Synthetic protein scaffolds to target and remove bacterial virulence pili

Abstract

Synthetic bacterial phage maturation (Mat) protein scaffolds, compositions, and methods of use to decrease bacterial virulence, treat and prevent bacterial infections, and lower the incidence of bacterial gene transfers to combat the spread of treatment-resistant bacterial strains. Scaffolds include at least a portion of an apical domain of a Mat protein and can decrease type IV pili (T4P) attachment for reduction of bacterial virulence.

Description

SYNTHETIC PROTEIN SCAFFOLDS TO TARGET AND REMOVE BACTERIAL VIRULENCE PILICROSS-REFERENCE(S) TO RELATED APPLICATION(S)

[0001] This PCT application claims the benefit of, and priority to, U.S. provisional application 63 / 583,763 filed September 19, 2023; the contents of which are incorporated herein in their entirety for all purposes.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3681-P109WO_Sequence_Listing.xml. The XML file is 30,955 bytes; was created on September 17, 2024; and is being submitted electronically via Patent Center with the filing of the specification.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with Government support under R01GM141659 awarded by National Institutes of Health; under MCB-1902392 awarded by the National Science Foundation; under U24GM116787 awarded by the National Institutes of Health; and under R21AH56846 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0004] Acinetobacter infections impose a significant clinical burden, affecting various body systems, including lungs, bloodstream, and urinary tract. The severity of these infections ranges from mild to acute, with immunocompromised individuals being most susceptible. The emergence of antibiotic-resistant strains, especially of the species A. baumannii. has significantly limited treatment options, necessitating the exploration of alternative therapeutic approaches, such as phage therapy.

[0005] Acinetobacter s have type IV pili (T4P), which are involved in twitching motility, surface adhesion, biofilm formation, and natural transformation in gram-negative bacteria. These thin, filamentous, and retractile appendages underlie the virulence and ability by which Acinetobacters acquire and incorporate genetic material from theenvironment. Consequently, this results in the facile spread of antibiotic resistance genes, substantially increasing the challenges for bacterial treatment.

[0006] Single-stranded RNA bacteriophages (ssRNA phages) are positive-sense RNA viruses that have shown specificity for gram-negative bacteria by exploiting host retractile pili, including T4P. The genomes of these ssRNA viruses, typically 3-4kb long, exhibit a conserved organization of three core genes. These are, from the 5’ end, mat, coat, and rep, encoding the maturation protein (Mat), the coat protein (Coat) and the P-subunit of an RNA-dependent RNA replicase (Rep), respectively. In addition, there is usually a gene for a lysis protein, but its location is variable and often embedded out of frame for one of the core genes. Recently, metatranscriptomic studies have discovered 65,814 sequences of these ssRNA viruses, with more than 12,000 sequences being complete or near-complete genomes, each containing three core genes.

[0007] While the structures of two model ssRNA phages, MS2 and QP, which infect A. coli via the conjugation F pilus, have been characterized, it is unclear whether all ssRNA phages share the same structural features. Recently, studies have demonstrated that MS2 has the ability to bind to and induce detachment of the conjugative F-pili from infected E. coli cells. As retractile pili play roles in the virulence of many bacteria, in principle, if the infection-dependent pili detachment is a general feature for ssRNA phages, these simple viruses could be used to suppress the deployment of retractile pili and thus reduce the virulence of many important bacterial infections. While a cryo-electron microscopy (cryo- EM) structure has revealed how MS2 adsorbs to the F- pilus, due to the high diversity of retractile pili in gram-negative bacteria, the structural mechanism obtained from one phagepilus pair cannot apply to another, and results for one are not necessarily predictive for results for another.

[0008] Accordingly, there is a need for compounds, compositions, and methods for treatment and prevention of bacterial infections, including but not necessarily limited to Acinetobacter infections, that can disrupt or interfere with pili function for inhibition of bacterial binding and gene transfer within groups of bacteria. The present disclosure addresses these and other long-felt and unmet needs in the art.SUMMARY

[0009] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summaryis not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0010] In an aspect, the disclosure provides a synthetic bacterial phage maturation (Mat) protein scaffold comprising an apical domain polypeptide sequence. In embodiments, the scaffold consists essentially of the apical domain polypeptide sequence. In embodiments, the scaffold consists of the apical domain polypeptide sequence.

[0011] In embodiments, the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species. In embodiments, the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2. In embodiments, the apical domain polypeptide sequence comprises 100% identity to the polypeptide sequence of SEQ ID NO:2.

[0012] In embodiments, the scaffold further comprises a tag polypeptide sequence that enables a fluorescence detection of the scaffold, an interaction of a tag binder with the tag polypeptide sequence, or both.

[0013] In an aspect, the disclosure provides a composition comprising a scaffold and a carrier, a diluent, a salt, a pH buffering agent, a detergent, a lipid, and / or a surfactant. In an aspect, the disclosure provides a kit comprising the composition and an instructional material configured to instruct an individual in a use of the composition in a method of decreasing bacterial virulence in a biological matrix.

[0014] In an aspect, the disclosure provides a pharmaceutical composition comprising a scaffold and a pharmaceutically acceptable carrier. In an aspect, the disclosure provides a kit comprising the pharmaceutical composition and an instructional material configured to instruct an individual in a use of the pharmaceutical composition in a method of treating or preventing bacterial infection in a subject.

[0015] In an aspect, the disclosure provides a method of decreasing bacterial virulence in a biological matrix, the method comprising contacting the biological matrix with a scaffold. In embodiments, the scaffold increases an infection-dependent pili detachment, suppresses the deployment of retractile pili, or both, within the biologicalmatrix. In embodiments, an increase in the infection-dependent pili detachment corresponds to a decrease in bacterial virulence.

[0016] In an aspect, the disclosure provides a method of treating or preventing bacterial infection in a subject, the method comprising administering to the subject an effective amount of a synthetic bacterial phage Mat protein scaffold comprising an apical domain polypeptide sequence. In embodiments, the scaffold consists essentially of the apical domain polypeptide sequence. In embodiments, the scaffold consists of the apical domain polypeptide sequence.

[0017] In embodiments, the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species.

[0018] In embodiments, the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2. In embodiments, the apical domain polypeptide sequence comprises 100% identity to the polypeptide sequence of SEQ ID NO:2.

[0019] In an aspect, the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding a scaffold. In embodiments, the polynucleotide sequence encoding the scaffold comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide sequence encoding the scaffold comprises 100% identity to the polynucleotide sequence of SEQ ID NO: 1.

[0020] In an aspect, the disclosure provides a nucleic acid expression vector comprising a polynucleotide sequence encoding a scaffold. In an aspect, the disclosure provides a recombinant host cell comprising the nucleic acid expression vector.DESCRIPTION OF THE DRAWINGS

[0021] The foregoing aspects and many of the attendant advantages of this disclosure will become more readily appreciated as the same become better understood byreference to the following detailed description, when taken in conjunction with the accompanying drawings.

[0022] FIGs 1A-1F show the complete structure of AP205 mature virion encompassing a Mat-dimer, according to aspects of the disclosure. (FIG. 1 A) Top and cutin views of AP205, showing Coat, inner Mat, and outer Mat in different shades of grey. The gRNA is indicated. The cage lattice (top right) indicates the location of the Mat-dimer in the capsid, which breaks the icosahedral symmetry at a two-fold axis of the Coat shell. (FIG. IB) The model of the Mat displaying three domains: apical, central, and basal. (FIG. 1C) The cartoon representation for the secondary structure of the Mat. The scissor sign indicates the truncation to generate the protein scaffold, Mat200. (FIGs ID- IF) Three subunit interfaces within the Mat-dimer. The boxes on the dimer model (lower left) indicate the zoomed-in regions for FIGs ID (viewing from left), IE (viewing from front), and IF (viewing from top), respectively.

[0023] FIGs 2A-2G show interactions between capsid proteins and the gRNA, according to aspects of the disclosure. (FIG. 2A) Electrostatic potential map of a single Mat (light grey: negatively charged; dark grey: positively charged). (FIG. 2B) Electrostatic potential map of the Mat-dimer inserted in the Coat shell (non-shaded transparent surface). (FIG. 2C) Locations of Stem-loops 79, 88, 51 (the operator, underlined), and the first 75 nucleotides of the 5’ end in the virion. (FIGs 2D, 2E) Models and secondary structures of Stem-loops 88 (FIG. 2D) and 79 (FIG. 2E), which interact with outer and inner Mat, respectively. (FIG. 2F) The model and secondary structure of Stem-loop 51, which interacts with one Coat-dimer. The start codon AUG, -5, and -18 positions are indicated and labeled. (FIG. 2G) The model and secondary structure of the first 75 nucleotides at the 5’ end of the gRNA, which interacts with three Coat-dimers.

[0024] FIGs 3A-3H show the structure of Acinetobacter T4P, according to aspects of the disclosure. (FIG. 3A) The cryo-EM density map of Acinetobacter T4P displayed at an isosurface threshold of 0.065c with diameter, helical rise and helical twist labeled (left column). The same density map displayed at a lower isosurface threshold of 0.025c, which shows the density for glycans (light shade) on the pilus (middle column). The atomic model of Acinetobacter T4P (right column). (FIG. 3B) The atomic model of one pilin. Boxes indicate the zoomed-in regions in FIGs 3D-3H. The distance between Gly78 and Ser79 is around 13 A. (FIG. 3C) The cartoon illustration for the secondary structure of the Acinetobacter Type IV pilin. (FIGs 3D, 3E) The model and map showingdisulfide bonds of Cys57 / Cys67 (FIG. 3D) and Cysl32 / Cysl45 (FIG. 3E), respectively. (FIG. 3F) The cryo-EM density around Ser79. (FIG. 3G) The cryo-EM density around Gly78. (FIG. 3H) The zoomed-in view of the glycosylation site at Serl47. The glycan density is weaker than the protein density and displayed at an isosurface threshold of 0.025G.

[0025] FIGs 4A-4E show binding of AP205 to Acinetobacter T4P and the design of Mat200, according to aspects of the disclosure. (FIG. 4A) Raw micrograph showing AP205 phage particles adsorbed to Acinetobacter T4P. The white arrowheads indicate two adjacent T4P. The scale bar denotes 300 A. (FIG. 4B) The cryo-EM structure of AP205 bound to two T4P. (FIG. 4C) The apical domain of each Mat directly interacts with three adjacent pilins (Pilin 1: upper left light grey; Pilin 2: middle right dark grey; Pilin 3: lower left light gray). The irregular shaded transparent region indicates the “footprint” of the apical domain on T4P. The dark and light shaded circles represent Ser79 and the C-terminal Serl47, respectively. The model in the bottom right comer indicates the locations of these three pilins involved in the interaction with a Mat. (FIG. 4D) The zoomed-in view of the interface between AP205 Mat and Acinetobacter T4P. (FIG. 4E) Fluorescent light microscopy showing A. gp!6 T4P specifically labeled by Mat200-sfGFP. Top row: The micrograph of Mat200-sfGFP binding to T4P of A. gp!6 cells. Middle row: The micrograph showing Coatcb5-sfGFP does not bind to A. gp!6 cells. Bottom row: The micrograph of A. gp!6 cells alone. The scale bar denotes 5 pm.

[0026] FIGs 5A-5C show cryo-EM data processing of AP205 virions and VLPs, according to aspects of the disclosure. (FIG. 5A) Cryo-EM data processing pipeline of the AP205 apo state with density map for the T=3 capsid (bottom left), for the T=4 capsid (bottom middle) and for the mature virion (bottom right). (FIG. 5B) A representative micrograph of the AP205 particles. The scale bar denotes 300 A. (FIG. 5C) Gold-Standard Fourier Shell Correlation (GSFSC) plot for cryo-EM reconstructions of the AP205 T=3 VLP, T=4 VLP, and the mature virion.

[0027] FIGs 6A-6B show a comparison of Mat models, according to aspects of the disclosure. (FIG. 6A) Comparison of Mat structures from ssRNA phage AP205, MS2 and QP. (FIG. 6B) Comparison of the inner (lower left of left portion of figure) and outer (lower right of left portion of figure) Mat of AP205, with apical, central, and basal domains labeled. Notably, the angles between the central and basal domains differ between the two Mat of each AP205 virion.

[0028] FIG. 7 shows an interaction between the Mat-dimer and the Coat shell, according to aspects of the disclosure. The central panel illustrates the Mat-dimer surrounded by ten copies of Coat-dimers, numbered from 1 to 10. Arrows indicate the viewing directions. Subpanels, at the four corners, depict the interactions between the Coat and Mat-dimer, which occurred in the corresponding boxes labeled on the model in the central panel. These interactions include: The 3’ end of the gRNA being sandwiched between the outer Mat and Coat-dimer 2 (Top left subpanel); Coat-dimer 3 presents Arg33 and Arg35 to interact with inner Mat residue Seri 03 through hydrogen bonds (top right subpanel); Local electrostatic interaction between Coat-dimer 8 residues ArglOl, Asp 104, and Glu97 and inner Mat residues Asp26 and Lysl69 (bottom left subpanel); Hydrogen bonding between inner Mat residue Cys501 and the backbone oxygen of Cys68 on Coatdimer 7 (bottom right subpanel).

[0029] FIG. 8 shows a secondary structure of the AP205 gRNA, according to aspects of the disclosure. The secondary structure of the entire 4269 nucleotides of the AP205 genome, visualized by Forna, is shown. The numbers label the 88 stem-loops in the AP205 genome.

[0030] FIGs 9A-9C show differences among the operators in AP205 (FIG. 9A), MS2 (FIG. 9B), and QP (FIG. 9C), according to aspects of the disclosure. Top row in each figure shows interactions with the Coat-dimer. Bottom row in each figures shows secondary structures of the operators. The numbers to the right of the forward slashes annotate the relative positions of corresponding residues to the start codon (AUG) of the rep gene.

[0031] FIGs 10A-10N show examples of gRNA-Coat interactions in the AP205 virion, according to aspects of the disclosure. (FIGs 10A-10L) The thirteen RNA stemloops which specifically interact with the Coat-dimers. The RNA secondary structures of these stem-loops are shown on the side. (FIG. 10M) The model and electrostatic potential surface of a Coat-dimer viewing from outside (left column) or inside (right column) of the capsid. (FIG. 10N) Two orthogonal views of the model for the first 75 nucleotides of the gRNA at the 5’ end interacting with three Coat-dimers. The Coat-dimers are shown as electrostatic potential surface.

[0032] FIGs 11A-11C show locations of the RNA stem-loops that specifically interact with the Coat shell, according to aspects of the disclosure. (FIG. 11 A) Cage model for the AP205 virion showing pentameric (gray) and hexameric (white) faces on the capsid.Each pentameric facet was labeled with Roman numerals from I-XII. Each thick solid black line represents a Coat-dimer. The Mat-dimer is located between pentameric faces III and IIX. (FIG. 1 IB) an unwrapped AP205 cage lattice into 2D with each thick solid black line represents a Coat-dimers on the capsid, while the thick dotted line represents the location of the Coat-dimer that was replaced by the Mat-dimer. The thirteen RNA stem-loops that specifically interact with Coat-dimers are circled and labeled on the 2D lattice. The Matdimer is shown as transparent to aid visualization of those stem-loops interacting with the Coat shell. Stems 61 and 62 interact with the same Coat- dimer. Stem 71 interacts with two Coat-dimers. The RNA domain formed by the first 75 nts is also labeled (5’, shaded circle) and interacts with three Coat-dimers. (FIG. 11C) The diagram of the AP205 genome shaded from the 5' end (dark grey) to the 3' end (medium gray). The core genes: lys, mat, coat(cp), and rep are denoted under the diagram. The numbers above the diagram denote the thirteen RNA stem-loops and the 5’ end domain plotted in FIG. 1 IB. The underlined stem-loop 51 is the actual RNA translational operator for the rep gene. FIGs 10A-10N show the sequence and model for each of these RNA stem-loops and domains.

[0033] FIG. 12 shows structures of AP205 T=3 and T=4 VLPs, according to aspects of the disclosure. The T=3 (left) and T=4 (right) VLP capsids are assembled by 180 and 240 copies of Coat, respectively.

[0034] FIGs 13A-13C show AP205 only binds to T4P instead of FilA pili, according to aspects of the disclosure. (FIG. 13 A) Cryo-EM shows T4P and FilA pili (No phage added). (FIG. 13B) Cryo-EM shows each AP205 can bind to one or two straight T4P. FilA pili are curved, without any phage bound. (FIG. 13C) Negative-stain EM shows phages adsorb to two T4P in situ. Here T4P are curved due to negative staining. White arrows indicate two T4P emanating from two pilus-assembly machines at the cell envelope. The scale bars denote 300 A in all micrographs.

[0035] FIGs 14A-14C show cryo-EM data processing of A. gpl6 T4P, according to aspects of the disclosure. (FIG. 14A) The cryo-EM data processing pipeline for the T4P is illustrated, with the final density map (lower right) shaded based on local resolutions. (FIG. 14B) A region of an exemplary micrograph from the T4P dataset. T4P is straight in vitreous ice. The scale bar denotes 300 A. (FIG. 14C) The resolution for the T4P reconstruction is estimated by the Gold-Standard Fourier Shell Correlation (GSFSC) plot.

[0036] FIGs 15A-15C show cryo-EM data processing of the AP205-T4P complex, according to aspects of the disclosure. (FIG. 15 A) The cryo-EM data processingpipeline for the AP205-T4P complex is depicted, with the final density maps shaded based on local resolutions (bottom row). (FIG. 14B) A representative micrograph of the AP205 adsorbed to purified T4P. The curved pili in the background are FilA pili. The scale bar denotes 300 A. (FIG. 14C) The Gold-Standard Fourier Shell Correlation (GSFSC) plot estimating resolutions for the reconstructions.

[0037] FIGs 16A-16D show AP205 Mat is adapted to the internally cleaved pilin, according to aspects of the disclosure. (FIG. 16A) The cleaved pilins are shaded for Pilins 1, 2, and 3, respectively, as in FIGs 4A-4E. The internal cleavage site at Ser79 of Pilin 3 is at the Mat-binding interface. The AlphaFold- modeled uncleaved Pilin 3 is overlaid onto the cleaved Pilin 3. The “footprint” of Mat is indicated. The right circle represents Ser79 of the cleaved pilin from the native T4P, while the left circle represents Ser79 of the uncleaved pilin modeled by AlphaFold. (FIG. 16B) Viewing the binding interface between Mat and Pilin 3. In the absence of the cleavage at Ser79, the interface would adopt a different conformation with Ser79 being 19A away from its current location, which can be incompatible for Mat binding. (FIGs 16C, 16D) The interface between Mat and pilins if they were not post-translationally cleaved (FIG. 16C), or if they are cleaved at Ser79 (FIG. 16D).

[0038] FIG. 17 shows an SDS PAGE for SUMO-Mat200-sfGFP Purification, according to aspects of the disclosure. Lane El & E2: eluted SUMO-Mat200-sfGFP using 100 mM and 300 mM Imidazole, respectively; Lane post 1 & post 2: after the sumo protease cleavage reactions, when sumo tags were cleaved, leaving Mat200-sfGFP; Lane E3: eluted SUMO protein using 500 mM Imidazole; Lane F 14- Fl 5: eluted SUMO-Mat200- sfGFP proteins from a Superdex 200 size-exclusion column.

[0039] FIGs 18A1-18B show a comparison of type IV pilins from different bacteria, according to aspects of the disclosure. (FIGs 18A1, 18A2, 18 A3) Sequence and (FIG. 18B) Structure comparison of type IV pilins from Acinetobacter baylyi, Acinetobacter baumannii (missing the first 21 residues), Acinetobacter gp!6, and Pseudomonas aeruginosa. The four pilins have relatively conserved N-terminal sequences though the C-termini are not conserved. The overall pilin structures are similar and consist of an a-helical domain and a P-sheet domain, forming a ‘lollipop’ shape. The shade in FIG. 18A follows the Jal View default scheme with each residue in the alignment assigned a shade if the amino acid profile of the alignment at that position meets minimum criteria specific for the residue type. Hydrophobic, Positive charge, Negative charge, Polar,Cysteines, Glycines, Prolines, Aromatic, and Unconserved are indicated. The bars represent conservation, alignment quality and consensus scores. The occupancy bars represent numbers of aligned positions.

[0040] FIGs 19A-19B show models of Mat-T4P complex fit into the locally refined cryo-EM density for T4P bound to the inner and outer Mat, respectively, according to aspects of the disclosure. (FIGs 19 A, 19B) Slice views showing the model fitted map of the T4P bound to inner (FIG. 19A) and outer (FIG. 19B) Mat, respectively.

[0041] FIGs 20A-20D show depictions of Mat200, including a size comparison of Mat protein and Mat200-sfGFP (FIGs 20A, 20B) and experimental results including fluorescence microscopy showing detachment (FIG. 20C) and quantification of detachment as pili detection per cell (FIG. 20D) demonstrating detachment of pili from cells in vitro, according to aspects of the disclosure.

[0042] FIGs 21A-21D show structures of the Mat and Mat200polypeptides, including a size comparison of Mat and Mat200, (FIG. 21 A), fluorescence microscopy (FIG. 2 IB) showing pili detachment from cells with Mat200(FIG. 21C), and quantification of pili detachment from cells with AP205, Mat200, and Mat200-sfGFP as compared with buffer control (FIG. 2 ID), according to aspects of the disclosure.DETAILED DESCRIPTION

[0043] Bacterial infections, and bacterial resistance to antibiotic treatments, remain a continuing problem in healthcare. Bacterial virulence, or the ability of a bacterium to thrive, reproduce, and / or cause symptoms of illness in a subject, relies at least in part on the function of pili, e.g. , Type IV pili of the bacterium. Pili can be considered as appendages that extend from the bacterium and facilitate attachment of the bacterium to other bacteria, host cells, and environmental surfaces, and facilitate the uptake of DNA by the bacterium which can increase the spread of treatment-resistant genetic alleles within a population of bacteria. The spread of treatment-resistant bacteria in an infection is serious and can be fatal.

[0044] The disclosure provides innovative anti-pili antibacterial treatments that interfere with pili function, which can be used either alone or in combination with one or more other treatments. The treatments leverage novel insights into the structures of ssRNA bacteriophages for the development of minimal protein scaffolds that can bind pili and disrupt pili function, thereby disrupting bacterial function. The disclosed approaches can,in various embodiments, weaken or sensitize a bacterial population, such that the population is susceptible to other treatments, such as antibiotic therapies.SYNTHETIC PROTEIN SCAFFOLDS

[0045] In various aspects, the disclosure provides synthetic protein scaffolds comprising polypeptide structures that are configured to bind or interact with bacterial pili and prevent or interfere with at least one pili function. In embodiments, a synthetic protein scaffold includes an apical domain polypeptide sequence of a bacterial phage maturation (Mat) protein, or a polypeptide sequence with a percent identity to the apical domain polypeptide sequence of the bacterial phage Mat protein.

[0046] A bacterial phage Mat protein, as described herein including the full- length Mat polypeptide sequence as set forth at SEQ ID NO:25, includes multiple domains with secondary and tertiary structures. In embodiments, a subset of the polypeptide sequence of SEQ ID NO:25 can be used for a synthetic protein scaffold. Such a subset can include an N-terminal portion of the polypeptide sequence of SEQ ID NO:25, such as the first 200 amino acid residues (z.e., Mat2oo).

[0047] While a specific example is disclosed of such a functional N-terminal portion of the polypeptide sequence of SEQ ID NO:25, as Mat2oo (including as set forth at SEQ ID NO:2), it should be understood that variations of the specific example can be implemented, such that the variations have the desired functionality, without departing from the scope and spirit of the disclosure. Such variations can include, but are not limited to, chemical, post-translational, or enzymatic modification of the polypeptide, use of nonstandard amino acid residues, linkage of linkers, drugs, tags, or other moieties to the polypeptide, as well as addition, subtraction, and substitution of amino acid residues. Such variations from the disclosed specific example can be tested for the desired functionality using the techniques disclosed herein.

[0048] In various embodiments, the synthetic protein scaffold does not include any or substantial polypeptide sequences of one or more other domains of the bacterial phage Mat protein; in this sense, the synthetic protein scaffold can be considered as a reduced or minimal set of polypeptides needed for pili binding and disruption. The advantages of using a shorter polypeptide sequence for the synthetic protein scaffold compared to a full-length bacterial phage Mat protein include, but are not limited to, a decreased molecular weight (MW) of the synthetic scaffold, an increased solubility of thesynthetic scaffold, an increased active / inactive ratio of polypeptides that bind pili, an increased potency per mass of the synthetic scaffold, an increased ease of synthesis and reproducibility of results with the synthetic scaffold, an increased variety of formulation options for compositions and pharmaceutical compositions that contain the synthetic scaffold, and an increased compatibility with other treatments that can be co-formulated and / or co-administered with the synthetic protein scaffold, including but not limited to antibiotic treatments.

[0049] Accordingly, in embodiments, the scaffold consists essentially of the apical domain polypeptide sequence. In such embodiments, the scaffold does not include additional polypeptide sequences that substantially alter the basic and novel anti-bacterial functions of the scaffold. In other embodiments, the scaffold consists of the apical domain polypeptide sequence. In such embodiments, the scaffold does not include any additional polypeptide sequences, other than the polypeptide sequence of the scaffold.

[0050] While specific polypeptide sequences are disclosed for a Mat protein of a bacteria, e.g., an Acinetobacter species, variations and deviations from the explicitly disclosed sequences can be implemented, in embodiments, without departing from the scope and spirit of the disclosure. In embodiments, such deviations can be expressed as polypeptide sequences having less than 100% identity to a reference sequence. For example, in embodiments, an apical domain polypeptide sequence of a synthetic scaffold has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species. In embodiments, the apical domain polypeptide sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2. In embodiments, the apical domain polypeptide sequence has 100% identity to the polypeptide sequence of SEQ ID NO:2.

[0051] In various aspects, the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding a scaffold. In embodiments, the polynucleotide sequence encoding the scaffold comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleotide sequence of SEQ ID NO: 1. In embodiments, the polynucleotide sequence encoding the scaffold comprises100% identity to the polynucleotide sequence of SEQ ID NO: 1. In various aspects, the disclosure provides a nucleic acid expression vector comprising a polynucleotide sequence encoding a scaffold. In various aspects, the disclosure provides a recombinant host cell comprising the nucleic acid expression vector.

[0052] As would be understood by the person having ordinary skill in the art, the phrase “at least” when used with reference to a specific percent identity includes the specified percent identity and percent identities that are greater than the specified percent identity, including decimals between the specified percent identity and a next specified percent identity.

[0053] Furthermore, while percent identity is one way of expressing such deviations from expressly disclosed sequences, in embodiments, such deviations can be expressed as polypeptide sequences having one or more alternate amino acid residues, one or more non-standard amino acid residues, one or more deleted amino acid residues, one or more added amino acid residues, one or more substituted amino acid residues, or any combination thereof. In embodiments, the scaffold further comprises an additional polypeptide sequence, such as a tag polypeptide sequence that enables a fluorescence detection of the scaffold, an interaction of a tag binder with the tag polypeptide sequence, or both. The tag polypeptide sequence can add a functionality to the scaffold that is in addition to its functionality of binding bacterial pili.COMPOSITIONS AND KITS

[0054] In various aspects, the disclosure provides compositions that include a synthetic scaffold. Compositions can comprise additional agents, including but not necessarily limited to a carrier (e.g., aqueous, non-aqueous), a diluent (e.g, water), a salt (e.g, ionic strength; NaCl, MgCh, CaCh, etc.), a pH buffering agent (e.g., Tris, HEPES, etc.), a detergent (e.g., Triton X-100, Tween-20, etc.), a lipid (e.g., fat, oil, triglyceride, phospholipid, wax, etc.), a surfactant (e.g., anionic surfactant, non-ionic surfactant, etc.), or any combination thereof. Compositions can be formulated for use of the synthetic scaffold as a laboratory or experimental reagent, e.g., for in vitro or in vivo research. A composition can be provided as part of a kit that also includes an instructional material configured to instruct an individual in a use of the composition in a method of decreasing bacterial virulence in a biological matrix, for example. The instructional material can be inprint and / or an electronic media format, such as a website, web article, video playback, audio playback, or the like.

[0055] In various aspects, the disclosure provides a pharmaceutical composition comprising a scaffold and a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition is formulated for a safe or clinically tolerable administration to a subject, such as a human or animal subject, for example, as part of a clinical trial or therapeutic treatment. The pharmaceutical composition can be suitably formulated and administered by any suitable route of administration, including but not necessarily limited to topical, dermal, oral, buccal, inhalation, ocular, nasal, aricular, rectal, ureteral, intramuscular (IM) injection, intraperitoneal (IP) injection, intravenous (IV) injection, infusion, blood or tissue transplantation, and the like. The pharmaceutical composition can be administered to a subject according to any established or experimental treatment regimen, for example, once daily, twice daily, three or more times daily, once weekly, twice weekly, three or more times weekly, or the like. The pharmaceutical composition can be administered to the subject as a standalone treatment or can be administered to the subject in combination with one or more established or experimental antibiotic treatments. The synthetic protein scaffold can be formulated as the only biologically active agent in the pharmaceutical composition, or can be formulated with one or more additional biologically active agents in the pharmaceutical composition, for example, as a combination therapy product. A pharmaceutical composition can be provided as part of a kit that includes an instructional material configured to instruct an individual in a use of the pharmaceutical composition in a method of treating or preventing bacterial infection in a subject.METHODS

[0056] In various aspects, the disclosure provides methods of decreasing bacterial virulence in a biological matrix. Methods include contacting the biological matrix with a scaffold of the disclosure, optionally formulated as a composition or a pharmaceutical composition, in various embodiments. Upon contact of the scaffold with bacteria or pili of the biological matrix, the scaffold increases an infection-dependent pili detachment, suppresses the deployment of retractile pili, or both, within the biological matrix. In embodiments, an increase in the infection-dependent pili detachment corresponds to a decrease in bacterial virulence. For example, the method can comprise administering the scaffold one or more times according to one or more dosages and / or treatment regimens,and monitoring bacterial virulence in the biological matrix as time passes with the treatment. In embodiments, an assay can measure pili attachment with the scaffold, DNA uptake by bacteria, prevalence of antibiotic-resistant alleles within a population of bacteria, signs or symptoms of illness in a subject, or the like.

[0057] Accordingly, in another aspect, the disclosure provides a method of treating or preventing bacterial infection in a subject. The method comprises administering to the subject an effective amount of a synthetic bacterial phage Mat protein scaffold comprising an apical domain polypeptide sequence, as disclosed herein either explicitly or implicitly, or by way of equivalents. Treatment methods can comprise administering to the subject an additional treatment, such as a treatment for management of fever, an antibiotic treatment, or the like. Since treatment with the scaffold can debilitate the bacteria, this can be combined with an established or experimental antibiotic treatment, according to embodiments.

[0058] In other aspects, the disclosure provides various industrial uses of synthetic scaffolds of the disclosure in the preparation of medicaments for the treatment or prevention of bacterial infections in subjects by administering the medicaments to the subjects.TERMINOLOGY

[0059] In the claims and for purposes of the present disclosure, the terms “a”, “an”, “the”, and the like, refer to the singular and the plural forms of the object or element referenced.

[0060] As used herein in the description and claims, the term “comprising”, is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The term “consisting of,” as used in a claim, excludes any element, step, or ingredient not specified in the claim. The term “consisting essentially of,” as used in a claim, limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

[0061] For the purposes of the present disclosure, the phrase “at least one of A, B, and C,” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C), including all further possible permutations when greater than three elements are listed. Likewise, as used herein, the term “A, B, and / or C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C), including all further possible permutations when greater than threeelements are listed. Unless otherwise stated, the term “or” is an inclusive “or”, and the phrase “A or B” means (A), (B), or (A and B). Unless otherwise stated, the term “and” requires both elements; for example, the phrase “A and B” means (A and B).

[0062] Unless stated otherwise, or recognized by a person having ordinary skill in the art as being otherwise for practicing the methods, the methods of the disclosure can be performed, in whole or in part, in any order and in any number of steps. Methods can be performed by individuals without substantial assistance from automated or semi-automated approaches, or can be performed by individuals with substantial assistance from automated or semi -automated approaches which can include but are not limited to computational devices and systems, including but not limited to computers, smart devices, remote servers, automated or semi -automated robotics systems, and the like. In at least some instances, methods can be performed mostly or completely with these automated or semi-automated approaches or their equivalents.

[0063] As used herein, the term “scaffold” refers to a peptide, polypeptide, or protein that includes a polypeptide sequence having a secondary or tertiary structure that can bind to and interfere with a bacterial pili function. As used herein, the term includes, but is not necessarily limited to, protein scaffolds that include a bacterial phage maturation (Mat) protein scaffold comprising an apical domain polypeptide sequence.

[0064] As used herein, the term “synthetic” refers to a molecule or substance produced by a human or human-facilitated process, such as a synthetic protein scaffold produced by recombinant DNA technology, and the like.

[0065] As used herein, the term “percent identity” refers to a percentage of positional bases (e.g., nucleotide bases) or residues (e.g., amino acid residues) of two or more optimally aligned sequences that are identical between the two or more optimally aligned sequences. Percent identity can be computed by aligning the sequences, counting the number of bases or residues that are positionally identical between the sequences as / / , dividing n by the total number of bases or residues T (i.e.,and multiplying the result by 100%. For example, the polypeptide sequence YDWPE has 80% identity to the polypeptide sequence YKWPE.

[0066] As used herein, the term “tag polypeptide sequence” refers to a polypeptide sequence that can serve as a label for detection of a polypeptide containing the tag polypeptide sequence with a detection assay, a biologically active moiety that imparts a biological function to a polypeptide containing the tag polypeptide sequence, a label forenrichment or purification of a polypeptide containing the tag polypeptide sequence, or the like. Non-limiting examples of a tag polypeptide sequence include a polyhistidine tag, a green fluorescent protein (GFP) tag, a SUMO tag, and the like.TABLES

[0067] Table 1. Cry o-EM imaging parameters.

[0068] Table 2. Cryo-EM data processing statistics.* 12,703 micrographs from the dataset of the AP205-T4P complex were added to the apo AP205 dataset to better resolve the gRNA,** These 244,426 particles were selected by Relion from the 12,703 micrographs of the AP205- T4P dataset.# 65,745 particles were further selected from the 244,426 particles.EXAMPLES

[0070] Example 1. Single-stranded RNA phage-derived protein to target and remove bacterial virulence pili

[0071] Introduction

[0072] Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of Acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target bacteria via retractile pili, including type IV. This example delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, structures of the AP205 virion are solved with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing an unprecedented post-translational pilin cleavage, and the pili-adsorbed AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ is developed, with practical applications in research, diagnostics, and preventative and treatment therapies.

[0073] The present disclosure investigates how the ssRNA phage, AP205, infects Acinetobacters via T4P. However, there is limited structural information. Previously, only the structure of AP205 Coat had been solved, the structure revealing a circular permutation compared to the Coat of MS2 and Qp. The complete structure of AP205, including the packaged genome, remains unknown. As for the receptor pili, although the recombinant globular domain of A. baumannii type IV pilin has been determined using X-ray crystallography, it may not accurately represent potential post-translational modifications of the native Acinetobacter T4P. Additionally, the molecular mechanism by which AP205 interacts with T4P is not yet understood.

[0074] In this example, cryo-EM structures of AP205 alone, Acinetobacter T4P alone, and AP205 in complex with T4P are determined, and these enabled the design of a small protein scaffold to effectively target and label T4P in live cells, at least a portion of which can be administered for inhibition of T4P binding in methods of treatment and prevention of bacterial infection.ResultsThe complete structure of the AP205 virion

[0075] About 8% of the vitrified AP205 particles show a well-defined three- dimensional (3D) conformation of the genomic RNA (gRNA) inside the capsid. The smaller proportion of particles exhibiting a defined RNA organization, in contrast to MS2 (>90%) and QP (-30%), may be due to the more heterogeneous RNA conformations inside the capsid. Nonetheless, a 3.1A-resolution asymmetric reconstruction of the mature AP205 virion was achieved (FIG. 5), enabling to construct a complete atomic model of both the capsid protein shell and the 4269-nt gRNA (FIG. 1A).

[0076] In the structure, 178 copies of the Coat self-assembled into a near-icosahedral shell (with the triangulation number, 7=3), measuring approximately 290A in diameter. Surprisingly in contrast to MS2 and QP, which have a single Mat per virion, AP205 possesses a distinctive Mat-dimer, resembling the shape of the letter “F” at a pseudo twofold axis of the capsid and increasing the length of the virion for an additional 70A. The 3’ region of the gRNA is proximal to the Mat-dimer on one side of the capsid, while the 5’ region resides on the opposite side. This is consistent with the previously reported structures of ssRNA coliphages. Notably, one RNA stem-loop originating from the 3’ end of the AP205 gRNA, which interacts with the Mat-dimer, is partially exposed outside the capsidshell (FIG. 1A bottom). This observation can suggest that this specific RNA fragment is the initial region to exit the capsid and enter the cell.

[0077] Compared to other ssRNA phages like MS2 and QP, Mat of AP205 is significantly larger (FIG. 6A), including 534 amino acids (393 aa in MS2 and 421 aa in QP). It can be divided into three domains, namely the apical, central, and basal domains (FIGs IB and C). The apical domain includes two small P-sheets, SI (involving strands P2, P3, P9, piO and pi 1) and S2 (involving strands pi, P4, P7, P8 and P 12) connected by an a-helix (al), while the central domain features a large P-sheet S3 (involving strands P5, P6, P 13, pi4, P 15, P 16, pi7 and P 18) and two short a-helices (a2 and a3). The basal domain comprises four a-helices (a4, a5, a6, and a7). The basal domains of the Mat-dimer insert into the capsid, while the apical domains are exposed on the surface. The two copies of Mat exhibit different conformations: One Mat (referred as outer Mat) extends further to elevate its apical domain away from the capsid shell while the other Mat (referred as inner Mat) is embedded deeper within the capsid and has a larger bending angle between the central and basal domains (FIG. 6B). These two Mat proteins intertwine to form hydrogen bonds and salt bridges at three sites. The first interaction occurs between the a4 helix from the outer Mat and the loop connecting P4 and al from the inner Mat, involving a hydrogen bond between Gln220 (outer Mat) and Glu67 (inner Mat) (FIG. ID). The second interaction involves a6 / a7 from the outer Mat and P 16 / p 17 from the inner Mat. It includes a hydrogen bond between Ser531 (outer Mat) and Arg402 (inner Mat), as well as two hydrogen bonds of Asn524 (outer Mat) and Ly s471 (inner Mat) with the protein backbones of the opposite Mat (FIG. IE). The third interaction is within a six-helix bundle between the two basal domains, connected by a salt bridge between Asp321 (a5 of the outer Mat) and Arg347 (a5 of the inner Mat) (FIG. IF). These interactions hold the two copies of AP205 Mat together, forming a dimer that nearly triples the volume and doubles the surface area compared to a single Mat in ssRNA coliphages.Interactions between the gRNA and capsid proteins

[0078] Each Mat monomer presents large negatively charged protein surface patches around the top half of the basal domain (FIG. 2A). These patches are formed by acidic amino acid sidechains present on Helices a6, a7, and the upper portion of Helices a4 and a5, which connect to the central domain. However, due to the negatively charged nature of the phage gRNA phosphate backbone, such an acidic surface is not favored to interact with the RNA, particularly for the inner Mat that penetrates deeper into the capsid. Thedimerization of the two Mat proteins resolves this issue by shielding the negatively charged surface of the inner Mat with the lower part of the basal domain from the outer Mat. This results in a positively charged Mat-dimer surface in the capsid-embedded regions of the basal domains (FIG. 2B). Concurrently, the corresponding negatively charged region in the outer Mat is protected by the coat protein shell (FIG. 2B). This arrangement creates a more energetically favorable Mat-dimer configuration that is compatible with virion assembly.

[0079] The AP205 gRNA is highly branched, presenting a total of 88 stem-loops (FIG. 8), two of which provide specific interactions with the Mat-dimer (FIG. 2C) via multiple 7t-stackings between aromatic amino acid sidechains and RNA bases. The first site for the 7t-stacking takes place between the central domain of the outer Mat and the last RNA helix, Stem- loop 88 of the gRNA. Here, the sidechains of Phel07 from a2 and Tyrl21 from the following loop stack with the bases G4249, C4250 and U4251 (FIG. 2D). Another 7t-stacking site occurs in the inner Mat at the corresponding location, between the central domain and Stem-loop 79. In this site, the corresponding Phel07 and Tyrl21 of the inner Mat stack with G3729, C3732 and U3733 (FIG. 2E). A comparison between Stemloops 88 and 79 reveals a remarkable similarity in RNA motifs, specifically at the tip of the RNA stems. Stem-loop 88 has the sequence “4249-GCUUAGC-4255”, while Stem-loop 79 features“3729-Gf7f7CUUUGC-3737”. Notably, the two extra uridines (italicized) in the above sequence of Stem-loop 79 are flipped out, enabling two base pairings between C and G, similar to the corresponding site in Stem-loop 88 (FIG. 2D right and FIG. 2E right). The G and U (bold front) in the above sequence are the two bases where aromatic sidechains Phel07 and Tyrl21 clamp onto. Without wishing to be bound by any particular theory, this can suggest the presence of a conserved “GCUUXGC” motif that has undergone evolutionary optimization to facilitate a strong interaction with each of the two Mats.

[0080] In ssRNA phages, the gRNA engages with the inner surface of the Coat shell through stem-loop structures. These Coat-RNA interactions are exemplified by the binding of a Coat-dimer to the RNA operator for the rep gene, where the base of adenosines, around the tip of the stem-loop, flips out to interact with the Coat-dimer. During later stages of ssRNA phage infection, Coat-dimers bind to the RNA operator to suppress the expression of the replicase protein, controlling the production of their progeny within the host cell, particularly when an excessive number of Coat proteins have been synthesized. The RNA operator stem-loop for AP205 (Stem-loop 51) stands out as notably longercompared to other known ssRNA phages (FIG. 9), resulting in a distinct interaction with the Coat-dimer. The structural analysis revealed two adenosines at positions -5 and -18, relative to the start codon of the rep gene, flip out to interact with a Coat-dimer, sequestering the start codon of rep from the translation machinery (FIG. 2F).

[0081] Among the 88 stem-loop structures found in the AP205 gRNA, 13 of them were clearly discernible in the cryo-EM map, indicating their strong affinity for the capsid shell (FIGs 10A-10L). These stem-loops, many of which exhibit flipped-out adenosine residues, potentially function as “packaging signals” that aid in the folding and encapsulation of the gRNA. Notably, in AP205, numerous interactions between gRNA and Coat deviate from the typical packaging signal forms. In some cases, there are no flipped adenosines to interact with the Coat. Particularly, the 5’ end of the gRNA (Residues 1-75) folds into a distinct three-way junction domain, wherein the last few nucleotides in this domain form a pseudoknot with the first RNA stem-loop (FIG. 2G). This RNA domain engages in interactions with three adjacent Coat-dimers and exhibits well-defined density, indicating a stable association with these Coat-dimers. However, no flipped adenosines are observed in this RNA structure. It is the unique shape of this RNA domain that allows it to fit precisely into the positively charged interface formed by the three Coat-dimers (FIGs 10M, ION) Given its location at the beginning of a newly synthesized gRNA, this RNA domain may recruit the initial Coat-dimers to seed the assembly of the capsid shell. Notably, the 5’ half of the gRNA has very few RNA stems that specifically interact with the capsid (FIG. 11). This may contribute to more heterogeneous gRNA conformations when being packaged, leading to a much lower percentage of the AP205 particles showing defined gRNA density as compared to other ssRNA phages such as MS2 and Qp. It is worth noting that approximately 1% of the particles from the dataset produce a reconstruction that have a larger diameter of 310A and exhibit a T=4 icosahedral symmetry (FIGs 5A-5C and FIG. 12), increasing the capsid volume by -20%.Structure of the Acinetobacter T4P

[0082] The host for AP205 is the Acinetobacter species genotype 16 (A. gp!6 which is homologous to A. baumannii. with a genome sequence similarity of 87% over 73% coverage of the A. baumannii TP1 chromosome. Electron microscopy shows that these Acinetobacter cells have two types of morphologies for the cell surface appendages: straight and curved (FIG. 13A). Density gradient-based purification is ineffective at separating these appendages, but mass-spectrometry analysis indicates that they are T4Pand a pilus of unknown functions (annotated as FilA), respectively. Cryo-EM images reveal that AP205 particles bind to the straight T4P, but not to the curved FilA filament (FIG. 13B). Using single-particle cryo-EM and helical reconstruction, a high-resolution structure of the purified straight T4P was obtained at 2.5A resolution (FIG. 3A, FIGs 14A-14C) and the mature pilin modeled into the cryo-EM density (FIG. 3B). The sequence of the signal peptide, including the first seven amino acids, is exclusively found in the prepilin and absent in the mature pilin.

[0083] The mature T4P pilin subunits have a distinct “lollipop” shape, presenting a globular C- terminal P-sheet domain and two N-terminal a-helices (FIGs 3B and 3C). They assemble into a 60A-wide filament, with a helical rise of 10.0 A and a twist angle of 91.9°. The P-sheet domain is exposed on the outside, while the a-helices are buried within the center of the filament.

[0084] In the related species A. baumannii. T4P pilins undergo glycosylation mediated by a tfpO- like O-oligosaccharyltransferase. Within the helical reconstruction of A. gp!6 T4P, additional density corresponding to potential glycans was observed (FIGs 3A and 3H). These glycans are added post-translationally to the last amino acid Serl47 of the mature pilin. Glycosylation of pili assists Acinetobacter cells in evading host immunity and certain dsDNA bacteriophages. Nevertheless, ssRNA phage AP205 retains the ability to bind to glycosylated T4P (FIGs 13B and 13C). The cryo-EM density of the glycan is weaker than the protein density. This might be attributed to the flexible attachment of the glycan to Serl47 and substoichiometric glycosylation of pilins in the T4P, leading to weakened glycan density upon applying the helical symmetry during cryo-EM map reconstructions.

[0085] Each pilin in the T4P has two disulfide bonds, involving cysteine / cysteine pairs (Cys57 / Cys67 shown in FIG. 3D and Cysl32 / Cysl45 shown in FIG. 3E). These disulfide bonds likely form when pilin subunits traverse through the oxidative periplasmic environment from the inner membrane pilin pool during pilus assembly. Surprisingly, a 13- A gap in the density of the polypeptide chain is observed between Gly78 and Ser79 (FIGs 3B, 3C, 3F, and 3G). This indicates that, in addition to the post-translational cleavage at the N-terminus to remove the signal peptide involving the first seven amino acids in the prepilin, there is another cleavage in the middle of the peptide chain, a phenomenon not reported for other pilins. While the precise mechanism behind such pilin cleavage remains undiscovered, the fact that it takes place on the external surface of T4P suggests a directinfluence on its interactions with the environment, such as its engagements with the immune system or phages.Structure of the AP205-T4P complex

[0086] To investigate the recognition mechanism between AP205 and T4P, singleparticle cryo-EM was employed to obtain a structure of the AP205 / T4P complex (FIGs 4A and 4B, FIGs 15A-15C). Both Mats within a virion can attach to a T4P individually through their apical domains, with a higher likelihood of binding observed for the outer Mat (FIG. 15A).

[0087] Within the T4P-Mat complex, the apical domain of each Mat specifically plugs into the groove formed by three adjacent pilin subunits within the T4P (referred to as Pilins 1, 2, and 3 in FIG. 4C). Notably, the C-terminal Serl47 of Pilins 1 and 2 are positioned near P3 and P7 of the Mat apical domain, respectively (FIG. 4D). Apparently, glycosylation at these two serines can prevent binding of AP205 Mat. It is likely that the substoichiometric glycosylation of pilins allows AP205 to bind to unglycosylated sites on the T4P. The smaller size of the apical domain further facilitates its fit into binding sites without potential hindrance from neighboring glycans on the surface of the pilus. It was also observed that the internal cleavage site of Pilin 3 at Ser79 is located at the binding interface for the apical domain. This cleavage allows Ser79 to move ~13A away from Gly78, allowing its interaction with P8 of the Mat. The Mat of AP205 has evolved to adapt to such a post-translational cleavage at the phage-pilus binding interface and may not be compatible with an uncleaved pilin anymore (FIGs 16A-16D).An AP205-derived protein scaffold targeting T4P

[0088] The structures suggest that the Mat apical domain could be a protein scaffold that binds specifically to native A. gpl6 T4P. To create this scaffold, the N- terminal fragment of the Mat was cloned, encompassing the entire apical domain as well as P5, P6, and a2 from the central domain (FIG. 1C). This resulted in a continuous 200 amino acid sequence from the N-terminus, designated as Mat20Q. Mat200was then recombinantly expressed in E. coli and purified to homogeneity (FIG. 17). To assess the pilus binding ability of Mat200, a super-fold green fluorescent protein was fused to the C-terminus of Mat200(Mat200-sfGFP). Using fluorescence microscopy, the binding of Mat200-sfGFP to T4P was visualized in live Acinetobacter cells (FIG. 4E). This labeling process occurred rapidly, taking less than 5 minutes, and displayed highly specific binding. As a control,Coatcb5-sfGFP, the coat protein from another ssRNA phage PhiCb5 fused to sfGFP, cannot bind to the same T4P.Discussion

[0089] Given that various bacterial T4P share a similar overall sequence and structure (FIGs 18A1-18B), the Mat200scaffold can be engineered to bind to other T4P from other bacteria. Taking advantage of the fast mutation rate of ssRNA phages, one can use an evolution-and- selection approach to direct the Mat200scaffold to target other T4P. This can be a promising diagnostic and therapeutic strategy for bacterial infections.

[0090] The presence of two Mat proteins within a single AP205 virion presents intriguing possibilities. An additional Mat could provide an expanded binding surface for the phage to attach to the receptor pili. Considering that Acinetobacter T4P exhibits substoichiometric glycosylation, the presence of an extra Mat protein could increase the chances of the phage locating an unglycosylated region on the pilus to bind. Furthermore, the gRNA is bound to each Mat via a conserved motif of “GCUUXGC” in Stem-loops 88 and 79 by forming similar 7t-stackings between RNA bases and aromatic sidechains of Mat. This suggests the gRNA has a similarly strong affinity for both Mats, with either of the two Mats capable of escorting the gRNA into the cell. Additionally, the binding of Mat- dimer to Stem-loops 88 and 79, which are approximately 500-nt apart in sequence, may facilitate the nucleation of the RNA, aiding the packaging process.

[0091] The retraction of the receptor pili is believed to guide the Mat-bound gRNA through the channel within the pilus-assembly machine for cellular entry. In the natural environment, during the interaction of AP205 with Acinetobacter, it is plausible that multiple T4P from the same cell come into close proximity. This allows the pair of Mat proteins from a single phage to simultaneously bind to two pili (FIG. 13C), as shown in the major class of the single-particle cryo-EM structure of the in-vitro reconstituted AP205- pili complex. The mechanism by which the gRNA enters the cell in such circumstances remains elusive. Since each phage-bound pilus originates from its respective pilusassembly machine at the cell envelope and will potentially compete for the bound phage particle. It remains to be determined under such circumstances, whether the gRNA enters through the channel of the pilus bound to the outer Mat or the channel of the pilus bound to the inner Mat. Further experiments are necessary to address this intriguing question.Materials and MethodsSample purifications and cryo-EM grid preparations

[0092] AP205 purification. 5 mLA gpl6 overnight cell culture was incubated and shaken at 30 °C and 200 rpm. The 5 mL overnight culture was then inoculated into one IL culture, grown at 30 °C and shaken at 200 rpm until the optical density (OD) ~ 0.1. 30 mL AP205 lysate (generated from previous purifications with a titer ~106) and 0.5 mM CaCh were added to the IL culture, which was incubated for 30 min at room temperature. The IL culture was then shaken and incubated at 30 °C and 200 rpm overnight, and spun down for 30 min at 4,000 rpm (4 °C). The cell pellet was resuspended in a buffer (20 mM Tris- HC1, 150 mM NaCl, pH 8.0). The resuspended cells were lysed by passing through a microfluidizer at 25,000 psi. The total lysate was spun down at 18,000 rpm for 30 min at 4°C and the supernatant was collected and concentrated to 1 mL. The 1 mL solution was added to a CsCl gradient solution (step gradient: 1.0, 1.2, 1.4, and 1.6 g / mL) and spun for 24 hr at 200,000 g using ultracentrifugation. The separated bands in the CsCl solution were drawn using syringes and dialyzed against a dialysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) to remove CsCl residues for 2 consecutive 24 hr at 4 °C. The fraction containing AP205 was confirmed by tittering against A. gpl6 cells, and the purity was checked by SDS-PAGE gel and negative-stain electron microscopy.

[0093] A. gpl6 T4P purification. 30 mL A. gpl6 overnight cell culture was incubated and shaken at 30 °C and 200 rpm. 100 pL of the overnight culture was added to each agar plate of the 100 plates made, spread using a cell spreader, and incubated at 30 °C overnight. The 100 overnight plates were then placed on ice and cells on plates were collected using a purification buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0) and went through needles (diameter: 18G + 25G) 3 times to shear off the pili. The cell solution was spun down at 4,000 rpm, 4 °C for 30 min to remove any remaining cells and the clear supernatant was collected and concentrated to 1 mL for a CsCl-gradient-based purification. The further purification steps were adopted from the AP205 purification protocol mentioned above, but the CsCl step gradient used was 1.0, 1.2, and 1.4 mg / mL. The presence and purity of AP205 pili were confirmed by negative-stain electron microscopy.

[0094] Cryo-EM grid preparations. 3 pL of each purified sample, apo AP205, apo T4P and AP205-T4P (by mixing AP205 and T4P at 1 : 1 volume ratio at 30° for 30 mins), was applied to a 300-mesh 2 / 1 c-flat copper grid and vitrified with a Vitrobot® Mark III (FEI Company) or a Vitrobot® Mark IV (ThermoFisher®) in vitreous ice.

[0095] Single-particle cryo-EM. The numbers and parameters of data collections and processing for apo AP205, apo T4P and AP205-T4P complex are summarized in Tables 1 and 2, respectively. The details are described below.

[0096] Data collection for the apo AP205. The samples of apo AP205 were imaged under a Titan® Krios® cryo-electron microscope (ThermoFisher®) at SLAC National Accelerator Laboratory operated at 300 kV. 41,648 movie stacks were collected using EPU with a Gatan K2® Summit direct detection camera (Gatan) at a nominal magnification of 130,000x in the counting mode, yielding a pixel size of 1.06 A. A total electron dose of 50 e' / A2was fractionated over 35 frames (0.2 s / frame, 7 s in total). A postcolumn energy filter was applied with a slit width of 30 eV.

[0097] Data processing of the AP205 virion, T=3, and T=4 VLPs. The movie stacks were first aligned and filtered by MotionCor2. Particles with good quality were semi- automatically picked by EMAN2 or automatically picked using Gautomatch. Gctf was used to estimate the contrast transfer function for each particle. After 2D classification using RELION, 1,745,007 particles were selected for 3D Classification, which yielded 86,946 good particles with a defined gRNA conformation. To better resolve the gRNA, 65,745 particles from the dataset of the AP205-T4P complex (see FIG. 5 and Table 2 for more details) were also combined in the final refinement by cryoSPARC to yield a reconstruction at a resolution of 3.1 A. Only 152,691 (~8%) particles of AP205, corresponding to the mature virions, showed a genome with a defined tertiary structure.

[0098] Most of the particles showed an icosahedral capsid without a defined tertiary structure of the gRNA in the 3D classification result. Icosahedral refinement was then applied to these particles, yielding a T=3 icosahedral structure of the capsid at 3.2-A resolution, which presented features consistent with previously published structures of AP205 VLPs. Around 1% of the particles had larger capsids (with a diameter of 310 A). 7,716 of these larger particles were refined with icosahedral symmetry and yielded a T=4 icosahedral capsid at 3.4 A resolution.

[0099] Data collection of A. gpl6 T4P. The samples of T4P were imaged under a Titan® Krios® G4 cryo-electron microscope (ThermoFisher®) at the Texas A&M University operated at 300 kV. 5,946 movie stacks were collected using EPU with a Biocontinuum image-filtered Gatan K3® direct detection camera (Gatan) at a nominal magnification of 105,000x in the super-resolution mode, yielding a pixel size of 0.86 Aafter binning by 2. A total electron dose of 50 e' / 2was fractionated over 40 frames (0.2 s / frame, 8 s in total). A post-column energy filter was applied with a slit width of 15 eV.

[0100] Data processing of A. gpl6 T4P. Initial 2D references were generated from 1,472 manually picked particles and these were used for the autopicking of 905,414 segments using CryoSPARC. From these segments, 2D class-averages with high- resolution details were identified, and the segments contributed to these averages were used for a helical reconstruction. Possible helical symmetries were estimated based on an averaged power spectrum generated from the raw particles and the outcome volumes were examined by trial and error. The data processing procedure is shown in FIGs 14A-14C.

[0101] Data collection of the AP205-T4P complex. Data were collected on the same cryo- electron microscope with the same set of parameters as apo AP205 except at different tilting angles to alleviate preferred orientations of the AP205-T4P complex on the grid. In total, 12,703 movie stacks were obtained, with 3,037, 1,780, 7,157, and 729 stacks collected at 0°, 15°, 30°, and 37° tilting, respectively.

[0102] Data processing of the AP205-T4P complex. These movie stacks were imported and processed using cryoSPARC. A total of 225,247 AP205 particles, regardless of T4P bound or not, were picked and subjected to multiple rounds of 2D classifications and heterogeneous refinements, yielding 94,095 particles classified as AP205-T4P complexes, which were subjected to one more round of 3D classification to generate three classes. Among these three classes, two classes showing two bound pili for each AP205 (~61K particles) were selected and used for a homogeneous refinement of the AP205- Two- T4P complex at 3.6-A resolution. Particles in the remaining class were subjected to an additional round of 3D classification into two classes. One of the two classes showing only one pilus bound to the outer Mat of AP205 (~17K particles) was reconstructed at a resolution of 4.2A. The other class shows very weak pilus density and was discarded for further processing.

[0103] In these density maps of the AP205-T4P complexes, the T4P was poorly resolved, likely due to the flexibility of the pilus-bound apical domain of the Mat relative to the rest of the phage, leading to a spread of orientations for each T4P in the image particle. This caused inaccurate image alignments in cryo-EM data processing. To improve the density map, especially around the T4P region, the ~94K particles were hierarchically classified. A generous mask was first applied around the regions of both outer and inner Mat, and the potentially associated T4P to locally classify all the particles into six classes.Another two tighter masks were then created: one for the region covering only the outer Mat and its associated T4P while the other for the region covering only the inner Mat and its associated T4P. The particles were locally refined within each of the six classes from the previous round of 3D classification using each of the two tighter masks and obtained twelve reconstructions: six for the outer Mat-T4P complex and six for the inner Mat-T4P complex. The best reconstructions for the inner Mat-T4P and outer Mat-T4P complexes (out of 12 reconstructions) reached overall resolutions of 8.2 A and 8.6 A, respectively, but showed much more complete density for the T4P and clearer secondary structures of the pilin subunits. The density for the pilus that bound to the inner Mat had a better resolution compared to the one that bound to the outer Mat, as the apical domain of the inner Mat was stabilized by the basal domain of outer Mat (Helix a4, FIG. ID) and the outer surface of the Coat shell (FIG. 7 bottom left panel). Combining particles in the six classes for either the inner or the outer Mat-T4P complexes did not improve the resolutions of the final refinements, which was probably due to the conformational heterogeneity in the long filamentous pili as for the MS2-bound F- pili. The data processing procedure is described in FIGs 15A-15CMolecular Modeling and Visualization

[0104] AP205 Mat modeling. The initial model of Mat was generated by I- TASSER and AlphaFold. The inner and outer Mat models were then both manually fixed using Coot and the ISOLDE plugin in ChimeraX. The final models were iteratively refined by RosettaCM and Phenix inside the cryo-EM density maps.

[0105] AP205 gRNA modeling. The RNA density was traced and segmented into fragments by UCSF Chimera. The model for each fragment was generated and then connected using DRAFFTER. The complete gRNA was then refined in the density using MDFF.

[0106] AP205 Coat shell modeling (including T=3 and T=4 VLPs). AP205 T=3 VLP structure (PDB ID: 5LQP) was used to generate the Coat shell of AP205 mature virion. One Coat- dimer was removed to accommodate the Mat-dimer. The same strategy was used to generate the initial models for the capsids of AP205 T=3 and T=4 VLPs. These AP205 capsids, virions and VLPs, were then fitted into the cryo-EM density maps using Chimera.

[0107] AP205 virion modeling. The models of AP205 Mat, Coat shell, and gRNA were combined in Chimera, iteratively refined using Phenix, and manually inspected. Modifications were made using Coot and ISOLDE.

[0108] A. gpl6 T4P modeling. A. gpl6 was whole-genome sequenced and the sequence of the type IV pilin was extracted (FIGs 18A, 18B). The first seven residues, corresponding to the signal peptide in the prepilin were removed. The remaining sequence was then submitted to AlphaFold to generate an initial model for the mature pilin, which was manually fitted into the density using Coot. The density of the first amino acid of the mature pilin (Phe8) is not complete in the cryo-EM map. Therefore, the model of the mature T4P pilin is from Thr9 to Serl47. In addition, the internal cleavage of the polypeptide was introduced between Gly78 and Ser79. This model was adjusted in ISOLDE and refined by Phenix iteratively.

[0109] The model of the T4P filament was then generated by applying the helical symmetry on the pilin subunit.

[0110] Mat-T4P complex modeling. Within the local map of the T4P -bound inner Mat, the high-resolution structures of the T4P and AP205 inner Mat from the virion were docked, and the model of the complex adjusted and refined using Coot, ISOLDE, and Phenix iteratively to model the details of the interaction between the Mat apical domain and the T4P (FIG. 19A). Such a model of the Mat-T4P complex is consistent with the locally refined density map of the T4P -bound outer Mat (FIG. 19B), even though at a lower resolution of 8.6 A.

[0111] AP205-T4P complex modeling. To generate the AP205-T4P complex, the models of outer Mat-T4P and inner Mat-T4P complexes, previously refined in the local maps, were combined with the model of the apo AP205 virion by superimposing the corresponding Mat models.T4P-labeling by Mat200-sfGFP

[0112] Mat200-sfGFP protein purification. The His SUMO Mat200-sfGFP sequence was codon optimized, synthesized, and inserted into a pET28a(+) vector by GenScript. Transformation of 100 ng pET28a(+) His SUMO Mat200-sfGFP plasmid was performed on E. Coli BL21 DE3 cells, and 25mL of overnight culture was added to IL of LB broth supplemented with Kanamycin (final concentration: 50 pg / mL) for large-scale expressions. The IL cell culture was grown at 37 °C for approximately 4 hours and then cooled to 18 °C when the OD reached 0.6, followed by another 1-hour incubation. Proteinexpressions were induced using a final concentration of 0.5 mM IPTG and cells were collected and centrifuged at 4,000 rpm for 30 minutes. The cell pellet was resuspended in a lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8) containing EDTA-free cocktail protease inhibitor tablet (ThermoFisher®). The resuspended cells were lysed by passing through a microfluidizer for three times at 25,000 psi. The total lysate was then centrifuged at 18,000 rpm for 30 minutes at 4 °C, and the supernatant was collected and injected into a Ni affinity column. The column was washed with 30 mL of washing buffer (20 mM Tris- HCl, 300 mM NaCl, pH 8, 5 mM Imidazole), and His SUMO Mat200-sfGFP was eluted with 15 mL of elution buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8, 300 mM Imidazole). The eluted protein was concentrated, and 500 pL of the concentrated protein was loaded onto a pre-equilibrated Superdex® 200 size-exclusion column using an equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8). The fraction which corresponds to the monomeric form of the protein was collected.

[0113] To remove the SUMO tag, SUMO protease was added at a concentration of 1 mg / mL to SUMO Mat200-sfGFP, and the volume was adjusted to 50 mL. This SUMO protease cleavage reaction was carried out at room temperature for 1 hour and success confirmed by SDS-PAGE gel analysis. The monomeric form of Mat200-sfGFP was further purified by the size exclusion chromatography (FIG. 17).

[0114] Fluorescence microscopy analysis. Mat200-sfGFP was adjusted to a working concentration of 1.5 pM. The over-day culture of A. gpl6 cells were grown to mid- exponential phase before being aliquoted and mixed with diluted Mat200-sfGFP at a 1 : 1 ratio. Samples were incubated at room temperature for 5 minutes, and a small drop (~1.5 pL) of the mixture was added onto a PBS-agarose pad before applying a coverslip on top for imaging. Images were taken as Z-series with 300 nm interval in both GFP and phase contrast channels.

[0115] Example 2. Mat200-mediated detachment of type 4 pili (T4P)Introduction

[0116] The rise of hospital -acquired infections, driven by the escalation of antimicrobial resistant (AMR) bacterial strains, has posed many serious challenges in healthcare. A prevalent source of AMR bacterial human diseases is attributed to Acinetobacter species (spp^) as evidenced by a 78% increase in nosocomial infections between 2019 and 2020. Acinetobacter baumannii demonstrated a marked increase in resistance to multiple antibiotic classes, with the proportion of isolates resistant to at leastthree classes rising from 4% to 55% over several years. Acinetobacter spp. are gramnegative bacteria that can survive in a variety of environments, such as soil and water, which enabled this pathogen to become an urgent threat to patients, especially those in hospital-like settings.

[0117] Acinetobacter spp. utilize extracellular appendages called type IV pili (T4P) which enhance the ability of the host cell to acquire and integrate genetic material which increases their virulence. These hair-like, filamentous structures display dynamic cycles of extension and retraction which facilitate T4P involvement in a variety of cellular processes such as motility, biofilm formation, host adhesion, and DNA uptake.

[0118] There are three types of T4P which are primarily classified based on major pilin subunit primary sequence and length. Type IVa pili (T4aP) are the most abundant T4P which are in various gram-negative species, including Acinetobacter spp. T4aP include four subcomplexes: first, the pilus subcomplex which is composed of the major pilin subunit, PilA, and minor pilin subunits; second, the inner membrane motor subcomplex which contains the ATPases required for pilus dynamics; third, the alignment subcomplex which incorporates protein complexes and channels necessary to align the major pilin subunits; fourth, the outer membrane subcomplex which includes channels needed for the pilus to extrude out beyond the cell. The T4P is commonly composed of the many copies of PilA, the major pilin subunit, which form helical polymers with few minor pilin subunits that cap the pilus. The assembly and disassembly of PilA allows for the pilus to extend and retract rapidly in conjunction with other subcomplexes which help the pilus span across the membranes.

[0119] Single-stranded RNA bacteriophages (ssRNA phages) utilize pili to infect host bacteria. ssRNA phage genomes are -4000 nucleotides and contain one positive-sense strand of viral RNA (vRNA) that encodes four proteins: the maturation protein (Mat), coat protein (coat), replicase, and a lysis protein. The virion comprises multiple copies of coat protein that encapsulates the vRNA which is typically bound to one Mat protein on the 3’ end which protrudes from the capsid. AP205 possesses two identical copies of the Mat protein, an inner and an outer Mat protein, allowing it to bind to two pili simultaneously. The dimerization of both Mat proteins aids in shielding the negatively charged surface of the inner Mat with the lower portion of the outer Mat. However, AP205 is commonly bound to a pilus via its outer Mat protein, which sticks out of the coat protein farther than the inner Mat protein. The infection process of ssRNA phages begins when the Mat protein of thephage binds to the side of a pilus. Pilus retraction then draws the pilus-bound phage towards the cell surface, enabling the delivery of vRNA from the phage capsid into the cell through an unidentified mechanism.

[0120] Without wishing to be bound by any particular theory, it can be hypothesized that a major function of the Mat protein is to assist in delivering vRNA from the phage into the host cell. This model can be furthered by showing that the ssRNA phages MS2 and QP can infect Escherichia coli (E. coli) cells via their F-pili and the mechanism by which vRNA enters the cell can also cause detachment of the pili. After the phage adsorbs to the pilus of the host cell, pilus retraction brings the phage close to the cell surface and near the basal body region. The affinity between Mat and the pilus causes bending as the pilus attempts to retract into the cell membrane and the detachment of the pilus allows the vRNA of the phage to enter the cell via an unknown entry mechanism.

[0121] Pilus detachment is associated with other ssRNA phage infections in Pseudomonas aeruginosa and Vibrio cholerae and the mechanism of entry is potentially universal across ssRNA phages. This example focuses on determining the minimal components of the ssRNA phage AP205 that are necessary and sufficient for pilus detachment in Acinetobacter higginsii (A. higginsii). The determination of the entry mechanism in this example can apply to other AMR bacteria that can be infected by ssRNA phages.Results

[0122] Reference is now made to FIGs 20A-20D. To visualize and quantify T4P in A. higginsii (NCBI: TXID70347) cells, a previously established fluorescent protein fusion was used, which uses the first 200 amino acids of the N-terminus of the AP205 Mat protein and superfolder-GFP (sfGFP), named Mat200-sfGFP. Using this fluorescent construct, one is able to determine the quantitative impact of AP205 binding on T4P activity in liquid media. It was observed that Mat200-sfGFP can bind to the T4P of A. higginsii (FIGs 20A-20D). A. higginsii cells were grown to exponential phase (OD600 ~ 0.4) and were treated for various lengths of time either with AP205 at a multiplicity of infection (MOI) of 5, or buffer, followed by the addition of Mat200-sfGFP in order to label the pili. Under these conditions, an increase in the number of free pili was observed. This detachment was observed after 20 min and plateaued at ~0.3 detached pili per cell. When the cells were treated solely with Mat200-sfGFP, an increase in free pili was also observed after 20 min; however, the number of detached pili per cell plateaued at -0.09.

[0123] Reference is now made to FIGs 21A-21D. The same experimental methods were used to determine if Mat200-sfGFP was sufficiently leading to an increase of free pili in the media. Mat200was purified alone to determine if the apical domain of the AP205 Mat protein played a role in the detachment of T4P. Interestingly, when cells were treated with Mat200alone, an increase in free pili was detected 20 min post-treatment and the number of detached pili per cell plateaued around ~0.1 (FIGs 21A-21D). This suggests that the first 200 residues of the Mat protein has an integral role in the adherence of AP205 to a T4P. Since free pili are detected when cells are treated with AP205, Mat200-sfGFP, and Mat200alone, the interactions between Mat200and a pilus are likely crucial for the delivery of vRNA.

[0124] QP maturation protein causes the detachment of E. coli F-pilus. To further test the broad-spectrum of detachment mechanisms, another ssRNA phage can be used, QP, which targets Escherichia coli (E. coli), to repeat the experiment in a different bacteria system. In various instances, saturated MBP-A2 concentration for percentage of pili detachment can be quantified.Discussion

[0125] Mat200alone can bind and interfere with pili, and at least these residues are effective for the binding of AP205 to a T4P. In addition, Mat200or Mat200-sfGFP can bind in abundance to the pili, but these may not generate enough force to detach pilus bound thereto. AP205 is significantly larger than Mat200and may require more force for the successful delivery of vRNA, which results in detachment.Methods

[0126] A. gpl6 Growth Conditions. GP16 cells were grown in LB medium in a test tube at 30°C with shaking (225 rpm) unless indicated otherwise.AP205 Purification

[0127] Fluorescence Microscopy Imaging. Mat200-sfGFP was adjusted to a working concentration of 1.5 pM. An over-day culture of A. gp!6 cells were grown to midexponential phase and an aliquot was mixed with Mat200-sfGFP (8: 1). A small drop (~1 pL) of the mixture was added onto a PBS agarose pad before a coverslip was added on top to allow for imaging. Images were taken in both the phase contrast and GFP channels with Z-stack intervals of 300 nm. Imaging was conducted at room temperature using a Nikon® Eclipse Ti2® inverted epifluorescence microscope with a lOOx objective lens (Plan Apochromat®, numerical aperture 1.45, oil immersion). Images were captured on a cooledEMCCD camera with mask (Princeton Instruments®). The cells were imaged under phase contrast and the fluorescent filter cube ET GFP (Chroma 96362).

[0128] Pilus Detachment Assay. An overnight culture containing a single colony of A. gpl6 cells was grown in 1 mL LB inside of a test tube at 30°C with shaking for roughly 16 hrs. The following morning, an over-day culture was grown by diluting the overnight culture 1 : 100 into 3 mL of fresh LB media. The cells were grown to OD600 ~ 0.4 (-3.5-4 hrs) and an aliquot of 40 pL was placed into a microcentrifuge tube. Then, 5 pL of AP205 phage (MOI = 5), or equivalent buffer, was added and incubated for varying time lengths (0, 2, 5, 10, 20, 30, 40, or 60 min) in a 30°C water bath without shaking. After, 5 pL of Mat200-sfGFP, or equivalent buffer, was added to label the pili for 2 min at room temperature in dark conditions. A small drop (-1 pL) of the mixture was added onto a 1 mm thick 1.5% PBS agarose pad on top of a small coverslip (18 by 18 mm) which was topped with a large coverslip (24 by 50 mm). Images in both the phase contrast (100 ms exposure) and GFP (10 ms exposure) channels were taken with Z-stacks in 300 nm intervals in 10-25 various stage positions. Images were analyzed using a combination of NIS- Elements software (Nikon®), OmniPose® for cell segmentation and counting, and Imaged for detached pili identification and counting.

[0129] Mat200-sfGFP Expression and Purification. Both the expression and purification of Mat200-sfGFP were performed based on previously established methods. Briefly, E. coli BL21 DE3 cells containing the pET28-His-SUMO-Mat200-sfGFP vector were grown overnight at 37°C with 25 mL LB media and 50 pg / mL of kanamycin. The overnight culture was added to 1 L of fresh LB and kanamycin for expression assays and was grown until the OD600 reached 0.4-0.6. The cells were chilled on ice and transferred to 18°C and induced with 0.5 mM IPTG for 20 hours. The cells were harvested using centrifugation at 4,000 x g for 30 min at 4°C followed by a resuspension in lysis buffer (50 mM Tris pH=8, 200 mM NaCl) containing a cocktail protease inhibitor tablet (ThermoFisher®). The cells were lysed using a microfluidizer at 25,000 psi and the supernatant was collected using centrifugation at 14,000 x g for 30 min at 4°C. The collected supernatant was injected into a Ni affinity column which was washed with washing buffer (20 mM Tris pH=8, 200 mM NaCl, 5 mM Imidazole) and His-SUMO- Mat200-sfGFP was eluted with elution buffer (20 mM Tris pH=8, 200 mM NaCl, 300 mM Imidazole). The eluted protein was concentrated and 500 pL was loaded into a Superdex®200 size-exclusion column using an equilibration buffer (50 mM Tris pH=8, 200 mM NaCl). The fraction that corresponds to the SUMO-Mat200-sfGFP protein was collected.

[0130] Bacterial strains. All E. coli strains were grown in LB medium containing related antibiotics. Cells were grown in baffled flasks at 37°C with shaking (265 rpm) unless other conditions indicated.

[0131] MBP-A2 expression and purification. The expression and purification of MBP-A2 were performed based on a previously reported method. pET28-MBP-A2 was transformed to BL21 strain, which originally contained pZA32-MurAA plasmid to prevent A2 cause cell lysis. Cells were grown at 37°C with aeration until OD600 reach 0.4 ~ 0.6, then chilled in ice bath for 1 h prior to induction with 1 mM IPTG and expression at 16°C for 18 h. Cells were harvested by centrifugation at 12 000 g for 15 min at 4°C and resuspended in lysis buffer [20 mM Tris-HCl (pH = 8), 150 mM NaCl, 1% glycerol, 1 mM EDTA, 20 pg ml-1 DNase, 10 pg ml-1 RNaseA and protease inhibitor cocktail (Sigma®)]. Cells were disrupted by passage through a SLM-Aminco French pressure cell (Spectronic Instruments®) at 16 000 psi, and lysate was clarified by centrifugation at 12 000 g for 15 min at 4°C, and then filtered through 0.45pm filter before loading onto amylose resin column (Amylose High Flow resin, New England Biolabs (NEB)). Protein was eluted with lOmM maltose in elution buffer [20 mM Tris-HCl (pH = 8), 150 mM NaCl, 1% glycerol],

[0132] Labeling of F-pilus. The visualization of F-pilus was performed by incubating with MS2-GFP. The expression and purification of MS2-GFP was performed as previous reported. LZ2619 cells with 1 mM IPTG treatment at 10 min post infection. Purification of the phages was achieved using ammonium sulfate precipitation and CsCl gradient centrifugation. In brief, 280 g per liter of ammonium sulfate was added to the lysates in small increments, followed by chilling at 4 °C for 4 h. The precipitates were sedimented by centrifugation at 8,000 rpm for 1 h and resuspended in MS2 buffer (150 mM NaCl, 5 mM EDTA, and 50 mM Tris pH 7.5). The suspensions were dialyzed in 3.5-10 kDa molecular weight cutoff cassettes three times at 2-h intervals with MS2 buffer at 4 °C, followed by treatment with DNase at 10 U / mL for 1 h at 4 °C. A clearing spin was performed, after which the phage preparations were mixed with 0.55 g CsCl per gram of phage solution to produce a final density of 1.38. The suspensions were centrifuged at 45,000 rpm for 24 h at 4 °C. The bands were collected and dialyzed against MS2 buffer, and the purified preparations were stored at 4 °C.

[0133] Pilus detachment assay. Overnight cultures of piliated cells were diluted 1 : 100 in LB containing antibiotics and 4 mM CaCh. The cells were grown to an OD600 of 0.4, then purified QP or MBP-A2 was added to the culture. Infections were performed at 37 °C without shaking to minimize pilus agitation. Samples were removed at regular time points using cut pipette tips and dispensed into prechilled tubes and allowed to equilibrate on ice. A small volume of MS2-GFP equating to an MOI of 20 was added to the samples and left on ice for 20 min to allow phage adsorption to the pili.

[0134] Samples were prepared for microscopy by spotting 1 pL of sample onto a large coverslip (No. 1, 24 x 50 mm; Thermo Fisher Scientific) and gently overlaying a small, 1-mm-thick 1.5% agarose pad dissolved in PBS on the sample before placing it under the microscope. Z stacks of 300 nm were used in the GFP channel to precisely measure pili. Cells and pili were imaged on multiple stage positions (between 10 and 20 per sample) in phase (100 ms exposure to detect cells) and GFP (100 ms to detect pili) channels. The images were analyzed using NIS-Elements software (Nikon®) and Fiji® for cell and pili counts and length measurements.NON-LIMITING EMBODIMENTS

[0135] While general features of the disclosure are described and shown and particular features of the disclosure are set forth in the claims, the following non-limiting embodiments relate to features, and combinations of features, that are explicitly envisioned as being part of the disclosure. The following non-limiting Embodiments contain elements that are modular and can be combined with each other in any number, order, or combination to form a new non-limiting Embodiment, which can itself be further combined with other non-limiting Embodiments.

[0136] Embodiment 1. A synthetic bacterial phage maturation (Mat) protein scaffold comprising an apical domain polypeptide sequence.

[0137] Embodiment 2. The scaffold of Embodiment 1 or any other Embodiment, wherein the scaffold consists essentially of the apical domain polypeptide sequence.

[0138] Embodiment s. The scaffold of Embodiment 2 or any other Embodiment, wherein the scaffold consists of the apical domain polypeptide sequence.

[0139] Embodiment 4. The scaffold of any one of Embodiments 1-3 or any other Embodiment, wherein the apical domain polypeptide sequence comprises at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species.

[0140] Embodiment 5. The scaffold of any one of Embodiments 1-3 or any other Embodiment, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2.

[0141] Embodiment 6. The scaffold of Embodiment 5 or any other Embodiment, wherein the apical domain polypeptide sequence comprises 100% identity to the polypeptide sequence of SEQ ID NO:2.

[0142] Embodiment 7. The scaffold of any one of Embodiments 1-6 or any other Embodiment, wherein the scaffold further comprises a tag polypeptide sequence that enables a fluorescence detection of the scaffold, an interaction of a tag binder with the tag polypeptide sequence, or both.

[0143] Embodiment 8. A composition comprising the scaffold of any one of Embodiments 1-7 or any other Embodiment and a carrier, a diluent, a salt, a pH buffering agent, a detergent, a lipid, and / or a surfactant.

[0144] Embodiment 9. A pharmaceutical composition comprising the scaffold of any one of Embodiments 1-7 or any other Embodiment and a pharmaceutically acceptable carrier.

[0145] Embodiment 10. A kit comprising the composition of Embodiment 8 or any other Embodiment and an instructional material configured to instruct an individual in a use of the composition in a method of decreasing bacterial virulence in a biological matrix.

[0146] Embodiment 11. A kit comprising the pharmaceutical composition of Embodiment 9 or any other Embodiment and an instructional material configured to instruct an individual in a use of the pharmaceutical composition in a method of treating or preventing bacterial infection in a subject.

[0147] Embodiment 12. A method of decreasing bacterial virulence in a biological matrix, the method comprising contacting the biological matrix with the scaffold of any one of Embodiments 1-7 or any other Embodiment.

[0148] Embodiment 13. The method of Embodiment 12 or any other Embodiment, wherein the scaffold increases an infection-dependent pili detachment, suppresses the deployment of retractile pili, or both, within the biological matrix.

[0149] Embodiment 14. The method of Embodiment 13 or any other Embodiment, wherein an increase in the infection-dependent pili detachment corresponds to a decrease in bacterial virulence.

[0150] Embodiment 15. A method of treating or preventing bacterial infection in a subject, the method comprising administering to the subject an effective amount of a synthetic bacterial phage Mat protein scaffold comprising an apical domain polypeptide sequence.

[0151] Embodiment 16. The method of Embodiment 15 or any other Embodiment, wherein the scaffold consists essentially of the apical domain polypeptide sequence.

[0152] Embodiment 17. The method of Embodiment 16 or any other Embodiment, wherein the scaffold consists of the apical domain polypeptide sequence.

[0153] Embodiment 18. The method of any one of Embodiments 15-17 or any other Embodiment, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species.

[0154] Embodiment 19. The method of any one of Embodiments 15-17 or any other Embodiment, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2.

[0155] Embodiment 20. The method of Embodiment 15 or any other Embodiment, wherein the apical domain polypeptide sequence comprises 100% identity to the polypeptide sequence of SEQ ID NO:2.

[0156] Embodiment 21. A nucleic acid comprising a polynucleotide sequence encoding the scaffold of any one of Embodiments 1-7.

[0157] Embodiment 22. The nucleic acid of Embodiment 21, wherein the polynucleotide sequence encoding the scaffold comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleotide sequence of SEQ ID NO: 1.

[0158] Embodiment 23. The nucleic acid of Embodiment 21, wherein the polynucleotide sequence encoding the scaffold comprises 100% identity to the polynucleotide sequence of SEQ ID NO: 1.

[0159] Embodiment 24. A nucleic acid expression vector comprising the polynucleotide sequence of any one of Embodiments 21-23.

[0160] Embodiment 25. A recombinant host cell comprising the nucleic acid expression vector of Embodiment 24.

[0161] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A synthetic bacterial phage maturation (Mat) protein scaffold comprising an apical domain polypeptide sequence.

2. The scaffold of claim 1, wherein the scaffold consists essentially of the apical domain polypeptide sequence.

3. The scaffold of claim 2, wherein the scaffold consists of the apical domain polypeptide sequence.

4. The scaffold of any one of claims 1-3, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species.

5. The scaffold of any one of claims 1-3, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2.

6. The scaffold of claim 5, wherein the apical domain polypeptide sequence comprises 100% identity to the polypeptide sequence of SEQ ID NO:2.

7. The scaffold of any one of claims 1-6, wherein the scaffold further comprises a tag polypeptide sequence that enables a fluorescence detection of the scaffold, an interaction of a tag binder with the tag polypeptide sequence, or both.

8. A composition comprising the scaffold of any one of claims 1-7 and a carrier, a diluent, a salt, a pH buffering agent, a detergent, a lipid, and / or a surfactant.

9. A pharmaceutical composition comprising the scaffold of any one of claims 1-7 and a pharmaceutically acceptable carrier.

10. A kit comprising the composition of claim 8 and an instructional material configured to instruct an individual in a use of the composition in a method of decreasing bacterial virulence in a biological matrix.

11. A kit comprising the pharmaceutical composition of claim 9 and an instructional material configured to instruct an individual in a use of the pharmaceutical composition in a method of treating or preventing bacterial infection in a subject.

12. A method of decreasing bacterial virulence in a biological matrix, the method comprising contacting the biological matrix with the scaffold of any one of claims 1-7.

13. The method of claim 12, wherein the scaffold increases an infectiondependent pili detachment, suppresses the deployment of retractile pili, or both, within the biological matrix.

14. The method of claim 13, wherein an increase in the infection-dependent pili detachment corresponds to a decrease in bacterial virulence.

15. A method of treating or preventing bacterial infection in a subject, the method comprising administering to the subject an effective amount of a synthetic bacterial phage Mat protein scaffold comprising an apical domain polypeptide sequence.

16. The method of claim 15, wherein the scaffold consists essentially of the apical domain polypeptide sequence.

17. The method of claim 16, wherein the scaffold consists of the apical domain polypeptide sequence.

18. The method of any one of claims 15-17, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of an apical domain of a Mat protein of an Acinetobacter species.

19. The method of any one of claims 15-17, wherein the apical domain polypeptide sequence comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polypeptide sequence of SEQ ID NO:2.

20. The method of claim 15, wherein the apical domain polypeptide sequence comprises 100% identity to the polypeptide sequence of SEQ ID NO:2.

21. A nucleic acid comprising a polynucleotide sequence encoding the scaffold of any one of claims 1-7.

22. The nucleic acid of claim 21, wherein the polynucleotide sequence encoding the scaffold comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleotide sequence of SEQ ID NO: 1.

23. The nucleic acid of claim 21 , wherein the polynucleotide sequence encoding the scaffold comprises 100% identity to the polynucleotide sequence of SEQ ID NO: 1.

24. A nucleic acid expression vector comprising the polynucleotide sequence of any one of claims 21-23.

25. A recombinant host cell comprising the nucleic acid expression vector of claim 24.

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