASEAFTLIDGAKAEVNTNLEGNSCTNATAAKNTIAGKYGSLVIAGTAASDASPTASTGCTLTYTFKGTGVSSQLASKVIGATLLNNGTLTKNATTTTVDADILPKSFT >Pil_CIP102159 (sequence number 96) MNTMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRSRVAEALTALSSAKATVSENIANNGGVIAAGACAGYTNQTTATANVASTSCTDTTGVVSATTTTKAGGFVITMTPTVNADKTVVWKCTVPAASFKYAPAECRGT >Pil_junii_65(Accession No. 97) MKSMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTTRAKMSEVVNFAAAAKSAVSECAISTGTLSDCNTNAKAGLEAAADLKSTYVESVTVGNNGVITLEIKGTNVTALDAANLTMTPTLDNKAGVSWVCKISSNTLNKYVPANCRST >Pil_YZS-X(Accession No. 98) MNTVQKGFTLIELMIVVAIIGILAAIAIPAYTDYTARAKVTEAVGALASAKTSVSEYFTSQGVMPTNATQAGINTAPAGQYVSAVAYTKTSDTVSKISATLANINSEANTKTIVLEGTGDTAKGVSWVCKGGTAPGKFLPANCRGT >Pil_T-3-2(Accession No. 99) MNAQKGFTLIELMIVIAIIGILAAIAIPAYQNYIAKAQVAEAFTLADGIKTSVGTNLQSGTCFASGAATAATTDTIEGKYGTATTVADTTTGSNGCGIKYTFKSSGVSNKLTSKVIGMAITENGVLKKSTVTTTDAPADLLPQSFT >Pil_CIP102637(Accession No. 100) MECNVLMIVVAIIGILAAIAIPAYTDYTARAKVTEAVGALASAKTSVSEFFTSQGVMPSDADAAGINKTPAGDYVAGVTYTRTDPTHAVVAVELKNINSDANGTTFQLNATGDTAKGVSWTCSSAGTKPTPEKFLPANCRGT >Pil_Mo_Δ28(Accession No. 58) AYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGASPMDATALAGNKNVASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGADGTTALNLSAASAGSQVNWRCGGTGTTVAAKFLPANCRGT >Pil 20 Sequon / Pil_Mo_DSL (SEQ ID NO: 60) CGGTGTTVAAKFLPANCRGT >EPA-Pil_Mo_Δ28 (SEQ ID NO: 101) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGASPMDATALAGNKNVASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGADGTTALNLSAAASAGSQVNWRCGGTGTTVAAKFLPANCRGT >EPA-Pil 20 (SEQ ID NO: 102) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAK LARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQV DQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIA GDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGLDPSSIPDKEQAISALPDYASQPGKPPREDLKGGGGCGGTGTTVAAKFLPANCRGT >PglS sequon (SEQ ID NO: 59) CTGVTQIASGASAATTNVASAQC >EPA_PglS Sequon_Pil 20 (SEQ ID NO: 103) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDACTGVTQIASGASAATTNVASAQCVRGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKGGGGCGGTGTTVAAKFLPANCRGT >EPA_PglS Sequence 2X (SEQ ID NO: 104) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDAT FFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPG SGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDA CTGVTQIASGASAATTNVASAQCVRGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEECTGVTQIASGASAATTNVASAQCGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Dsba secretion signal sequence (SEQ ID NO: 142) MKKIWLALAGLVLAFSASA >Dsba_EPA_Exotoxin_A_ΔE553 (SEQ ID NO: 143) (EPA carrier without sequon. This "bare" version has the non-native N-terminal DsbA secretion signal sequence and lacks residue E553, which inactivates the toxin. MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMG DELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVA LYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGG VRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK FlgI secretion signal sequence (SEQ ID NO: 144) MIKFLSALILLLVTTAAQA >FlgI_CRM197_G52E (SEQ ID NO: 145) (an inactivated (G52E) form of CRM197 with a non-native FlgI secretion signal sequence at the N-terminus) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS >DsbA_Tetanus toxin fragment C_TTc (SEQ ID NO: 146) MKKIWLALAGLVLAFSASAAKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKV SASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNN NNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIK LYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND >DsbA_cholera toxin_B_subunit (SEQ ID NO: 147) MKKIWLALAGLVLAFSASANGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 4-3]

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Claims

1. A glycoconjugate comprising an oligosaccharide or polysaccharide covalently attached to a receptor protein, the receptor protein comprises or consists of a TfpM-associated pilin-like protein or a glycosylated fragment thereof, and the oligosaccharide or polysaccharide is covalently bound to the TfpM-associated pilin-like protein or a glycosylated fragment thereof; the TfpM-associated pilin-like protein or glycosylated fragment thereof comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently linked to the C-terminal serine or threonine; Optionally, the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin-like protein; Optionally, the receptor protein is a fusion protein comprising the TfpM-associated pilin-like protein or glycosylated fragment thereof translationally fused to a heterologous carrier protein, wherein the TfpM-associated pilin-like protein or glycosylated fragment thereof is the most C-terminal sequence of the receptor protein, such that the receptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently linked to the C-terminal serine or threonine; Optionally, the oligosaccharide or polysaccharide comprises glucose at its reducing end; and / or Optionally, said glycoconjugate is immunogenic.

2. the length of the glycosylated fragment of the TfpM-related pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; The glycoconjugate of claim 1 , wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

3. (a) the TfpM-related pilin-like protein or glycosylated fragment thereof is (i) Pil Ｍｏ (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1-28 Ｍｏ (Pil Ｍｏ Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) the TfpM-related pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), and Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), (c) the TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin; and / or (d) the glycosylated fragment of the pilin-like protein is Pil Ｍｏ Pilrin disulfide loop region (Pil ２０ Also known as Pil Ｍｏ _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of said pilin or a variant thereof, wherein said C-terminal threonine is replaced with serine (SEQ ID NO: 148); Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil ２０ (SEQ ID NO: 60), Pil １９ (SEQ ID NO: 133), Pil １８ (SEQ ID NO: 134), Pil １７ (SEQ ID NO: 135), Pil １６ (SEQ ID NO: 136), Pil １５ (SEQ ID NO: 109), Pil １４ (SEQ ID NO: 137), Pil １３ (SEQ ID NO: 110), Pil １２ (SEQ ID NO: 138), Pil １１ (SEQ ID NO: 139), Pil １０ (SEQ ID NO: 112), Pil ９ (SEQ ID NO: 140), Pil ８ (SEQ ID NO: 141), Pil ７ (SEQ ID NO: 113), Pil ６ (SEQ ID NO: 114), Pil ５ (SEQ ID NO: 115), Pil ４ (SEQ ID NO: 116), or Pil ３ (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０［Ａ］ (SEQ ID NO: 166), Pil １９［Ａ］ (SEQ ID NO: 167), Pil １８［Ａ］ (SEQ ID NO: 168), Pil １７［Ａ］ (SEQ ID NO: 169), Pil １６［Ａ］ (SEQ ID NO: 170), Pil １５［Ａ］ (SEQ ID NO: 171), Pil １４［Ａ］ (SEQ ID NO: 172), Pil １３［Ａ］ (SEQ ID NO: 173), Pil １２［Ａ］ (SEQ ID NO: 174), Pil １１［Ａ］ (SEQ ID NO: 175), Pil １０［Ａ］ (SEQ ID NO: 176), Pil ９［Ａ］ (SEQ ID NO: 177), Pil ８［Ａ］ (SEQ ID NO: 178), Pil ７［Ａ］ (SEQ ID NO: 179), Pil ６［Ａ］ (SEQ ID NO: 180), Pil ５［Ａ］ (SEQ ID NO: 181), or Pil ４［Ａ］ (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil ２０Ｓ (SEQ ID NO: 148), Pil １９Ｓ (SEQ ID NO: 149), Pil １８Ｓ (SEQ ID NO: 150), Pil １７Ｓ (SEQ ID NO: 151), Pil １６Ｓ (SEQ ID NO: 152), Pil １５Ｓ (SEQ ID NO: 153), Pil １４Ｓ (SEQ ID NO: 154), Pil １３Ｓ (SEQ ID NO: 155), Pil １２Ｓ (SEQ ID NO: 156), Pil １１Ｓ (SEQ ID NO: 157), Pil １０Ｓ (SEQ ID NO: 158), Pil ９Ｓ (SEQ ID NO: 159), Pil ８Ｓ (SEQ ID NO: 160), Pil ７Ｓ (SEQ ID NO: 161), Pil ６Ｓ (SEQ ID NO: 162), Pil ５Ｓ (SEQ ID NO: 163), Pil ４Ｓ (SEQ ID NO: 164), or Pil ３Ｓ (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０Ｓ［Ａ］ (SEQ ID NO: 183), Pil １９Ｓ［Ａ］ (SEQ ID NO: 184), Pil １８Ｓ［Ａ］ (SEQ ID NO: 185), Pil １７Ｓ［Ａ］ (SEQ ID NO: 186), Pil １６Ｓ［Ａ］ (SEQ ID NO: 187), Pil １５Ｓ［Ａ］ (SEQ ID NO: 188), Pil １４Ｓ［Ａ］ (SEQ ID NO: 189), Pil １３Ｓ［Ａ］ (SEQ ID NO: 190), Pil １２Ｓ［Ａ］ (SEQ ID NO: 191), Pil １１Ｓ［Ａ］ (SEQ ID NO: 192), Pil １０Ｓ［Ａ］ (SEQ ID NO: 193), Pil ９Ｓ［Ａ］ (SEQ ID NO: 194), Pil ８Ｓ［Ａ］ (SEQ ID NO: 195), Pil ７Ｓ［Ａ］ (SEQ ID NO: 196), Pil ６Ｓ［Ａ］ (SEQ ID NO: 197), Pil ５Ｓ［Ａ］ (SEQ ID NO: 198), or Pil ４Ｓ［Ａ］ (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

4. 2. The glycoconjugate of claim 1, wherein the receptor protein is a fusion protein comprising a heterologous carrier protein, and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof.

5. the receptor protein is a fusion protein, which, in addition to the glycosylation fragment of the TfpM-related pilin-like protein located at its C-terminus, further comprises an additional glycosylation sequence of an oligosaccharyltransferase (OTase) other than the TfpM OTase; optionally, said additional glycosylation sequence is an internal glycosylation fragment of CompP, and further optionally, said glycosylation fragment of CompP comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO:59), or a fragment thereof comprising amino acids ASA at least positions 11-13; 2. The glycoconjugate of claim 1, wherein optionally, the additional glycosylation sequence is also covalently attached to the oligosaccharide or polysaccharide.

6. the fusion protein comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more additional glycosylation sequences; Optionally, the fusion protein does not comprise more than 2, more than 3, more than 5, more than 10, more than 15, more than 20, or more than 25 additional glycosylation sequences; Optionally, said additional glycosylation sequences are identical; Optionally, said additional glycosylation sequences are different from each other, and / or Optionally, at least three, at least four, or at least five of said additional glycosylation sequences are all different from one another; and / or 6. The glycoconjugate of claim 5, wherein optionally, none of the additional glycosylation sequences are the same.

7. 2. The glycoconjugate of claim 1, wherein the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least three repeating units of the oligosaccharide or polysaccharide structure and / or a size of at least ten monosaccharides.

8. (i) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, and the polysaccharide is a capsular polysaccharide; Optionally, Streptococcus pneumoniae (S. pneumoniae) or Streptococcus agalactiae (S. agalactiae), and Optionally, the S. agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; (ii) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella, and the polysaccharide is a capsular polysaccharide or an O-antigen polysaccharide; Optionally, the bacterium is Klebsiella pneumoniae; or (iii) the oligosaccharide or polysaccharide is produced by bacteria of the genus Salmonella, and the polysaccharide is an O-antigen polysaccharide; Optionally, the bacterium is Salmonella enterica (S. enterica) and the Salmonella enterica (S. enterica) polysaccharide is a group B O antigen.

9. The complex carbohydrates are Optionally, within a bacterial cell, Optionally, in Escherichia coli, Optionally produced in bacteria of the genus Klebsiella, and / or 2. The glycoconjugate of claim 1, wherein optionally the bacterial species is K. pneumoniae, K. varicola, K. michinganenis, or K. oxytoca.

10. the bioconjugate is a conjugate vaccine that elicits an immune response when administered to a subject; Optionally, the immune response induces long-term memory (memory B cells and T cells) and is an antibody response, optionally a serotype-specific antibody response; Optionally, the antibody response is an IgG or IgM response; Optionally, the antibody response is an IgG response, optionally an IgG1 response; and / or Optionally, the conjugate vaccine generates immune memory in a subject administered the vaccine.

11. A glycosylated fragment of a pilin-like protein comprising or consisting of an isolated fragment of a TfpM-related pilin-like protein, (a) the TfpM-related pilin-like protein or glycosylated fragment thereof is (i) Pil Ｍｏ (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1-28 Ｍｏ (Pil Ｍｏ Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) the TfpM-related pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), and Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), (c) the TfpM-related pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin; and / or (d) the glycosylated fragment of the pilin-like protein is Pil Ｍｏ Pilrin disulfide loop region (Pil ２０ Also known as Pil Ｍｏ _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids or variants from the C-terminus of said pilin, wherein said C-terminal threonine is replaced with serine (SEQ ID NO: 148); Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil ２０ (SEQ ID NO: 60), Pil １９ (SEQ ID NO: 133), Pil １８ (SEQ ID NO: 134), Pil １７ (SEQ ID NO: 135), Pil １６ (SEQ ID NO: 136), Pil １５ (SEQ ID NO: 109), Pil １４ (SEQ ID NO: 137), Pil １３ (SEQ ID NO: 110), Pil １２ (SEQ ID NO: 138), Pil １１ (SEQ ID NO: 139), Pil １０ (SEQ ID NO: 112), Pil ９ (SEQ ID NO: 140), Pil ８ (SEQ ID NO: 141), Pil ７ (SEQ ID NO: 113), Pil ６ (SEQ ID NO: 114), Pil ５ (SEQ ID NO: 115), Pil ４ (SEQ ID NO: 116), or Pil ３ (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０［Ａ］ (SEQ ID NO: 166), Pil １９［Ａ］ (SEQ ID NO: 167), Pil １８［Ａ］ (SEQ ID NO: 168), Pil １７［Ａ］ (SEQ ID NO: 169), Pil １６［Ａ］ (SEQ ID NO: 170), Pil １５［Ａ］ (SEQ ID NO: 171), Pil １４［Ａ］ (SEQ ID NO: 172), Pil １３［Ａ］ (SEQ ID NO: 173), Pil １２［Ａ］ (SEQ ID NO: 174), Pil １１［Ａ］ (SEQ ID NO: 175), Pil １０［Ａ］ (SEQ ID NO: 176), Pil ９［Ａ］ (SEQ ID NO: 177), Pil ８［Ａ］ (SEQ ID NO: 178), Pil ７［Ａ］ (SEQ ID NO: 179), Pil ６［Ａ］ (SEQ ID NO: 180), Pil ５［Ａ］ (SEQ ID NO: 181), or Pil ４［Ａ］ (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil ２０Ｓ (SEQ ID NO: 148), Pil １９Ｓ (SEQ ID NO: 149), Pil １８Ｓ (SEQ ID NO: 150), Pil １７Ｓ (SEQ ID NO: 151), Pil １６Ｓ (SEQ ID NO: 152), Pil １５Ｓ (SEQ ID NO: 153), Pil １４Ｓ (SEQ ID NO: 154), Pil １３Ｓ (SEQ ID NO: 155), Pil １２Ｓ (SEQ ID NO: 156), Pil １１Ｓ (SEQ ID NO: 157), Pil １０Ｓ (SEQ ID NO: 158), Pil ９Ｓ (SEQ ID NO: 159), Pil ８Ｓ (SEQ ID NO: 160), Pil ７Ｓ (SEQ ID NO: 161), Pil ６Ｓ (SEQ ID NO: 162), Pil ５Ｓ (SEQ ID NO: 163), Pil ４Ｓ (SEQ ID NO: 164), or Pil ３Ｓ (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０Ｓ［Ａ］ (SEQ ID NO: 183), Pil １９Ｓ［Ａ］ (SEQ ID NO: 184), Pil １８Ｓ［Ａ］ (SEQ ID NO: 185), Pil １７Ｓ［Ａ］ (SEQ ID NO: 186), Pil １６Ｓ［Ａ］ (SEQ ID NO: 187), Pil １５Ｓ［Ａ］ (SEQ ID NO: 188), Pil １４Ｓ［Ａ］ (SEQ ID NO: 189), Pil １３Ｓ［Ａ］ (SEQ ID NO: 190), Pil １２Ｓ［Ａ］ (SEQ ID NO: 191), Pil １１Ｓ［Ａ］ (SEQ ID NO: 192), Pil １０Ｓ［Ａ］ (SEQ ID NO: 193), Pil ９Ｓ［Ａ］ (SEQ ID NO: 194), Pil ８Ｓ［Ａ］ (SEQ ID NO: 195), Pil ７Ｓ［Ａ］ (SEQ ID NO: 196), Pil ６Ｓ［Ａ］ (SEQ ID NO: 197), Pil ５Ｓ［Ａ］ (SEQ ID NO: 198), or Pil ４Ｓ［Ａ］ (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

12. the length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 12. The glycosylated fragment of a TfpM-associated pilin-like protein of claim 11, wherein the glycosylated fragment of the TfpM-associated pilin-like protein comprises a C-terminal serine or threonine residue.

13. 1. A fusion protein comprising a TfpM-related pilin-like protein or a glycosylated fragment thereof translationally fused to a heterologous carrier protein, the TfpM-related pilin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue; the TfpM-related pilin-like protein or glycosylated fragment is the most C-terminal sequence of the fusion protein; and the fusion protein comprises a C-terminal serine or threonine residue; Optionally, the fusion protein is glycosylated with an oligo- or polysaccharide covalently attached to the C-terminal serine or threonine; Optionally, said fusion protein is glycosylated with an oligo- or polysaccharide comprising a glucose at its reducing end covalently linked to said C-terminal serine or threonine.

14. the length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; The fusion protein of claim 13 , wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

15. (a) the TfpM-related pilin-like protein or glycosylated fragment thereof is (i) Pil Ｍｏ (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1-28 Ｍｏ (Pil Ｍｏ Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) the TfpM-related pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), and Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), (c) the TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin; and / or (d) the glycosylated fragment of the pilin-like protein is Pil Ｍｏ Pilrin disulfide loop region (Pil ２０ Also known as Pil Ｍｏ _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of said pilin or a variant thereof, wherein said C-terminal threonine is replaced with serine (SEQ ID NO: 148); Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil ２０ (SEQ ID NO: 60), Pil １９ (SEQ ID NO: 133), Pil １８ (SEQ ID NO: 134), Pil １７ (SEQ ID NO: 135), Pil １６ (SEQ ID NO: 136), Pil １５ (SEQ ID NO: 109), Pil １４ (SEQ ID NO: 137), Pil １３ (SEQ ID NO: 110), Pil １２ (SEQ ID NO: 138), Pil １１ (SEQ ID NO: 139), Pil １０ (SEQ ID NO: 112), Pil ９ (SEQ ID NO: 140), Pil ８ (SEQ ID NO: 141), Pil ７ (SEQ ID NO: 113), Pil ６ (SEQ ID NO: 114), Pil ５ (SEQ ID NO: 115), Pil ４ (SEQ ID NO: 116), or Pil ３ (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０［Ａ］ (SEQ ID NO: 166), Pil １９［Ａ］ (SEQ ID NO: 167), Pil １８［Ａ］ (SEQ ID NO: 168), Pil １７［Ａ］ (SEQ ID NO: 169), Pil １６［Ａ］ (SEQ ID NO: 170), Pil １５［Ａ］ (SEQ ID NO: 171), Pil １４［Ａ］ (SEQ ID NO: 172), Pil １３［Ａ］ (SEQ ID NO: 173), Pil １２［Ａ］ (SEQ ID NO: 174), Pil １１［Ａ］ (SEQ ID NO: 175), Pil １０［Ａ］ (SEQ ID NO: 176), Pil ９［Ａ］ (SEQ ID NO: 177), Pil ８［Ａ］ (SEQ ID NO: 178), Pil ７［Ａ］ (SEQ ID NO: 179), Pil ６［Ａ］ (SEQ ID NO: 180), Pil ５［Ａ］ (SEQ ID NO: 181), or Pil ４［Ａ］ (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil ２０Ｓ (SEQ ID NO: 148), Pil １９Ｓ (SEQ ID NO: 149), Pil １８Ｓ (SEQ ID NO: 150), Pil １７Ｓ (SEQ ID NO: 151), Pil １６Ｓ (SEQ ID NO: 152), Pil １５Ｓ (SEQ ID NO: 153), Pil １４Ｓ (SEQ ID NO: 154), Pil １３Ｓ (SEQ ID NO: 155), Pil １２Ｓ (SEQ ID NO: 156), Pil １１Ｓ (SEQ ID NO: 157), Pil １０Ｓ (SEQ ID NO: 158), Pil ９Ｓ (SEQ ID NO: 159), Pil ８Ｓ (SEQ ID NO: 160), Pil ７Ｓ (SEQ ID NO: 161), Pil ６Ｓ (SEQ ID NO: 162), Pil ５Ｓ (SEQ ID NO: 163), Pil ４Ｓ (SEQ ID NO: 164), or Pil ３Ｓ (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０Ｓ［Ａ］ (SEQ ID NO: 183), Pil １９Ｓ［Ａ］ (SEQ ID NO: 184), Pil １８Ｓ［Ａ］ (SEQ ID NO: 185), Pil １７Ｓ［Ａ］ (SEQ ID NO: 186), Pil １６Ｓ［Ａ］ (SEQ ID NO: 187), Pil １５Ｓ［Ａ］ (SEQ ID NO: 188), Pil １４Ｓ［Ａ］ (SEQ ID NO: 189), Pil １３Ｓ［Ａ］ (SEQ ID NO: 190), Pil １２Ｓ［Ａ］ (SEQ ID NO: 191), Pil １１Ｓ［Ａ］ (SEQ ID NO: 192), Pil １０Ｓ［Ａ］ (SEQ ID NO: 193), Pil ９Ｓ［Ａ］ (SEQ ID NO: 194), Pil ８Ｓ［Ａ］ (SEQ ID NO: 195), Pil ７Ｓ［Ａ］ (SEQ ID NO: 196), Pil ６Ｓ［Ａ］ (SEQ ID NO: 197), Pil ５Ｓ［Ａ］ (SEQ ID NO: 198), or Pil ４Ｓ［Ａ］ (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

16. 14. The fusion protein of claim 13, wherein the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof.

17. the fusion protein further comprises, in addition to the glycosylation fragment of the TfpM-related pilin-like protein located at its C-terminus, an additional glycosylation sequence of an oligosaccharyltransferase (OTase) other than the TfpM OTase; optionally, said additional glycosylation sequence is an internal glycosylation fragment of CompP, and further optionally, said glycosylation fragment of CompP comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO:59), or a fragment thereof comprising amino acids ASA at least positions 11-13; Optionally, the additional glycosylation sequence is also covalently attached to an oligosaccharide or polysaccharide.

18. the fusion protein comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more additional glycosylation sequences; Optionally, the fusion protein does not comprise more than 2, more than 3, more than 5, more than 10, more than 15, more than 20, or more than 25 additional glycosylation sequences; Optionally, said additional glycosylation sequences are identical; Optionally, said additional glycosylation sequences are different from each other, and / or Optionally, at least three, at least four, or at least five of said additional glycosylation sequences are all different from one another; and / or Optionally, none of the additional glycosylation sequences are the same.

19. 14. The fusion protein of claim 13, wherein the oligosaccharide or polysaccharide covalently attached to the pilin-like protein or glycosylated fragment thereof has a size of at least three repeating units of an oligosaccharide or polysaccharide structure and / or a size of at least ten monosaccharides.

20. (i) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, and the polysaccharide is a capsular polysaccharide; Optionally, Streptococcus pneumoniae (S. pneumoniae) or Streptococcus agalactiae (S. agalactiae), and Optionally, the S. agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; (ii) the oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella, and the polysaccharide is a capsular polysaccharide or an O-antigen polysaccharide; Optionally, the bacterium is Klebsiella pneumoniae; or (iii) the oligosaccharide or polysaccharide is produced by bacteria of the genus Salmonella, and the polysaccharide is an O-antigen polysaccharide; Optionally, the bacterium is Salmonella enterica (S. enterica) and the S. enterica polysaccharide is a group B O antigen.

21. The complex carbohydrates are Optionally, within a bacterial cell, Optionally, in Escherichia coli, Optionally produced in bacteria of the genus Klebsiella, and / or 14. The fusion protein of claim 13, wherein optionally the bacterial species is K. pneumoniae, K. varicola, K. michinganenis, or K. oxytoca.

22. the fusion protein is a vaccine that induces an immune response when administered to a subject; Optionally, the immune response induces long-term memory (memory B cells and T cells) and is an antibody response, optionally a serotype-specific antibody response; Optionally, the antibody response is an IgG or IgM response; Optionally, the antibody response is an IgG response, optionally an IgG1 response; and / or Optionally, the fusion protein generates immunological memory in a subject administered the vaccine.

23. 1. A method for producing glycoconjugates, said method comprising covalently attaching an oligosaccharide or polysaccharide to an acceptor protein comprising or consisting of a TfpM-associated pilin-like protein or a glycosylated fragment thereof using a TfpM oligosaccharyltransferase (OTase); the pyrin-like protein or glycosylated fragment comprises a C-terminal serine or threonine residue, the receptor protein comprises a C-terminal serine or threonine residue, and the oligosaccharide or polysaccharide is covalently bound to the C-terminal serine or threonine residue of the receptor protein; Optionally, the oligosaccharide or polysaccharide comprises glucose at its reducing end; Optionally, the receptor protein is a fusion protein according to any one of claims 13 to 22; Optionally, the method is a method for in vivo conjugation of an oligosaccharide or polysaccharide to a receptor protein; and / or Optionally, the glycoconjugate is immunogenic.

24. The TfpM OTase is Ｍｏ (SEQ ID NO: 56), TfpM ＤＳＭ１６６１７ (SEQ ID NO: 63), TfpM ＺＺＣ３ (SEQ ID NO: 64), TfpM ＴＵＭ１５０６９ (SEQ ID NO: 65), TfpM ＡＩ７ (SEQ ID NO: 66), TfpM ＶＥ－Ｃ３ (SEQ ID NO: 67), TfpM ＹＨ０１０２６ (SEQ ID NO: 68), TfpM ＣＩＰ１０２１４３ (SEQ ID NO: 69), TfpM ＡＩ４０ (SEQ ID NO: 70), TfpM Ｆ７８ (SEQ ID NO: 71), TfpM Ｓ７１ (SEQ ID NO: 72), TfpM ＡＮＣ４２８２ (SEQ ID NO: 73), TfpM ＣＩＰ１０２１５９ (SEQ ID NO: 74), TfpM ｊｕｎｉｉ－６５ (SEQ ID NO: 75), TfpM ＹＺＳ－Ｘ (SEQ ID NO: 76), TfpM ＣＩＰ１０２６３７ (SEQ ID NO: 77), TfpM Ｔ－３－２ (SEQ ID NO: 78), TfpM ＢＩ７３０ (SEQ ID NO: 79), TfpM Ａ３Ｋ９１ (SEQ ID NO: 80), and / or TfpM ７２－Ｏ－ｃ (SEQ ID NO: 81), comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to Optionally, said TfpM OTase is TfpM Ｍｏ (SEQ ID NO: 56), TfpM ＤＳＭ１６６１７ (SEQ ID NO: 63), TfpM ＺＺＣ３ (SEQ ID NO: 64), TfpM ＴＵＭ１５０６９ (SEQ ID NO: 65), TfpM ＡＩ７ (SEQ ID NO: 66), TfpM ＶＥ－Ｃ３ (SEQ ID NO: 67), TfpM ＹＨ０１０２６ (SEQ ID NO: 68), TfpM ＣＩＰ１０２１４３ (SEQ ID NO: 69), TfpM ＡＩ４０ (SEQ ID NO: 70), TfpM Ｆ７８ (SEQ ID NO: 71), TfpM Ｓ７１ (SEQ ID NO: 72), TfpM ＡＮＣ４２８２ (SEQ ID NO: 73), TfpM ＣＩＰ１０２１５９ (SEQ ID NO: 74), TfpM ｊｕｎｉｉ－６５ (SEQ ID NO: 75), TfpM ＹＺＳ－Ｘ (SEQ ID NO: 76), TfpM ＣＩＰ１０２６３７ (SEQ ID NO: 77), TfpM Ｔ－３－２ (SEQ ID NO: 78), TfpM ＢＩ７３０ (SEQ ID NO: 79), TfpM Ａ３Ｋ９１ (SEQ ID NO: 80), or TfpM ７２－Ｏ－ｃ (SEQ ID NO: 81), Optionally, said TfpM OTase is TfpM Ｍｏ (SEQ ID NO: 56).

25. the length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 24. The method of claim 23, wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

26. (a) the TfpM-related pilin-like protein or glycosylated fragment thereof is (i) Pil Ｍｏ (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1-28 Ｍｏ (Pil Ｍｏ Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) the TfpM-related pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), and Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), (c) the TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin; and / or (d) the glycosylated fragment of the pilin-like protein is Pil Ｍｏ Pilrin disulfide loop region (Pil ２０ Also known as Pil Ｍｏ _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids or variants from the C-terminus of said pilin, wherein said C-terminal threonine is replaced with serine (SEQ ID NO: 148); Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil ２０ (SEQ ID NO: 60), Pil １９ (SEQ ID NO: 133), Pil １８ (SEQ ID NO: 134), Pil １７ (SEQ ID NO: 135), Pil １６ (SEQ ID NO: 136), Pil １５ (SEQ ID NO: 109), Pil １４ (SEQ ID NO: 137), Pil １３ (SEQ ID NO: 110), Pil １２ (SEQ ID NO: 138), Pil １１ (SEQ ID NO: 139), Pil １０ (SEQ ID NO: 112), Pil ９ (SEQ ID NO: 140), Pil ８ (SEQ ID NO: 141), Pil ７ (SEQ ID NO: 113), Pil ６ (SEQ ID NO: 114), Pil ５ (SEQ ID NO: 115), Pil ４ (SEQ ID NO: 116), or Pil ３ (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０［Ａ］ (SEQ ID NO: 166), Pil １９［Ａ］ (SEQ ID NO: 167), Pil １８［Ａ］ (SEQ ID NO: 168), Pil １７［Ａ］ (SEQ ID NO: 169), Pil １６［Ａ］ (SEQ ID NO: 170), Pil １５［Ａ］ (SEQ ID NO: 171), Pil １４［Ａ］ (SEQ ID NO: 172), Pil １３［Ａ］ (SEQ ID NO: 173), Pil １２［Ａ］ (SEQ ID NO: 174), Pil １１［Ａ］ (SEQ ID NO: 175), Pil １０［Ａ］ (SEQ ID NO: 176), Pil ９［Ａ］ (SEQ ID NO: 177), Pil ８［Ａ］ (SEQ ID NO: 178), Pil ７［Ａ］ (SEQ ID NO: 179), Pil ６［Ａ］ (SEQ ID NO: 180), Pil ５［Ａ］ (SEQ ID NO: 181), or Pil ４［Ａ］ (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil ２０Ｓ (SEQ ID NO: 148), Pil １９Ｓ (SEQ ID NO: 149), Pil １８Ｓ (SEQ ID NO: 150), Pil １７Ｓ (SEQ ID NO: 151), Pil １６Ｓ (SEQ ID NO: 152), Pil １５Ｓ (SEQ ID NO: 153), Pil １４Ｓ (SEQ ID NO: 154), Pil １３Ｓ (SEQ ID NO: 155), Pil １２Ｓ (SEQ ID NO: 156), Pil １１Ｓ (SEQ ID NO: 157), Pil １０Ｓ (SEQ ID NO: 158), Pil ９Ｓ (SEQ ID NO: 159), Pil ８Ｓ (SEQ ID NO: 160), Pil ７Ｓ (SEQ ID NO: 161), Pil ６Ｓ (SEQ ID NO: 162), Pil ５Ｓ (SEQ ID NO: 163), Pil ４Ｓ (SEQ ID NO: 164), or Pil ３Ｓ (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０Ｓ［Ａ］ (SEQ ID NO: 183), Pil １９Ｓ［Ａ］ (SEQ ID NO: 184), Pil １８Ｓ［Ａ］ (SEQ ID NO: 185), Pil １７Ｓ［Ａ］ (SEQ ID NO: 186), Pil １６Ｓ［Ａ］ (SEQ ID NO: 187), Pil １５Ｓ［Ａ］ (SEQ ID NO: 188), Pil １４Ｓ［Ａ］ (SEQ ID NO: 189), Pil １３Ｓ［Ａ］ (SEQ ID NO: 190), Pil １２Ｓ［Ａ］ (SEQ ID NO: 191), Pil １１Ｓ［Ａ］ (SEQ ID NO: 192), Pil １０Ｓ［Ａ］ (SEQ ID NO: 193), Pil ９Ｓ［Ａ］ (SEQ ID NO: 194), Pil ８Ｓ［Ａ］ (SEQ ID NO: 195), Pil ７Ｓ［Ａ］ (SEQ ID NO: 196), Pil ６Ｓ［Ａ］ (SEQ ID NO: 197), Pil ５Ｓ［Ａ］ (SEQ ID NO: 198), or Pil ４Ｓ［Ａ］ (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

27. 24. The method of claim 23, wherein the receptor protein is a fusion protein comprising a heterologous carrier protein, and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and any fragment thereof.

28. the receptor protein is a fusion protein comprising, in addition to the glycosylation fragment of the TfpM-related pilin-like protein located at its C-terminus, an additional glycosylation sequence of an oligosaccharyltransferase (OTase) other than TfpM OTase, and the method further comprises covalently attaching an oligosaccharide or polysaccharide to the additional glycosylation sequence using an OTase other than TfpM OTase; Optionally, the receptor protein is a fusion protein comprising a ComP glycosylation fragment, and the method further comprises covalently attaching an oligosaccharide or polysaccharide to the ComP glycosylation fragment using PglS OTase; 24. The method of claim 23, optionally wherein the CompP glycosylated fragment is an internal glycosylated fragment of CompP, and further optionally wherein the CompP glycosylated fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least amino acids ASA at positions 11 to 13.

29. 24. The method of claim 23, wherein the binding occurs within a host cell.

30. the host cell is a bacterial cell; Optionally, in Escherichia coli; Optionally, in a bacterium from the genus Klebsiella, or 30. The method of claim 29, wherein optionally the bacterial species is K. pneumoniae, K. varicola, K. michinganenis, or K. oxytoca.

31. 30. The method of claim 29, comprising culturing a host cell containing (a) a gene cluster encoding the proteins necessary to synthesize the oligosaccharide or polysaccharide, (b) TfpM OTase, and (3) the receptor protein.

32. 24. The method of claim 23, wherein the method produces a conjugate vaccine.

33. A host cell comprising (a) a gene cluster encoding the proteins necessary to synthesize the oligosaccharide or polysaccharide, (b) a TfpM OTase, and (3) a receptor protein comprising a TfpM-related pilin-like protein or a glycosylated fragment thereof.

34. 34. The host cell of claim 33, wherein the receptor protein is a fusion protein.

35. the host cell comprises a nucleic acid encoding the TfpM OTase; and / or the host cell comprises a nucleic acid encoding the receptor protein; Optionally, the TfpM OTase and the receptor protein are encoded by the same nucleic acid.

36. An isolated nucleic acid encoding a glycosylated fragment of a pilin-like protein according to claim 11 or 12 and / or a fusion protein according to any one of claims 13 to 22.

37. 37. The isolated nucleic acid of claim 36, wherein the nucleic acid is a vector.

38. 37. A host cell comprising the isolated nucleic acid of claim 36, Optionally, the host cell is a bacterial cell, and further comprising: Optionally, the host cell is Escherichia coli; Optionally, the host cell is from the genus Klebsiella; or Optionally, the host cell is a K. pneumoniae, K. varicola, K. michinganenis, or K. oxytoca host cell.

39. 23. A composition comprising the conjugate vaccine of claim 10 or the fusion protein of claim 22, and an adjuvant and / or carrier.

40. 40. A method for inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need thereof an effective amount of the conjugate vaccine of claim 10, the fusion protein of claim 22, or the composition of claim 39.

41. the immune response is an antibody response; the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, a cellular response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system crosstalk, and a combination of two or more of the foregoing immune responses; and / or 41. The method of claim 40, wherein the immune response is selected from the group consisting of an innate response, a humoral response, an antibody response, a T cell response, and a combination of two or more of the immune responses.

42. 40. A method for preventing or treating a bacterial disease and / or infection in a subject, comprising administering to a subject in need thereof the conjugate vaccine of claim 10, the fusion protein of claim 22, or the composition of claim 39; Optionally, the subject is a human.

43. the infection is a local or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune; the disease is pneumonia, and / or 43. The method of claim 42, wherein the infection is a systemic infection and / or a blood infection.

44. 44. The method of any one of claims 40 to 43, wherein the conjugate vaccine, the fusion protein, or the composition is administered by intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

45. 1. A method for producing a pneumococcal conjugate vaccine against pneumococcal infection, said method comprising: (a) isolating a glycoconjugate according to any one of claims 1 to 10 or a glycosylated fusion protein according to any one of claims 13 to 22; (b) combining said isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and / or carrier.

46. 10. A glycoconjugate, glycosylated fusion protein or conjugate vaccine according to any one of the preceding claims for use in inducing a host immune response against a bacterial pathogen and / or for preventing or treating bacterial diseases and / or infections in a subject.

47. A recombinant nucleic acid construct comprising a nucleotide sequence encoding the TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence.

48. The TfpM OTase is Ｍｏ (SEQ ID NO: 56), TfpM ＤＳＭ１６６１７ (SEQ ID NO: 63), TfpM ＺＺＣ３ (SEQ ID NO: 64), TfpM ＴＵＭ１５０６９ (SEQ ID NO: 65), TfpM ＡＩ７ (SEQ ID NO: 66), TfpM ＶＥ－Ｃ３ (SEQ ID NO: 67), TfpM ＹＨ０１０２６ (SEQ ID NO: 68), TfpM ＣＩＰ１０２１４３ (SEQ ID NO: 69), TfpM ＡＩ４０ (SEQ ID NO: 70), TfpM Ｆ７８ (SEQ ID NO: 71), TfpM Ｓ７１ (SEQ ID NO: 72), TfpM ＡＮＣ４２８２ (SEQ ID NO: 73), TfpM ＣＩＰ１０２１５９ (SEQ ID NO: 74), TfpM ｊｕｎｉｉ－６５ (SEQ ID NO: 75), TfpM ＹＺＳ－Ｘ (SEQ ID NO: 76), TfpM ＣＩＰ１０２６３７ (SEQ ID NO: 77), TfpM Ｔ－３－２ (SEQ ID NO: 78), TfpM ＢＩ７３０ (SEQ ID NO: 79), TfpM Ａ３Ｋ９１ (SEQ ID NO: 80), and / or TfpM ７２－Ｏ－ｃ (SEQ ID NO: 81), comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to Optionally, said TfpM OTase is TfpM Ｍｏ (SEQ ID NO: 56), TfpM ＤＳＭ１６６１７ (SEQ ID NO: 63), TfpM ＺＺＣ３ (SEQ ID NO: 64), TfpM ＴＵＭ１５０６９ (SEQ ID NO: 65), TfpM ＡＩ７ (SEQ ID NO: 66), TfpM ＶＥ－Ｃ３ (SEQ ID NO: 67), TfpM ＹＨ０１０２６ (SEQ ID NO: 68), TfpM ＣＩＰ１０２１４３ (SEQ ID NO: 69), TfpM ＡＩ４０ (SEQ ID NO: 70), TfpM Ｆ７８ (SEQ ID NO: 71), TfpM Ｓ７１ (SEQ ID NO: 72), TfpM ＡＮＣ４２８２ (SEQ ID NO: 73), TfpM ＣＩＰ１０２１５９ (SEQ ID NO: 74), TfpM ｊｕｎｉｉ－６５ (SEQ ID NO: 75), TfpM ＹＺＳ－Ｘ (SEQ ID NO: 76), TfpM ＣＩＰ１０２６３７ (SEQ ID NO: 77), TfpM Ｔ－３－２ (SEQ ID NO: 78), TfpM ＢＩ７３０ (SEQ ID NO: 79), TfpM Ａ３Ｋ９１ (SEQ ID NO: 80), and / or TfpM ７２－Ｏ－ｃ (SEQ ID NO: 81), Optionally, said TfpM OTase is TfpM Ｍｏ 48. The recombinant construct of claim 47, wherein the construct is (SEQ ID NO: 56).

49. 49. The recombinant construct of claim 47 or 48, wherein the heterologous transcriptional regulatory sequence is a promoter sequence.

50. a nucleotide sequence encoding a TfpM-associated pilin-like protein or a glycosylated fragment thereof, or a fusion protein comprising a TfpM-associated pilin-like protein or a glycosylated fragment thereof operably linked to said nucleotide sequence encoding said TfpM OTase, Optionally, the coding sequence for the TfpM-associated pilin-like protein or glycosylated fragment thereof, or a fusion protein comprising the TfpM-associated pilin-like protein or glycosylated fragment thereof, is within 2, 5, 10, 20, 30, 40, or 50 nucleotides of the sequence encoding the TfpM OTase; 50. The recombinant construct of any one of claims 47 to 49, wherein optionally the coding sequence for the TfpM-associated pilin-like protein or glycosylated fragment thereof, or a fusion protein comprising the TfpM-associated pilin-like protein or glycosylated fragment thereof, overlaps with an operably linked nucleotide sequence encoding TfpM OTase.

51. the length of the glycosylated fragment of the pilin-like protein is 3 to 139 amino acids, 20 to 139 amino acids, 116 to 139 amino acids, 3 to 22 amino acids, 10 to 22 amino acids, 11 to 22 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, or 11 to 21 amino acids; 51. The recombinant construct of claim 50, wherein the glycosylated fragment of the TfpM-related pilin-like protein comprises a C-terminal serine or threonine residue.

52. (a) the TfpM-related pilin-like protein or glycosylated fragment thereof is (i) Pil Ｍｏ (SEQ ID NO: 57), (ii) Pil lacking amino acids corresponding to residues 1-28 Ｍｏ (Pil Ｍｏ Δ28, SEQ ID NO:58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:57 or SEQ ID NO:58, wherein the TfpM-related pilin-like protein contains a C-terminal serine or threonine residue, and optionally the C-terminal threonine is replaced with serine; (b) the TfpM-related pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), and Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), (c) the TfpM-associated pilin-like protein or the glycosylated fragment of the pilin-like protein is Pil ＤＳＭ１６６１７ (SEQ ID NO: 82), Pil ＺＺＣ３－９ (SEQ ID NO: 83), Pil ＴＵＭ１５０６９ (SEQ ID NO: 84), Pil ＡＩ７ (SEQ ID NO: 85), Pil ＶＥ－Ｃ３ (SEQ ID NO: 86), Pil ＹＨ０１０２６ (SEQ ID NO: 87), Pil ＣＩＰ１０２１４３ (SEQ ID NO: 88), Pil ＡＩ４０ (SEQ ID NO: 89), Pil Ｆ７８ (SEQ ID NO: 90), Pil Ｓ７１ (SEQ ID NO: 91), Pil ＡＮＣ４２８２ (SEQ ID NO: 92), Pil ７２－Ｏ－ｃ (SEQ ID NO: 93), Pil ＢＩ７３０ (SEQ ID NO: 94), Pil Ａ３Ｋ９１ (SEQ ID NO: 95), Pil ＣＩＰ１０２１５９ (SEQ ID NO: 96), Pil ｊｕｎｉｉ－６５ (SEQ ID NO: 97), Pil ＹＺＳ－Ｘ (SEQ ID NO: 98), Pil Ｔ－３－２ (SEQ ID NO: 99), Pil ＣＩＰ１０２６３７ (SEQ ID NO: 100), and any fragment or variant thereof containing a C-terminal serine or threonine residue, wherein the C-terminal threonine is replaced with serine, and optionally the glycosylated fragment of the TfpM-related pilin-like protein comprises at least the last three amino acids from the C-terminus of the pilin; and / or (d) the glycosylated fragment of the pilin-like protein is Pil Ｍｏ Pilrin disulfide loop region (Pil ２０ Also known as Pil Ｍｏ _DSL, SEQ ID NO: 60) or a truncated derivative thereof comprising at least the last three amino acids from the C-terminus of said pilin or a variant thereof, wherein said C-terminal threonine is replaced with serine (SEQ ID NO: 148); Optionally, (e) the glycosylated fragment of the pilin-like protein is Pil ２０ (SEQ ID NO: 60), Pil １９ (SEQ ID NO: 133), Pil １８ (SEQ ID NO: 134), Pil １７ (SEQ ID NO: 135), Pil １６ (SEQ ID NO: 136), Pil １５ (SEQ ID NO: 109), Pil １４ (SEQ ID NO: 137), Pil １３ (SEQ ID NO: 110), Pil １２ (SEQ ID NO: 138), Pil １１ (SEQ ID NO: 139), Pil １０ (SEQ ID NO: 112), Pil ９ (SEQ ID NO: 140), Pil ８ (SEQ ID NO: 141), Pil ７ (SEQ ID NO: 113), Pil ６ (SEQ ID NO: 114), Pil ５ (SEQ ID NO: 115), Pil ４ (SEQ ID NO: 116), or Pil ３ (SEQ ID NO: 117), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０［Ａ］ (SEQ ID NO: 166), Pil １９［Ａ］ (SEQ ID NO: 167), Pil １８［Ａ］ (SEQ ID NO: 168), Pil １７［Ａ］ (SEQ ID NO: 169), Pil １６［Ａ］ (SEQ ID NO: 170), Pil １５［Ａ］ (SEQ ID NO: 171), Pil １４［Ａ］ (SEQ ID NO: 172), Pil １３［Ａ］ (SEQ ID NO: 173), Pil １２［Ａ］ (SEQ ID NO: 174), Pil １１［Ａ］ (SEQ ID NO: 175), Pil １０［Ａ］ (SEQ ID NO: 176), Pil ９［Ａ］ (SEQ ID NO: 177), Pil ８［Ａ］ (SEQ ID NO: 178), Pil ７［Ａ］ (SEQ ID NO: 179), Pil ６［Ａ］ (SEQ ID NO: 180), Pil ５［Ａ］ (SEQ ID NO: 181), or Pil ４［Ａ］ (SEQ ID NO: 182), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal threonine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin; or Optionally, (f) the glycosylated fragment of the pilin-like protein is Pil ２０Ｓ (SEQ ID NO: 148), Pil １９Ｓ (SEQ ID NO: 149), Pil １８Ｓ (SEQ ID NO: 150), Pil １７Ｓ (SEQ ID NO: 151), Pil １６Ｓ (SEQ ID NO: 152), Pil １５Ｓ (SEQ ID NO: 153), Pil １４Ｓ (SEQ ID NO: 154), Pil １３Ｓ (SEQ ID NO: 155), Pil １２Ｓ (SEQ ID NO: 156), Pil １１Ｓ (SEQ ID NO: 157), Pil １０Ｓ (SEQ ID NO: 158), Pil ９Ｓ (SEQ ID NO: 159), Pil ８Ｓ (SEQ ID NO: 160), Pil ７Ｓ (SEQ ID NO: 161), Pil ６Ｓ (SEQ ID NO: 162), Pil ５Ｓ (SEQ ID NO: 163), Pil ４Ｓ (SEQ ID NO: 164), or Pil ３Ｓ (SEQ ID NO: 165), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, and further optionally, the glycosylated fragment comprises at least the last 3 amino acids from the C-terminus of the pilin, and further optionally, the glycosylated fragment of the pilin-like protein comprises Pil ２０Ｓ［Ａ］ (SEQ ID NO: 183), Pil １９Ｓ［Ａ］ (SEQ ID NO: 184), Pil １８Ｓ［Ａ］ (SEQ ID NO: 185), Pil １７Ｓ［Ａ］ (SEQ ID NO: 186), Pil １６Ｓ［Ａ］ (SEQ ID NO: 187), Pil １５Ｓ［Ａ］ (SEQ ID NO: 188), Pil １４Ｓ［Ａ］ (SEQ ID NO: 189), Pil １３Ｓ［Ａ］ (SEQ ID NO: 190), Pil １２Ｓ［Ａ］ (SEQ ID NO: 191), Pil １１Ｓ［Ａ］ (SEQ ID NO: 192), Pil １０Ｓ［Ａ］ (SEQ ID NO: 193), Pil ９Ｓ［Ａ］ (SEQ ID NO: 194), Pil ８Ｓ［Ａ］ (SEQ ID NO: 195), Pil ７Ｓ［Ａ］ (SEQ ID NO: 196), Pil ６Ｓ［Ａ］ (SEQ ID NO: 197), Pil ５Ｓ［Ａ］ (SEQ ID NO: 198), or Pil ４Ｓ［Ａ］ (SEQ ID NO: 199), or a variant thereof having 1, 2, 3, 4, or 5 amino acid substitutions and maintaining the C-terminal serine, wherein the glycosylated fragment comprises at least the last 4 amino acids from the C-terminus of the pilin.

53. A recombinant construct according to any one of claims 50 to 52, wherein the fusion protein is a fusion protein according to any one of claims 13 to 22.

54. further comprising a nucleotide sequence encoding a PglS OTase operably linked to the TpfM OTase; Optionally, the PglS OTase coding sequence is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the TfpM OTase encoding sequence.

55. A vector comprising the recombinant nucleic acid construct of any one of claims 47 to 54.

56. A host cell comprising a recombinant nucleic acid construct according to any one of claims 47 to 54 or a vector according to claim 55, Optionally, the host cell is a bacterial cell, and further comprising: Optionally, the host cell is Escherichia coli; Optionally, the host cell is from the genus Klebsiella; or Optionally, the host cell is a K. pneumoniae, K. varicola, K. michinganenis, or K. oxytoca host cell.

57. 57. A method for producing TfpM OTase, comprising culturing the host cell of claim 56, wherein the vector of claim 54 is an expression vector, and recovering the TfpM OTase.

58. A glycoconjugate comprising an oligosaccharide or polysaccharide covalently attached to a receptor protein, A glycoconjugate comprising means by which said receptor protein is covalently bound to said oligosaccharide or polysaccharide by TfpM OTase.

59. The complex carbohydrate according to claim 58, wherein the complex carbohydrate is a complex carbohydrate according to any one of claims 1 to 10.

60. A fusion protein comprising a means for covalently binding to an oligosaccharide or polysaccharide by TfpM OTase, said means being translationally fused to a heterologous carrier protein; A fusion protein, wherein said means is located in the most C-terminal sequence of said fusion protein.

61. The fusion protein according to claim 60, wherein the fusion protein is a fusion protein according to any one of claims 13 to 22.

62. 62. A glycoconjugate according to claim 58 or 59, or a fusion protein according to claim 60 or 61, for use in any of the above methods.

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