Clebicine for the control of klebsiella

Abstract

To provide a protein having cytotoxic activity against Klebsiella.SOLUTION: Provided is a composition for use in therapy comprising one or more proteins or said proteins having cytotoxic activity against Klebsiella wherein said proteins comprise or consist of a first amino acid sequence segment and a second amino acid sequence segment, the first segment having a cytotoxic activity against Klebsiella having at least 90% sequence identity to a specified sequence or amino acid sequence thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides proteins having cytotoxic activity against Klebsiella. The present invention also provides compositions, including pharmaceutical compositions, comprising one or more of the proteins. The present invention further provides proteins having cytotoxic activity against Klebsiella and compositions comprising the proteins for use in therapy. The present invention also provides proteins or compositions comprising the proteins for use in methods for treating a subject's infection with Klebsiella. Furthermore, oral enteric formulations for delivering proteins or compositions to the small or large intestine are provided. Furthermore, pulmonary formulations for delivering proteins or compositions to the lungs are provided. Also provided are methods for preventing or reducing infection or contamination of objects with Klebsiella, methods for treating a Klebsiella infection in a subject or patient in need thereof, and methods for producing compositions comprising the proteins. The present invention also provides methods for producing compositions comprising the proteins of the present invention. The present invention further provides nucleic acid molecules encoding proteins having cytotoxic activity against Klebsiella, plants, plant tissues, or plant cells comprising the proteins, and plants, plant tissues, or plants comprising the nucleic acid molecules. [Background technology]

[0002] Klebsiella are non-motile, rod-shaped, Gram-negative bacteria enveloped by capsular polysaccharides that confer resistance to many host defense mechanisms. Klebsiella are opportunistic pathogens present in the environment and on mammalian mucosal surfaces. Three species of the genus Klebsiella are commonly associated with human disease: K. pneumoniae, K. oxytoca, and K. granulomatis. Recently, two additional Klebsiella species, K. variicola and K. quasipneumoniae, have been found to be capable of causing fatal infections (Long et al. 2017). The primary sources of infection with pathogenic bacteria include the gastrointestinal tract of patients and the hands of hospital personnel. Outside of hospitals, Klebsiella infection typically occurs in the lungs. The disease typically affects middle-aged and elderly men with debilitating conditions such as alcoholism, diabetes, or chronic bronchopulmonary spirochetosis (Chan et al., 2009). This patient population is thought to have impaired respiratory host defense mechanisms. The organism enters after the host inhales oropharyngeal microorganisms that colonize the lower respiratory tract (Hirsche et al., 2005).

[0003] In recent years, Klebsiella has become a serious pathogen in hospital-acquired infections. Common sites of nosocomial infection include the urinary tract, lower respiratory tract, bile duct, and surgical wound sites. Clinical syndromes include pneumonia, bacteremia, thrombophlebitis, urinary tract infection (UTI), cholecystitis, diarrhea, upper respiratory tract infection, wound infection, osteomyelitis, and meningitis (Miftode et al., 2008). The presence of invasive devices, contamination of respiratory support equipment, use of urinary catheters, and use of antibiotics are factors that increase the likelihood of nosocomial infections with Klebsiella species (Weisenberg et al., 2009). K. pneumoniae is one of six pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter) that constitute hospital-acquired ESKAPE infections that rapidly develop antibiotic resistance. In 2016, an outbreak of nosocomial pneumonia was reported in China, resulting in the deaths of five surgical patients due to infection with the hypervirulent, carbapenem-resistant K. pneumoniae (CRPK) ST11 strain (Gu et al., 2017). The strain can be called a "superbug" because it is both hypervirulent and hyperresistant. ST11 CR HvKP strains infect relatively healthy populations with normal immunity. They are mucoid strains and adhere to all surfaces in intensive care units. Colistin is the antibiotic of last resort against carbapenem-resistant Enterobacteriaceae, but it is largely ineffective against such strains. For the time being, ceftazidime / avibactam can be used to treat such infections, but resistance to these antibiotics can also quickly develop.

[0004] The increasing drug resistance of pathogens is a global challenge, necessitating the urgent development of a new generation of antibacterial agents. Bacteria produce toxic proteins called bacteriocins to compete for niches with each other. Bacteriocins typically kill only closely related bacteria belonging to the same species or genus. Their mechanisms of action are diverse, including pore formation, DNase and RNase activity, and inhibition of protein synthesis or DNA replication. Bacteriocins produced by Gram-positive bacteria are typically referred to as bacteriocins of a specific class according to their properties, while bacteriocins produced by Gram-negative strains are classified as colicin-type bacteriocins (high molecular weight, 25–80,000 Da) or microcins (low molecular weight, <10,000 Da) (Lagos et al., 2009). Antimicrobial peptides are not only produced by bacteria but also by various organisms when faced with bacterial infection. Antimicrobial peptides are used in medicine as peptide antibiotics, such as colistin (a polymyxin derived from Paenibacillus polymyxa) and vancomycin (derived from Amycolatopsis orientalis). However, such antibiotics are mostly used for topical administration or as last-resort drugs. Another challenge with peptides is the inefficiency and high cost of purification from natural sources. Chemical synthesis can overcome this, but it remains expensive. Recombinant peptide production in heterologous hosts is also difficult due to toxicity to host cells (Li, 2011).

[0005] To date, there are no registered colicin-like antibiotics. However, there are studies in the scientific literature on the use of colicin-like bacteriocins as potential antibacterial agents against Gram-negative pathogens. Colicins have been the most studied, with several research groups working with pyocins (Grinter et al., 2013; Ghequire and de Mot, 2014). Meanwhile, bacteriocins belonging to the nuclease class from Klebsiella have received little attention, with only a few published studies (James et al., 1987; Riley et al., 2001; Chavan et al., 2005). Detailed studies on the expression, purification, and activity testing of klebicins are lacking. Notwithstanding the prior art, it is an object of the present invention to provide agents that are active against Klebsiella. It is also an object of the present invention to provide agents or compositions that can be used to treat Klebsiella infections in a subject, particularly infections caused by antibiotic-resistant Klebsiella. It is a further object of the present invention to provide methods for preventing or reducing contamination of objects (e.g., food products with one or more Klebsiella species). Summary of the Invention

[0006] The present inventors have discovered a novel bacteriocin that is active against Klebsiella. 1) A protein having cytotoxic activity against Klebsiella, preferably a protein having lipid II cleavage activity or pore-forming ability in the cell membrane of Klebsiella cells.

[0007] 2) The protein according to Item 1, comprising or consisting of a first amino acid sequence segment and a second amino acid sequence segment, wherein the first amino acid sequence segment is capable of binding to a component of a Klebsiella cell, and the second amino acid sequence segment has lipid II cleavage activity or pore-forming activity in the cell membrane of a Klebsiella cell. 3) A protein having cytotoxic activity against Klebsiella, comprising or consisting of a first amino acid sequence segment and a second amino acid sequence segment, wherein the first segment is preferably the N-terminal segment of the protein and the second segment is the C-terminal segment of the protein.

[0008] 4) (A) The first segment is: (Ai) amino acid residues 1 to 128 of SEQ ID NO: 1 (KpneM); (A-ii) amino acid residues 1 to 127 of SEQ ID NO: 2 (KvarM); (A-iii) amino acid residues 1 to 123 of SEQ ID NO: 3 (KpneM2); (A-iv) amino acid residues 1 to 118 of SEQ ID NO: 4 (KaerM); (Av) amino acid residues 1 to 170 of SEQ ID NO: 5 (KpneA); (A-vi) amino acid residues 1 to 172 of SEQ ID NO: 6 (KaerA); (A-vii) amino acid residues 1 to 255 of SEQ ID NO: 7 (Koxy); (A-viii) amino acid residues 1 to 288 of SEQ ID NO: 8 (KpneIa), or (A-ix) Amino acid residues 1 to 236 of SEQ ID NO: 9 (KvarIa) comprising or consisting of the amino acid sequence of or (B) a first segment; (Bi) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (B-ii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (B-iii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (B-iv) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Bv) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (B-vi) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (B-vii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (B-viii) has at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8, or (B-ix) has at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9 Does it contain an amino acid sequence? or (C) The first segment is (Ci) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (C-ii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (C-iii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (C-iv) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Cv) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (C-vi) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (C-vii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (C-viii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8; or (C-ix) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9 4. The protein of any one of items 2 or 3, comprising the amino acid sequence

[0009] 5) (A) The amino acid sequence of the first segment is (Ai) amino acid residues 1 to 128 of SEQ ID NO: 1 (KpneM); (A-ii) amino acid residues 1 to 127 of SEQ ID NO: 2 (KvarM); (A-iii) amino acid residues 1 to 123 of SEQ ID NO: 3 (KpneM2); (A-iv) amino acid residues 1 to 118 of SEQ ID NO: 4 (KaerM); (Av) amino acid residues 1 to 170 of SEQ ID NO: 5 (KpneA); (A-vi) amino acid residues 1 to 172 of SEQ ID NO: 6 (KaerA); (A-vii) amino acid residues 1 to 255 of SEQ ID NO: 7 (Koxy); (A-viii) amino acid residues 1 to 288 of SEQ ID NO: 8 (KpneIa), or (A-ix) Amino acid residues 1 to 236 of SEQ ID NO: 9 (KvarIa) Is the amino acid sequence of or (B) The amino acid sequence of the first segment is (Bi) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (B-ii) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (B-iii) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (B-iv) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Bv) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (B-vi) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (B-vii) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (B-viii) at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8, or (B-ix) At least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9 Do you have; or (C) The amino acid sequence of the first segment is (Ci) a substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (C-ii) substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (C-iii) substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (C-iv) substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Cv) substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (C-vi) substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (C-vii) substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (C-viii) a substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8, or (C-ix) Substitution, addition, insertion and / or deletion of 1 to 40 amino acids compared to the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9 4. The protein according to any one of items 2 or 3, having the following structure:

[0010] 6) In item (B), any one of the sequence identities is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%; and / or 6. The protein according to item 4 or 5, wherein in item (C), the number of amino acid substitutions, additions, insertions, and / or deletions is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably at least 1 to 5, compared with any one of the amino acid sequences.

[0011] 7) (D) The second segment is (Di) amino acid residues 129 to 278 of SEQ ID NO: 1 (KpneM); (D-ii) amino acid residues 128 to 276 of SEQ ID NO: 2 (KvarM); (D-iii) amino acid residues 124 to 272 of SEQ ID NO: 3 (KpneM2); (D-iv) amino acid residues 119 to 266 of SEQ ID NO: 4 (KaerM); (Dv) amino acid residues 171 to 377 of SEQ ID NO: 5 (KpneA); (D-vi) amino acid residues 173 to 379 of SEQ ID NO: 6 (KaerA); (D-vii) amino acid residues 256 to 452 of SEQ ID NO: 7 (Koxy); (D-viii) amino acid residues 289 to 466 of SEQ ID NO: 8 (KpneIa), or (D-ix) Amino acid residues 237 to 414 of SEQ ID NO: 9 (KvarIa) comprising or consisting of the amino acid sequence of or (E) the second segment is (Ei) having at least 70% sequence identity with the amino acid sequence of amino acid residues 129 to 278 of SEQ ID NO: 1; (E-ii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 128 to 276 of SEQ ID NO: 2; (E-iii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 124 to 272 of SEQ ID NO: 3; (E-iv) having at least 70% sequence identity with the amino acid sequence of amino acid residues 119 to 266 of SEQ ID NO: 4; (Ev) having at least 70% sequence identity with the amino acid sequence of amino acid residues 171 to 377 of SEQ ID NO: 5; (E-vi) having at least 70% sequence identity with the amino acid sequence of amino acid residues 173 to 379 of SEQ ID NO: 6; (E-vii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 256 to 452 of SEQ ID NO: 7; (E-viii) has at least 70% sequence identity with the amino acid sequence of amino acid residues 289 to 466 of SEQ ID NO: 8, or (E-ix) has at least 70% sequence identity with the amino acid sequence of amino acid residues 237 to 414 of SEQ ID NO: 9 Does it contain an amino acid sequence? or (F) the second segment is (Fi) having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 129 to 278 of SEQ ID NO: 1; (F-ii) having 1 to 30 amino acid substitutions, additions, insertions or deletions compared to the amino acid sequence of amino acid residues 128 to 276 of SEQ ID NO: 2; (F-iii) having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 124 to 272 of SEQ ID NO: 3; (F-iv) having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 119 to 266 of SEQ ID NO: 4; (Fv) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 171 to 377 of SEQ ID NO: 5; (F-vi) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 173 to 379 of SEQ ID NO: 6; (F-vii) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 256 to 452 of SEQ ID NO: 7; (F-viii) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 289 to 466 of SEQ ID NO: 8; (F-ix) has 1 to 35 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acid residues 237 to 414 of SEQ ID NO: 9. 7. The protein according to any one of items 2 to 6, comprising an amino acid sequence.

[0012] 8) (D) The amino acid sequence of the second segment is (Di) amino acid residues 129 to 278 of SEQ ID NO: 1 (KpneM); (D-ii) amino acid residues 128 to 276 of SEQ ID NO: 2 (KvarM); (D-iii) amino acid residues 124 to 272 of SEQ ID NO: 3 (KpneM2); (D-iv) amino acid residues 119 to 266 of SEQ ID NO: 4 (KaerM); (Dv) amino acid residues 171 to 377 of SEQ ID NO: 5 (KpneA); (D-vi) amino acid residues 173 to 379 of SEQ ID NO: 6 (KaerA); (D-vii) amino acid residues 256 to 452 of SEQ ID NO: 7 (Koxy); (D-viii) amino acid residues 289 to 466 of SEQ ID NO: 8 (KpneIa), or (D-ix) Amino acid residues 237 to 414 of SEQ ID NO: 9 (KvarIa) or comprising or consisting of the amino acid sequence or (E) the amino acid sequence of the second segment is (Ei) at least 80% sequence identity with the amino acid sequence of amino acid residues 129 to 278 of SEQ ID NO: 1; (E-ii) at least 80% sequence identity with the amino acid sequence of amino acid residues 128 to 276 of SEQ ID NO: 2; (E-iii) at least 80% sequence identity with the amino acid sequence of amino acid residues 124 to 272 of SEQ ID NO: 3; (E-iv) at least 80% sequence identity with the amino acid sequence of amino acid residues 119 to 266 of SEQ ID NO: 4; (Ev) at least 80% sequence identity with the amino acid sequence of amino acid residues 171 to 377 of SEQ ID NO: 5; (E-vi) at least 80% sequence identity with the amino acid sequence of amino acid residues 173 to 379 of SEQ ID NO: 6; (E-vii) at least 80% sequence identity with the amino acid sequence of amino acid residues 256 to 452 of SEQ ID NO: 7; (E-viii) at least 80% sequence identity with the amino acid sequence of amino acid residues 289 to 466 of SEQ ID NO: 8, or (E-ix) At least 80% sequence identity with the amino acid sequence of amino acid residues 237 to 414 of SEQ ID NO: 9 have or or (F) The amino acid sequence of the second segment is (Fi) substitution, addition, insertion and / or deletion of 1 to 30 amino acids compared to the amino acid sequence of amino acid residues 129 to 278 of SEQ ID NO: 1; (F-ii) substitution, addition, insertion and / or deletion of 1 to 30 amino acids compared to the amino acid sequence of amino acid residues 128 to 276 of SEQ ID NO: 2; (F-iii) substitution, addition, insertion and / or deletion of 1 to 30 amino acids compared to the amino acid sequence of amino acid residues 124 to 272 of SEQ ID NO: 3; (F-iv) substitution, addition, insertion and / or deletion of 1 to 30 amino acids compared to the amino acid sequence of amino acid residues 119 to 266 of SEQ ID NO: 4; (Fv) 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 171 to 377 of SEQ ID NO: 5; (F-vi) substitution, addition, insertion and / or deletion of 1 to 35 amino acids compared to the amino acid sequence of amino acid residues 173 to 379 of SEQ ID NO: 6; (E-vii) substitution, addition, insertion and / or deletion of 1 to 35 amino acids compared to the amino acid sequence of amino acid residues 256 to 452 of SEQ ID NO: 7; (F-viii) 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 289 to 466 of SEQ ID NO: 8, or (F-ix) Substitution, addition, insertion and / or deletion of 1 to 35 amino acids compared to the amino acid sequence of amino acid residues 237 to 414 of SEQ ID NO: 9 8. The protein according to any one of items 2 to 7, having the following structure:

[0013] 9) The protein according to any one of items 7 and 8, wherein the first segment is any one of items Ai to A-iv, Bi to B-iv, or Ci to C-iv, and the second segment is any one of items Di to D-iv, Ei to E-iv, or Fi to F-iv.

[0014] 10) The protein according to Item 9, wherein the first segment is one or more of items Ai to A-iv, and the second segment is one of items Di to D-iv; or the first segment is one of items Bi to B-iv, and the second segment is one of items Ei to E-iv; or the first segment is one of items Ci to C-iv, and the second segment is one of items Fi to F-iv. 11) The protein according to any one of items 7 or 8, wherein the first segment is any one of items Av to A-ix, Bv to B-ix, or Cv to C-ix, and the second segment is any one of items Dv to D-ix, Ev to E-ix, or Fv to F-ix.

[0015] 12) The protein according to Item 11, wherein the first segment is any one of items Av to A-ix and the second segment is any one of items Dv to D-ix; or the first segment is any one of items Bv to B-ix and the second segment is any one of items Ev to E-ix; or the first segment is any one of items Cv to C-ix and the second segment is any one of items Fv to F-ix. 13) the first segment is any one of items Av to A-vi, Bv to B-vi, or Cv to C-vi, and the second segment is any one of items Dv to D-vi, Ev to E-vi, or Fv to F-vi, respectively; and / or The protein according to Item 11 or 12, wherein the first segment is any one of items A-viii to A-ix, B-viii to B-ix, or C-viii to C-ix, and the second segment is any one of items D-viii to D-ix, E-viii to E-ix, or F-viii to F-ix, respectively.

[0016] 14) The protein according to any one of items 4 to 13, wherein the cytotoxic activity of the protein and a comparative protein having the amino acid sequence of SEQ ID NO: 1 is such that spotting 20 microliters of each of a solution of the protein and the comparative protein onto a lawn of a susceptible Klebsiella strain on an agar plate and then incubating the agar plate at 37°C for 16 hours produces spots of at least the same diameter that are free of viable bacteria of susceptible Klebsiella quasipneumoniae subsp. similipneumoniae SB30 (DSM 28212), and the concentration of the protein in the solution is at most 5 times the concentration of the solution of the respective comparative protein.

[0017] 15) (a) (ai) SEQ ID NO: 1 (KpneM); (a-ii) SEQ ID NO: 2 (KvarM); (a-iii) SEQ ID NO: 3 (KpneM2); (a-iv) SEQ ID NO: 4 (KaerM); (av) SEQ ID NO: 5 (KpneA), (a-vi) SEQ ID NO: 6 (KaerA); (a-vii) SEQ ID NO: 7 (Koxy); (a-viii) SEQ ID NO: 8 (KpneIa), or (a-ix) SEQ ID NO: 9 (KvarIa) The amino acid sequence of or (b)(bi) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; (b-ii) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b-iii) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 3; (b-iv) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 4; (bv) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 5; (b-vi) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 6; (b-vii) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; (b-viii) has at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 8; or (b-ix) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 9 amino acid sequence; or (c) (ci) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 1; (c-ii) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 2; (c-iii) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 3; (c-iv) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 4; (cv) having 1 to 110 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 5; (c-vi) having 1 to 110 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 6; (c-vii) having 1 to 130 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 7; (c-viii) having 1 to 130 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 8; or (c-ix) having 1 to 120 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of SEQ ID NO: 9 Amino acid sequence 15. The protein according to any one of items 1 to 14, comprising or consisting of an amino acid sequence comprising or consisting of:

[0018] 16) (a) the amino acid sequence of the protein is (ai) SEQ ID NO: 1 (KpneM), (a-ii) SEQ ID NO: 2 (KvarM); (a-iii) SEQ ID NO: 3 (KpneM2); (a-iv) SEQ ID NO: 4 (KaerM); (av) SEQ ID NO: 5 (KpneA), (a-vi) SEQ ID NO: 6 (KaerA); (a-vii) SEQ ID NO: 7 (Koxy); (a-viii) SEQ ID NO: 8 (KpneIa), or (a-ix) SEQ ID NO: 9 (KvarIa) Is the amino acid sequence of or (b) the amino acid sequence of the protein is (bi) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; (b-ii) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b-iii) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 3; (b-iv) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 4; (bv) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 5; (b-vi) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 6; (b-vii) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; (b-viii) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 8 ,or (b-ix) at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 9 Do you have; or (c) the amino acid sequence of the protein is (ci) a substitution, addition, insertion and / or deletion of 1 to 80 amino acids compared to the amino acid sequence of SEQ ID NO: 1; (c-ii) substitution, addition, insertion and / or deletion of 1 to 80 amino acids compared to the amino acid sequence of SEQ ID NO: 2; (c-iii) substitution, addition, insertion and / or deletion of 1 to 80 amino acids compared to the amino acid sequence of SEQ ID NO: 3; (c-iv) substitution, addition, insertion and / or deletion of 1 to 80 amino acids compared to the amino acid sequence of SEQ ID NO: 4; (cv) substitution, addition, insertion and / or deletion of 1 to 110 amino acids compared to the amino acid sequence of SEQ ID NO: 5; (c-vi) substitution, addition, insertion and / or deletion of 1 to 110 amino acids compared to the amino acid sequence of SEQ ID NO: 6; (c-vii) substitution, addition, insertion and / or deletion of 1 to 130 amino acids compared to the amino acid sequence of SEQ ID NO: 7; (c-viii) a substitution, addition, insertion and / or deletion of 1 to 130 amino acids compared to the amino acid sequence of SEQ ID NO: 8, or (c-ix) substitution, addition, insertion and / or deletion of 1 to 120 amino acids compared to the amino acid sequence of SEQ ID NO: 9 16. The protein according to item 1 or 15, having the following structure:

[0019] 17) In item (b), any one of the sequence identities is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%; and / or 17. The protein according to any one of Items 15 and 16, wherein in item (c), the number of amino acid substitutions, additions, insertions, and / or deletions is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably at least 1 to 5, compared with any one of the amino acid sequences.

[0020] 18) The protein according to any one of items 15, 16 or 17, wherein the cytotoxic activity of the protein of any one of items (bi) to (b-ix) or (ci) to (c-ix) is such that spotting 20 microliters of a solution of the protein and a comparative protein on a lawn of a susceptible Klebsiella strain on an agar plate and incubating the agar plate at 37°C for 16 hours produces a spot of at least the same diameter that is free of viable bacteria of susceptible Klebsiella quasipneumoniae subsp. similipneumoniae SB30 (DSM 28212), and the concentration of the protein in solution is at most 5 times the concentration of a solution of the respective comparative protein.

[0021] 19) The protein according to any one of items 1 to 17, which has cell wall biosynthesis inhibitory activity, thereby enabling the protein to degrade peptidoglycan precursors linked with undecaprenyl phosphate. 20) The protein according to any one of items 2 to 17, wherein the first segment comprises a translocation and a receptor binding domain. 21) The protein according to any one of items 1 to 20, which has bactericidal or bacteriostatic activity against Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella granulomatis, Klebsiella quasipneumoniae, Klebsiella aerogenes, and / or Klebsiella variicola. 22) A protein having cytotoxic activity against Klebsiella, optionally a protein according to any one of items 1 to 21, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 24, wherein each X represents one of the 20 standard amino acid residues or the absence of an amino acid residue, and J represents either L (leucine) or I (isoleucine).

[0022] 23) The protein according to any one of items 1 to 22, wherein the reference sequence is any one of SEQ ID NOs: 1 to 4, 7, 8, or 22, preferably any one of SEQ ID NOs: 1 to 4 or 22. 24) The protein according to item 1 or 23, wherein the cytotoxic activity of the protein and a comparative protein having the amino acid sequence of SEQ ID NO: 1 is such that spotting 20 microliters of each of a solution of the protein and the comparative protein onto a lawn of a susceptible Klebsiella strain on an agar plate and subsequently incubating the agar plate at 37°C for 16 hours produces a spot of at least the same diameter that is free of viable bacteria of susceptible Klebsiella quasipneumoniae subsp. similipneumoniae SB30 (DSM 28212), and the concentration of the protein in the solution is at most 5 times the concentration of the solution of the comparative protein. 25) A composition comprising one or more proteins as defined in any one of items 1 to 24.

[0023] 26) The composition according to Item 25, wherein the cytotoxic activity of the composition and a comparative composition containing a comparative protein having the amino acid sequence of SEQ ID NO: 1 is such that spotting 20 microliters of each of a solution of the composition and a comparative solution of the comparative composition onto a lawn of a susceptible Klebsiella strain on an agar plate and then incubating the agar plate at 37°C for 16 hours produces spots of at least the same diameter that are free of viable bacteria of susceptible Klebsiella quasipneumoniae subsp. similipneumoniae SB30 (DSM 28212), and the concentration of the protein in the solution of the composition is at most 5 times the concentration of the protein in the comparative solution. 27) The composition according to item 25 or 26, further comprising at least one colicin and / or at least one salmosin. 28) The composition according to any one of items 25 to 27, which is a pharmaceutical composition. 29) The composition according to any one of items 25 to 28, which is a plant material or an extract thereof, wherein the plant material is a material derived from a plant expressing the one or more proteins, preferably an edible plant expressing the one or more proteins.

[0024] 30) The composition according to item 29, wherein the plant material is material derived from a plant selected from the group consisting of spinach, chard, beetroot, carrot, sugar beet, beet greens, amaranth, tobacco, preferably Nicotiana benthamiana, and / or the plant material is one or more leaves, roots, tubers or seeds, or crushed, powdered or ground products of said leaves, roots, tubers or seeds. 31) The composition according to any one of items 25 to 30, which is an aqueous solution containing the protein in dispersed form, preferably in dissolved form. 32) A protein or composition according to any one of items 1 to 31 for use in therapy, preferably for use in a method for treating an infection in a subject caused by Klebsiella, such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella variicola. 33) The protein for use according to item 32, wherein the Klebsiella is antibiotic-resistant, for example, carbapenem-resistant. 34) The protein for use according to item 32 or 33, wherein the reference sequence is any one of SEQ ID NOs: 1 to 4, 7, 8 or 22, preferably any one of SEQ ID NOs: 1 to 4 or 22.

[0025] 35) A method for preventing or reducing infection or contamination of an object by one or more Klebsiella species, comprising contacting the object with a protein as defined in any one of items 1 to 24 or a composition as defined in any one of items 25 to 31. 36) A method for treating a Klebsiella infection in a subject in need thereof, comprising administering to the subject a protein defined in any one of items 1 to 24 or a composition defined in any one of items 25 to 31. 37) The method according to item 35 or 36, wherein the Klebsiella includes Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella quasipneumoniae, Klebsiella aerogenes, and / or Klebsiella variicola. 38) A method for producing a composition comprising a protein as defined in any one of items 1 to 24, comprising: (i) expressing the protein in a plant, preferably an edible plant or tobacco; (ii) recovering plant material containing the expressed protein from said plant; (iii) extracting the protein from the plant material using an aqueous buffer to obtain a composition containing the protein; (iv) optionally removing undesirable contaminants from the composition. A method comprising:

[0026] 39) The composition of any one of items 25 to 31, wherein the one or more proteins are formulated for oral delivery to the small intestine or large intestine. 40) An oral formulation comprising the protein according to any one of items 1 to 24 or the composition according to any one of items 25 to 31, wherein the formulation can protect the protein from gastric conditions and release the protein in the small intestine or large intestine. 41) A nucleic acid molecule encoding a protein defined in any one of items 1 to 24. 42) A nucleic acid molecule or a nucleic acid construct encoding a protein as defined in any one of items 1 to 24, preferably a protein as defined in any one of items 15 to 23, preferably comprising a transcription promoter active in plant cells and a nucleotide sequence encoding the protein for expressing the nucleotide sequence under the control of the promoter in a cell, preferably a plant cell.

[0027] 43) A nucleic acid molecule or nucleic acid construct encoding a protein as defined in any one of items 1 to 24, preferably a protein as defined in any one of items 15 to 23, which is or encodes a viral (DNA or RNA) replicon comprising a nucleotide sequence encoding said protein for expressing said nucleotide sequence in a cell, preferably in a plant cell; wherein said replicon may contain a subgenomic promoter for expressing said nucleotide sequence in a plant cell or a cell of a plant under the control of said subgenomic promoter. 44) A plant, plant tissue or plant cell containing a protein as defined in any one of items 1 to 24. 45) A plant, plant tissue or plant cell containing a nucleic acid molecule or nucleic acid construct as defined in item 42 or 43. [Brief explanation of the drawings]

[0028] [Figure 1]FIG. 1 shows a schematic representation of the T-DNA region of a TMV-based vector for the expression of klebicins. RB: right T-DNA border, Act2: Arabidopsis actin promoter, RdRp: RNA-dependent RNA polymerase, 3'NTR: 3' untranslated region, T: nos terminator, LB: left T-DNA border, KpneM-cat1: coding sequence of klebicin KpneM (K. pneumoniae EWD35590.1) with the first intron of the catalase gene (cat-1) from castor bean (Ricinus communis), KpneM2: coding sequence of klebicin KpneM2 (Klebsiella sp. WP_047066220), KvarM: coding sequence of klebicin KvarM (K. variicola CTQ17225.1), KaerM: coding sequence of klebicin KaerM (K. aerogenes WP_015367360.1), KpneA: klebicin KpneA (K. pneumoniae SAV78255.1) coding sequence, KaerA: coding sequence for the klebicin KaerA (K. aerogenes WP_063414841.1), KoxyY: coding sequence for the klebicin KoxyY (K. oxytoca WP_024273778), KvarIa: coding sequence for the klebicin KvarIa (K. variicola KDL88409), KpneIa: coding sequence for the klebicin KpneIa (K. pneumoniae BAS34675). [Figure 2]Figure 2 shows SDS-PAGE analysis of klevicin expression in N. benthamiana leaves. Plant material (50 mg) 5 or 7 days post-spray (dps) (pooled three leaf samples, klevicin KaerA at 4 dps, and all remaining klevicins at 5 dps) was harvested, crushed in liquid nitrogen, extracted with 50 mM Tris-HCl, 300 mM NaCl, 15 mM sodium acetate, and 3 mM DTT (pH 7.5), and denatured at 98°C for 10 min. A solution containing 5 μg of protein was separated on a 12% polyacrylamide gel and Coomassie stained. M: PageRulerPrestained Protein Ladder (ThermoFisher Scientific Baltics); WT: crude extract from unsprayed N. benthamiana leaves; KvarIa, KpneIa, KpneA, KaerA, KoxyY, KpneM, KpneM2, KvarA, KaerM: extract from N. benthamiana leaves sprayed with klebicin expression constructs. The band corresponding to the recombinant klebicin is indicated by an asterisk. [Figure 3A]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3B]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3C]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3D]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3E]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3F]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3G]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3H]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3I]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3J]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3K]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 3L]Figure 3 illustrates the purification of klevicins from Nicotiana benthamiana leaf biomass. A, C, E, G, I, K: Purification scheme for KpneM (A), KpneM2 (C), KvarM (E), KpneA (G), KaerA (I), and KvarIa (K). B, D, F, H, J, L: SDS-PAGE analysis of protein samples obtained from different purification steps for KpneM (B), KpneM2 (D), KvarM (F), KpneA (H), KaerA (J), and KvarIa (L). Solutions containing 5 μg of protein were separated on a 12% polyacrylamide gel and stained with Coomassie. B: Lanes 1 and 7—PageRuler™ Prestained protein ladder; lane 2—crude extract; lane 3—total soluble protein loaded onto Phenyl Sepharose; lane 4—Phenyl Sepharose flow-through; lane 5—KpneM eluate (post-Phenyl Sepharose); lane 6—impurity eluate (post-Phenyl Sepharose); lane 8—protein loaded onto Q Sepharose; lane 9—KpneM flow-through on Q Sepharose; lane 10—impurity eluate (post-Q Sepharose); D: Lanes 1 and 6—PageRuler™ Prestained protein ladder; lane 2—total soluble protein loaded onto Phenyl Sepharose; lane 3—Phenyl Sepharose flow-through; lane 4—KpneM2 eluate (after Phenyl Sepharose), lane 5 - impurity eluate (after Phenyl Sepharose), lane 7 - protein loaded onto Q Sepharose, lane 8 - KpneM2 flow-through from Q Sepharose, lane 9 - impurity eluate (after Q Sepharose), F: Lanes 1 and 7 - PageRuler™ Prestained protein ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarM eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded onto Q Sepharose, lane 9 - KvarM flow-through from Q Sepharose, lane 10 - impurity eluate (after Q Sepharose),H: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KpneA eluate (after Phenyl Sepharose), lane 6 - impurity eluate (after Phenyl Sepharose), lane 8 - protein loaded on SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KpneA eluate (after SP Sepharose). J: Lanes 1 and 6 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded on SP Sepharose, lane 4 - SP Sepharose flow-through, lane 5 - KaerA eluate (after SP Sepharose), lane 7 - protein loaded on Q Sepharose, lane 8 - KaerA flow-through on Q Sepharose. L: Lanes 1 and 7 - PageRuler™ Prestained Protein Ladder, lane 2 - crude extract, lane 3 - total soluble protein loaded onto Phenyl Sepharose, lane 4 - Phenyl Sepharose flow-through, lane 5 - KvarIa eluate (post-Phenyl Sepharose), lane 6 - impurity eluate (post-Phenyl Sepharose), lane 8 - protein loaded onto SP Sepharose, lane 9 - SP Sepharose flow-through, lane 10 - KvarIa eluate (post-SP Sepharose). Arrows indicate recombinant proteins. [Figure 4] Figure 4 shows purified klebicin on a single gel. 0.5 μg of purified klebicin was separated on a 12% SDS-PAGE gel and Coomassie stained. M: PageRuler Unstained Protein Ladder (ThermoFisher Scientific Baltics). [Figure 5]Figure 5 illustrates the evaluation of klevicin activity against Klebsiella strains in a soft agar overlay assay. Overnight bacterial cultures were grown in CAA medium, homogenized to OD595 = 1.0, diluted 100-fold with molten top CAA agar, and poured onto CAA agar plates. A 20 μL drop of crude protein extract was placed on a 6 mm Whatman disk, and the Petri plate was incubated overnight at 30°C or 37°C. [Figure 6] Figure 6 shows the susceptibility of clinical Klebsiella isolates to six plant-expressed klebicins. One hundred clinical Klebsiella isolates (89 K. pneumoniae and 11 K. oxytoca) were tested in the plate drop assay. Strains susceptible to each klebicin are classified by the size of the zone of inhibition. [Figure 7] Figure 7 shows the cytotoxicity assay of klevicins in liquid culture. Overnight cultures of Klebsiella were diluted to OD600 = 0.3 in CAA medium and treated with 5 μg mL of either klevicin, and the bacteria were further cultured for 5 hours with shaking (200 rpm). The antibacterial activity of the klevicins was assessed by counting the colony-forming units of the tested cultures. Bars represent standard deviations. [Figure 8] Figure 8 shows the activity of klevicin against biofilms. Biofilms of K. quasipneumoniae, K. oxytoca, K. variicola, and K. aerogenes grown in CAA medium for 1 day were treated with 5 μg mL of either klevicin. The antibacterial activity of klevicin was assessed by counting the colony-forming units of the tested cultures. Bars represent standard deviation. [Figure 9]Figure 9 shows the impact of KvarIa treatment on the survival of Galleria mellonella (Galleria mellonella) larvae after challenge with K. quasipneumoniae DSM 28212. G. mellonella larvae were infected with 12,000-32,000 CFU of K. pneumoniae DSM 28212 and treated with 10 μg of KvarIa 2 hours post-infection. Larvae were incubated in Petri dishes at 37°C for up to 68 hours. Twenty larvae were used for each treatment time point. [Figure 10A] Figure 10 shows the predicted consensus sequences shown in SEQ ID NOS: 22 to 24. The "one-letter code" refers to the 20 standard amino acids, "X" indicates insufficient conservation at the respective positions to predict a consensus amino acid, and "-" indicates an amino acid omitted in the predicted consensus sequence due to low conservation. J represents L (leucine) or I (isoleucine). Highly conserved amino acids are highlighted in black, and moderately conserved amino acids are highlighted in gray. The consensus sequences were predicted using the software "Geneious Prime Clustal W" with standard settings. Figure 10A shows the predicted sequence of SEQ ID NOS: 22. Figure 10B shows the predicted sequence of SEQ ID NOS: 23. Figure 10C shows the predicted sequence of SEQ ID NOS: 24. [Figure 10B] Figure 10 shows the predicted consensus sequences shown in SEQ ID NOS: 22 to 24. The "one-letter code" refers to the 20 standard amino acids, "X" indicates insufficient conservation at the respective positions to predict a consensus amino acid, and "-" indicates an amino acid omitted in the predicted consensus sequence due to low conservation. J represents L (leucine) or I (isoleucine). Highly conserved amino acids are highlighted in black, and moderately conserved amino acids are highlighted in gray. The consensus sequences were predicted using the software "Geneious Prime Clustal W" with standard settings. Figure 10A shows the predicted sequence of SEQ ID NOS: 22. Figure 10B shows the predicted sequence of SEQ ID NOS: 23. Figure 10C shows the predicted sequence of SEQ ID NOS: 24. [Figure 11A]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11B]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11C]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11D]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11E]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11F]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11G]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11H]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 11I]Figure 11 shows the assessment of the activity and concentrations of the klebicins KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of the klebicins against susceptible bacteria was assessed in liquid culture or by radial diffusion assay. Antibacterial activity was expressed as CFU / mL Δlog10 when the activity was assessed in liquid culture, or as specific activity units (AU) when the radial diffusion assay was used for assessment. Klebicin concentrations were measured by Bradford assay. Data are the mean ± SD of three independent experiments. (A) KvarIa activity of klebicins KpneM and KpneM2 after storage at -20°C. (B) KvarIa activity of klebicins KpneM and KpneM2 after storage at 5°C. (C) KvarIa activity of klebicins KpneM and KpneM2 after storage at room temperature. (D) Activity of klevicins KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of klevicins KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of klevicins KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of klevicin concentration after storage at -20°C. (H) Trend line of klevicin concentration after storage at 5°C. (I) Trend line of klevicin concentration after storage at room temperature. [Figure 12A]Figure 12 shows the residual activity of KvarIa and Eudragit S100-coated KvarIa after in vitro gastric digestion in a soft agar overlay assay, as well as SDS-PAGE analysis of pepsin-digested KvarIa fragments. (A) Evaluation of the residual activity of KvarIa and Eudragit S100-coated KvarIa after simulated in vitro gastric digestion in a soft agar overlay assay. Protein samples were digested with pepsin for 0.5, 5, 10, 20, 30, and 60 minutes (pepsin:protein ratio = 1:40). Dilutions of all samples were prepared in a 1:2 ratio with distilled water, and 5 μL aliquots of the diluted samples were spotted onto MHA plates containing a lawn of K. quasipneumoniae DSM28212. (B) Tricine SDS-PAGE analysis of KvarIa digestion. Coomassie staining was used to visualize protein degradation and estimate the MW of the peptide products. The presence or absence of pepsin and KvarIa is indicated. Times are shown in minutes and correspond to (A). [Figure 12B]Figure 12 shows the residual activity of KvarIa and Eudragit S100-coated KvarIa after in vitro gastric digestion in a soft agar overlay assay, as well as SDS-PAGE analysis of pepsin-digested KvarIa fragments. (A) Evaluation of the residual activity of KvarIa and Eudragit S100-coated KvarIa after simulated in vitro gastric digestion in a soft agar overlay assay. Protein samples were digested with pepsin for 0.5, 5, 10, 20, 30, and 60 minutes (pepsin:protein ratio = 1:40). Dilutions of all samples were prepared in a 1:2 ratio with distilled water, and 5 μL aliquots of the diluted samples were spotted onto MHA plates containing a lawn of K. quasipneumoniae DSM28212. (B) Tricine SDS-PAGE analysis of KvarIa digestion. Coomassie staining was used to visualize protein degradation and estimate the MW of the peptide products. The presence or absence of pepsin and KvarIa is indicated. Times are shown in minutes and correspond to (A). [Figure 13] FIG. 13 shows a standard curve for the detection of K. quassipneumoniae obtained by real-time PCR based on the amplification of the khe gene. [Figure 14] The real-time PCR results in Figure 14 show that K. quasipneumoniae colonized mouse fecal samples before and after klebicin treatment. Fecal samples from three mice were used for each experimental time point. 18d: Fecal samples were collected on day 18 of the experiment, before the start of klebicin treatment; 22d: Fecal samples were collected on day 22 of the experiment (the day after the last klebicin oral administration). DETAILED DESCRIPTION OF THE INVENTION

[0029] The present inventors have identified proteins that have bactericidal or bacteriostatic activity against Klebsiella. Such proteins are referred to herein as "klevicins." Preferably, the proteins or klevicins of the present invention have lipid II cleavage activity or pore-forming activity in bacterial cell membranes. The proteins or klebicins of the present invention broadly comprise at least two amino acid sequence segments (sometimes simply referred to herein as "segments"). As used herein, an amino acid sequence segment refers to a number of adjacent amino acid residues in the primary structure of a protein or polypeptide, where the protein or polypeptide has a greater number of amino acid residues in its primary structure than the segment. The proteins of the present invention broadly comprise or consist of a first segment and a second segment. The first segment broadly provides the protein with the ability to bind to a component of Klebsiella cells (e.g., bind to a receptor) and / or it provides the protein with the ability to be introduced or internalized (translocated intracellularly) by Klebsiella cells. The second segment may have lipid II cleavage activity or the ability to form pores in the bacterial cell membrane. Thus, the second segment provides the protein with its cytotoxic activity. In one embodiment, the first segment is located at the N-terminus of the primary structure of the protein, and the second segment is located at the C-terminus, or vice versa, with the former being preferred. Thus, the protein of the present invention may comprise or consist of an N-terminal first segment and a C-terminal second segment. In another embodiment, the second segment is located at the N-terminus of the primary structure of the protein, and the first segment is located at the C-terminus. Thus, the protein of the present invention may comprise or consist of an N-terminal second segment and a C-terminal first segment.

[0030] First segment of the protein of the invention The protein of the present invention may comprise a first segment comprising any one of the amino acid sequences of items (Ai) to (A-ix) defined above. The amino acid sequences of SEQ ID NOs: 1 to 9 detailed in these items are the amino acid sequences of klevicins identified by the present inventors. Preferably, the amino acid sequence of the first segment is any one of the amino acid sequences of items (Ai) to (A-ix) defined above.

[0031] However, the present invention is not limited to klevicins having the specific klevicin first segment identified by the present inventors. Instead of the amino acid sequences of items (Ai) to (A-ix), the first segment may each comprise any one of the amino acid sequences of items (Bi) to (B-ix) defined above. The phrase "the first segment comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence from amino acid residue x to y of SEQ ID NO: Z" (x and y refer to the start and end sites, and Z refers to the number of the SEQ ID NO) means that the amino acid sequence of the first segment preferably has at least the same number of amino acid residues as the sequence from amino acid residue x to y of SEQ ID NO: Z and has the sequence identity indicated above over at least the entire length from residue x to y of SEQ ID NO: Z. This principle applies to all of the identities of items (Bi) to (B-ix) and other sequences defined herein. Herein, sequence identity is determined using Clustal Omega (CLUSTAL O 1.2.4) based on standard parameters. Preferably, the amino acid sequence of the first segment is any one of items (Bi) to (B-ix). The phrase "the amino acid sequence of the first segment has at least 70% sequence identity with the amino acid sequence from amino acid residue x to y of SEQ ID NO: Z" means that the amino acid sequence of the first segment has at least the same number of amino acid residues as the sequence from amino acid residue x to y of SEQ ID NO: Z, and has the sequence identity shown above over the entire length from residue x to y of SEQ ID NO: Z. This applies to items (Bi) to (B-ix) and all other sequence identities defined herein.

[0032] In another embodiment, the first segment comprises the amino acid sequence of any one of items (Ci) to (C-ix) defined above. Preferably, the amino acid sequence of the first segment is defined as any one of items (Ci) to (C-ix) defined above. The definitions of items (Ci) to (C-ix) mean that the amino acid sequence is that of the indicated amino acid residue range of the indicated SEQ ID NO:, except for the indicated number of substitutions, additions, insertions and / or deletions. When a protein is defined herein by a specified number or range of amino acid substitutions, additions, insertions, and / or deletions, these amino acid substitutions, additions, insertions, or deletions may be combined, but the specified number or range refers to the sum of all amino acid substitutions, additions, insertions, and deletions. Among amino acid substitutions, additions, insertions, and deletions, amino acid substitutions, additions, and deletions are preferred. The term "insertion" refers to an insertion into the amino acid sequence of a reference sequence (i.e., excluding additions to the C-terminus or N-terminus). The term "addition" refers to an addition to the C-terminus or N-terminus of the amino acid sequence of a reference sequence. Deletions can be deletions of amino acid residues at the terminal or within the reference sequence. The term "reference sequence" is used herein to refer to the amino acid sequence in the sequence listing that defines the amino acid sequence of the protein of the present invention. For example, in item (Ai), the reference sequence is SEQ ID NO: 1.

[0033] In item (B), any one of the sequence identities may be at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 97%. In item (C), the number of said amino acid substitutions, additions, insertions, and / or deletions is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5, compared to any one of the said amino acid sequences.

[0034] Therefore, the following items (i) to (ix) of each of items (B) and (C) define preferred embodiments of the first segment of the protein of the present invention. The first segment of the protein of the present invention preferably has any of the following amino acid sequences: (Bi) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (B-ii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (B-iii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (B-iv) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Bv) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (B-vi) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (B-vii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (B-viii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8, or (B-ix) an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9; or, (Ci) has 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 1 to 128 of SEQ ID NO: 1; (C-ii) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 1 to 127 of SEQ ID NO: 2; (C-iii) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 1 to 123 of SEQ ID NO: 3; (C-iv) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 1 to 118 of SEQ ID NO: 4; (Cv) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 1 to 170 of SEQ ID NO: 5; (C-vi) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 1 to 172 of SEQ ID NO: 6; (C-vii) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 1 to 255 of SEQ ID NO: 7; (C-viii) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 1 to 288 of SEQ ID NO: 8, or (C-ix) An amino acid sequence having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 1 to 236 of SEQ ID NO: 9.

[0035] In another embodiment, the amino acid sequence of the first segment of the protein of the present invention preferably has the following: (Bi) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (B-ii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (B-iii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (B-iv) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Bv) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (B-vi) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (B-vii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (B-viii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8; or (B-ix) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity with the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9; or, (Ci) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (C-ii) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (C-iii) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (C-iv) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Cv) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (C-vi) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (C-vii) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (C-viii) substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8, or (C-ix) Substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9.

[0036] The second segment of the protein of the invention Because the protein (klevicin) of the present invention has both a first and a second segment, the protein can be defined by both its first and second segments. Thus, the protein can be defined by a combination of any one of the first segments described above and a second segment defined herein. The second segment of the protein of the invention provides the protein with its bactericidal or bacteriostatic activity against Klebsiella. Preferably, the second segment has lipid II cleavage activity or pore-forming ability in the bacterial cell membrane. Such activities are known from other bacteriostatic or bactericidal bacterial proteins (e.g., colicins from E. coli).

[0037] The second segment may comprise or consist of any one of the amino acid sequences of items (Di) to (D-ix) defined above. Preferably, the amino acid sequence of the first segment is an amino acid sequence of items (Di) to (D-ix) defined above. When the definition of the first segment is combined with that of the second segment, preferably, the first and second segments defined based on the same SEQ ID NO: as the reference sequence are combined. For example, a protein may be defined by having a first segment based on item (A-ii) and a second segment based on item (D-ii).

[0038] However, the present invention is not limited to klevicins having the specific klevicin second segment identified by the present inventors. Instead of the amino acid sequence of items (Di) to (D-ix), the second segment can comprise or consist of the amino acid sequence of any one of items (Ei) to (Eix). Preferably, the amino acid sequence of the second segment is that of any one of items (Ei) to (Eix). In another embodiment, the second segment may comprise or consist of the amino acid sequence of any one of items (Fi) to (F-ix) defined above. Preferably, the amino acid sequence of the first segment is the amino acid sequence of any one of items (Fi) to (F-ix) defined above.

[0039] The following items (i) to (ix) of each of items (E) and (F) define preferred second segments. In one embodiment, the second segment of the protein of the invention preferably has the following amino acid sequence: (Ei) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 129 to 278 of SEQ ID NO: 1; (E-ii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 128 to 276 of SEQ ID NO: 2; (E-iii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 124 to 272 of SEQ ID NO: 3; (E-iv) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 119 to 266 of SEQ ID NO: 4; (Ev) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 171 to 377 of SEQ ID NO: 5; (E-vi) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 173 to 379 of SEQ ID NO: 6; (E-vii) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 256 to 452 of SEQ ID NO: 7; (E-viii) has at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 289 to 466 of SEQ ID NO: 8, or (E-ix) having at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 237 to 414 of SEQ ID NO: 9; or, (Fi) has 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 129 to 278 of SEQ ID NO: 1; (F-ii) having 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 128 to 276 of SEQ ID NO: 2; (F-iii) having 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 124 to 272 of SEQ ID NO: 3; (F-iv) having 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 119 to 266 of SEQ ID NO: 4; (Fv) has 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 171 to 377 of SEQ ID NO: 5; (F-vi) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 173 to 379 of SEQ ID NO: 6; (F-vii) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 256 to 452 of SEQ ID NO: 7; (F-viii) having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 289 to 466 of SEQ ID NO: 8, or (F-ix) Includes an amino acid sequence having 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acids 237 to 414 of SEQ ID NO: 9.

[0040] In another embodiment, the amino acid sequence of the second segment of the protein of the invention preferably has the following: (Ei) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 129 to 278 of SEQ ID NO: 1; (E-ii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 128 to 276 of SEQ ID NO: 2; (E-iii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 124 to 272 of SEQ ID NO: 3; (E-iv) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 119 to 266 of SEQ ID NO: 4; (Ev) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 171 to 377 of SEQ ID NO: 5; (E-vi) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 173 to 379 of SEQ ID NO: 6; (E-vii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 256 to 452 of SEQ ID NO: 7; (E-viii) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 289 to 466 of SEQ ID NO: 8; (E-ix) at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a segment from amino acid residues 237 to 414 of SEQ ID NO: 9; or, (Fi) substitution, addition, insertion and / or deletion of 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 129 to 278 of SEQ ID NO: 1; (F-ii) substitution, addition, insertion, and / or deletion of 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 128 to 276 of SEQ ID NO: 2; (F-iii) substitution, addition, insertion, and / or deletion of 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 124 to 272 of SEQ ID NO: 3; (F-iv) substitution, addition, insertion and / or deletion of 1 to 20, preferably 1 to 15, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 119 to 266 of SEQ ID NO: 4; (Fv) 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions, and / or deletions compared to the amino acid sequence of amino acids 171 to 377 of SEQ ID NO: 5; (F-vi) substitution, addition, insertion, and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 173 to 379 of SEQ ID NO: 6; (F-vii) substitution, addition, insertion, and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 256 to 452 of SEQ ID NO: 7; (F-viii) substitution, addition, insertion, and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 289 to 466 of SEQ ID NO: 8; (F-ix) Substitution, addition, insertion and / or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acids compared to the amino acid sequence of amino acids 237 to 414 of SEQ ID NO: 9.

[0041] In the present invention, any of the above-mentioned first segments can be combined with any of the second segments to form a protein of the present invention. In one embodiment, a protein of the present invention comprises a first segment of any one of items (i) to (iv) and a second segment of any one of items (i) to (iv), regardless of whether the first segment belongs to item (A), (B), or (C), and whether the second segment belongs to item (D), (E), or (F). However, in one embodiment, a protein of the present invention comprises a first segment of any one of items (Ci) to (C-iv) and a second segment of any one of items (Fi) to (F-iv). In another embodiment, the protein of the invention comprises a first segment of any one of items (v) to (ix) and a second segment of any one of items (v) to (ix), regardless of whether the first segment belongs to item (A), (B) or (C) and whether the second segment belongs to item (D), (E) or (F). However, in one embodiment, the protein of the invention comprises a first segment of any one of items (Cv) to (C-ix) and a second segment of any one of items (Fv) to (F-ix).

[0042] Other embodiments are as follows: In one embodiment, the protein of the present invention, in its broadest and most preferred embodiment, may have a first segment comprising or consisting of any of the amino acids of items (Ai) to (A-iv), (Bi) to (B-iv), or (Ci) to (C-iv), and a second segment comprising or consisting of any one of the amino acid sequences of items (Di) to (D-iv), (Ei) to (Eiv), or (Fi) to (F-iv). Preferably, the first segment comprises or consists of any one of the amino acid sequences of items (Ai) to (A-iii), (Bi) to (B-iii), or (Ci) to (C-iii), and the second segment comprises any one of the amino acids of (Di) to (D-iii), (Ei) to (Eiii), or (Fi) to (F-iii). More preferably, the first segment comprises the amino acid sequence of any one of (A-ii), (B-ii) or (C-ii), and the second segment comprises the amino acid sequence of any one of (D-ii), (E-ii) or (F-ii).More preferably, the first segment comprises the amino acid sequence of (A-ii), and the second segment comprises the amino acid sequence of (D-ii).

[0043] In another embodiment, the first segment comprises the amino acid sequence of any one of items (Av) to (A-ix), (Bv) to (B-ix), or (Cv) to (C-ix), and the second segment comprises the amino acid sequence of any one of items (Dv) to (D-ix), (Ev) to (Eix), or (Fv) to (F-ix). In a preferred embodiment, the first segment comprises the amino acid sequence of any one of items (Av), (A-vi), (Bv), (B-vi), (Cv) and (C-vi), and the second segment comprises the amino acid sequence of any one of items (Dv), (D-vi), (Ev), (Evi), (Fv) and (F-vi). In a more preferred embodiment, the first segment comprises or consists of the amino acid sequence of item (Av) or (A-vi) and the second segment comprises or consists of the amino acid sequence of item (Dv) or (D-vi). In another preferred embodiment, the first segment comprises or consists of the amino acid sequence of item (A-vii) and the second segment comprises or consists of the amino acid sequence of item (D-vii).

[0044] In a further alternative embodiment, the first segment comprises the amino acid sequence of any one of items (A-viii), (A-ix), (B-viii), (B-ix), (C-viii) or (C-ix) and the second segment comprises the amino acid sequence of any one of items (D-viii), (D-ix), (Eviii), (Eix), (F-viii) or (F-ix). Preferably, the first segment comprises or consists of the amino acid sequence of item (A-viii) or (A-ix) and the second segment comprises or consists of the amino acid sequence of item (D-viii) or (D-ix).

[0045] Further preferred embodiments of the proteins of the present invention are as defined in paragraphs (ai) to (a-ix), (bi) to (b-ix), and (ci) to (c-ix) above. In paragraph (b), "an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: Z" (where Z represents the number of a SEQ ID NO) means that the amino acid sequence has at least the same number of amino acid residues as the sequence of SEQ ID NO: Z and has the indicated sequence identity over at least the entire length of SEQ ID NO: Z. This applies to all paragraphs (bi) to (b-ix) and to corresponding sequence identities defined herein. The definitions in paragraphs (ci) to (c-ix) mean that the amino acid sequence is that of the entire amino acid sequence of the indicated SEQ ID NO, except for the indicated number of substitutions, additions, insertions, or deletions.

[0046] Alternatively, the amino acid sequence of the protein of the present invention can be defined as follows: (b') In one embodiment, the amino acid sequence of the protein of the invention is (b'-i) is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 1; (b'-ii) is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 2; (b'-iii) is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 3; (b'-iv) is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 4; (b'-v) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 5; (b'-vi) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 6; (b'-vii) is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 7; (b'-viii) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 8; or (b'-ix) is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to the amino acid sequence of SEQ ID NO: 9; (b'') In one embodiment, the amino acid sequence of the protein of the invention is (b''-i) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1; (b''-ii) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b''-iii) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3; (b''-iv) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4; (b''-v) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5; (b''-vi) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6; (b''-vii) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 7; (b''-viii) at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 8; or (b''-ix) shares at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:9.

[0047] Cytotoxic activity of the protein of the present invention The klevicins of the present invention can exert a cytotoxic effect against Klebsiella bacteria (e.g., K. pneumoniae, K. granulomatis, K. oxytoca, K. aerogenes, K. quasipneumoniae, and K. variicola). Preferably, the klevicins of the present invention are active against K. pneumoniae and K. oxytoca. K. pneumoniae is most preferred. The cytotoxic effect can be a bacteriostatic or bactericidal effect. Whether the protein has a cytotoxic effect can be experimentally tested, for example, using the assay of Example 4. In one embodiment, the Klebsiella has antibiotic resistance, such as carbapenem resistance, and the protein is a protein selected from items (i) to (iv), (vii), and (viii), preferably any of embodiments (i) to (iv) defined above. The protein selected from items (i) to (iv), (vii), and (viii) is a protein defined using SEQ ID NO: 1 to 4, 7, or 8 as a reference sequence.

[0048] The proteins of the present invention may have pore-forming activity in the cell membrane of Klebsiella cells. In the present invention, the proteins of items (av) to (a-ix) of SEQ ID NOs: 5 to 9, and their respective derivatives of items (bv) to (b-ix) and (cv) to (c-ix), have pore-forming activity. It is assumed that the pore-forming activity is due to the presence of a second amino acid sequence segment in these proteins.

[0049] Another class of proteins of the present invention is hypothesized to have lipid II cleavage activity, by analogy with the activity of E. coli colicin M. We hypothesize that this class of proteins has peptidoglycanase activity that specifically cleaves the bond between the lipid moiety and the pyrophosphoryl group of peptidoglycan lipid I and lipid II intermediates (located on the periplasmic side of the inner membrane), as observed in E. coli colicin M (Gross and Braun, Mol. Gen. Genet. 251 (1996) 388-396; Barreteau et al., Microbial Drug Resistance 18 (2012), 222-229). The released C55 polyisoprenols no longer allow translocation of MurNAc-pentapeptide-GlcNAc through the cell membrane. These klebicins are therefore hypothesized to exert toxicity on Klebsiella cells after they are imported into the periplasm through the outer cell wall. This property of the proteins of the invention can be analyzed according to a standard assay for colicin M activity using lipid I as a substrate, as described in El Ghachi et al., J. Biol. Chem. 281 (2006) 22761-22772. According to the present invention, the proteins of items (ai) to (a-iv) of SEQ ID NOs: 1 to 4, as well as their respective derivatives of items (bi) to (b-iv) and (ci) to (c-iv), have peptidoglycanase or lipid II cleavage activity. This activity is assumed to be due to the presence of a second amino acid sequence segment in these proteins.

[0050] The cytotoxic activity of the protein of the present invention and a reference protein having the amino acid sequence of SEQ ID NO: 1 is preferably such that, when 20 μL of each solution of the protein and the reference protein is spotted onto a lawn of susceptible Klebsiella pneumoniae subsp. similipneumoniae SB30 (DSM 28212) on an agar plate and the agar plate is then incubated at 37° C. for 16 hours, a spot of at least the same diameter is produced that does not contain viable bacteria of the susceptible Klebsiella strain, and the concentration of the protein in the solution is up to 5 times that of the solution of the reference protein. The solution is an aqueous solution. This test can be performed as described in Reference Example 1. As described in Reference Example 1, the protein concentration is measured as weight per volume.

[0051] In one embodiment, the protein of the present invention is any one of items (bi) to (b-ix) or (ci) to (c-ix) above, and the protein and a comparison protein having the amino acid sequence of the reference sequence of the SEQ ID NO: in items (bi) to (b-ix) or (ci) to (c-ix) have cytotoxic activity such that, when 20 μL of each solution of the protein and the comparison protein is spotted onto a lawn of susceptible Klebsiella pneumoniae subsp. similipneumoniae SB30 (DSM 28212) on an agar plate and the agar plate is then incubated at 37° C. for 16 hours, a spot of at least the same diameter is produced that does not contain viable bacteria of the susceptible Klebsiella strain, and the concentration of the protein in the solution is up to five times that of a solution of the comparison protein. The solution is an aqueous solution. This test can be performed as described in Reference Example 1. As also described in Reference Example 1, the protein concentration is measured as weight per volume.

[0052] Consensus sequence of the protein of the present invention The protein of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 22 to 24, wherein each X represents any one of the 20 standard amino acid residues or the absence of an amino acid residue, and J represents L (leucine) or I (isoleucine). Preferably, X is any one of the 20 standard amino acid residues, and J represents either L (leucine) or I (isoleucine). The 20 standard amino acid residues are as follows: A (alanine), C (cysteine), D (aspartic acid), E (glutamic acid), F (phenylalanine), G (glycine), H (histidine), I (isoleucine), K (lysine), L (leucine), M (methionine), N (asparagine), P (proline), Q (glutamine), R (arginine), S (serine), T (threonine), V (valine), W (tryptophan), and Y (tyrosine).

[0053] In a preferred embodiment, the cytotoxic activity of the protein is preferably such that the protein and a reference protein having the amino acid sequence of SEQ ID NO: 1 produce a spot of at least the same diameter that does not contain viable bacteria of the susceptible Klebsiella strain when 20 μL of each solution of the protein and the reference protein are spotted onto a lawn of susceptible Klebsiella pneumoniae subsp. similipneumoniae SB30 (DSM 28212) on an agar plate and the agar plate is then incubated at 37° C. for 16 hours, and the concentration of the protein in the solution is up to 5 times that of a solution of the reference protein. The cytotoxic activity assay is performed as described in Reference Example 1. In proteins of the present invention that exhibit a similar mode of action against Klebsiella, the amino acid sequences of the first and second segments preferably have conserved positional relationships and / or amino acid sequence stretches. The conserved positional relationships and / or amino acid sequence stretches are more likely to be important for the function of the proteins of the present invention. In the first amino acid sequence segment, the conserved positional relationships or stretches are usually associated with receptor binding and translocation functions. In the second amino acid sequence segment, the conserved positional relationships or stretches are usually associated with cytotoxicity against Klebsiella. The klebicins KpneM2, KvarM, KpneM, and KaerM share the consensus sequence of SEQ ID NO: 22. The klebicins KaerA and KpneA share the consensus sequence of SEQ ID NO: 23. The klebicins KpneIa and KvarIa share the consensus sequence of SEQ ID NO: 24. Each X in SEQ ID NOs: 22, 23 and 24 represents any one of the 20 standard amino acid residues or the absence of an amino acid residue at this position, and J represents L (leucine) or I (isoleucine).

[0054] Specifically, klevicins having lipid II cleavage activity (e.g., those defined with respect to SEQ ID NOs: 1-4) preferably have amino acid residues or amino acid sequence stretches corresponding to the following amino acid residues or amino acid sequence stretches: 1M, 2S / T, 3D / E, 4T, 5L / M, 7V, 9A, 22G, 24G, 50S, 91T, 97P, 132P, 138H, 139Y, 142G, 144G, 155G, 156L, 176G, 184F, 199L, 200G, 202I, 203T, 206TEGTL210 (SEQ ID NO: 25), 212I, 216G, 218W, 220YNGV223 (SEQ ID NO: 26), 225RAFNDTYD232 (SEQ ID NO: 27), 234N, 239R, 243A, 247T, 255G, 258Y, 260I, 262P, 262G, 271S, 272G; In the sequences, the number before the one-letter code of an amino acid residue indicates its position in SEQ ID NO: 22, and the number after the one-letter code of an amino acid residue indicates the position of the preceding amino acid residue in SEQ ID NO: 22 in the case of a stretch of two or more amino acid residues. Two or more amino acid residues separated by " / " means that any one of the indicated residues is present at the position corresponding to the indicated position in SEQ ID NO: 22. The expression "amino acid residue or amino acid sequence stretch corresponding to ..." means that the position in a given protein may differ from the position in SEQ ID NO: 22. However, the corresponding position can be determined by aligning the amino acid sequence of the protein with those of SEQ ID NOs: 1-4 and 22 (as shown in Figure 10A) and determining the corresponding position by counting the residues in the amino acid sequence of the protein starting from the first amino acid at the N-terminus.

[0055] Klevicins with pore-forming activity, as defined with respect to SEQ ID NOs: 5 and 6, preferably have amino acid residues or amino acid sequence stretches corresponding to the following amino acid residues or amino acid sequence stretches: 1M, 4E, 9V, 11G, 13N, 18V, 20WGG22, 25GNGNNGGAG33 (SEQ ID NO: 28), 36G, 39G, 45G, 47T, 52L, 65P, 67N, 68P, 72GAPW75 (SEQ ID NO: 29), 80S, 82K, 84A, 90AN91, 94KP95, 97KFKANIQN 104 (SEQ ID NO: 30), 106K, 111GSL113, 115SP116, 188V, 120KS121, 123SSGDVDTY130 (SEQ ID NO: 31), 132VSFGKEKYNV141 (SEQ ID NO: 32), 143YNRKKDSFT151 (SEQ ID NO: 33), 154YVDGGA159 (SEQ ID NO: 34), 161KPEHSMKDQAIAVV174 (SEQ ID NO: 35), 176LYLLNE181 (SEQ ID NO: 36), 186VI187, 189T, 193II194, 197SG198, 200T, 202SGKLG206 (SEQ ID NO: 37), 208KY209, 212LA213, 217A, 220I, 222NFQGKK227 (SEQ ID NO: 38), 229RSF231, 233DAM235, 237S, 244NP245, 247MKL249, 251QADK254 (SEQ ID NO: 39), 259NAL261, 263Q, 266LS267, 269LADRFKGL277 (SEQ ID NO: 40), 279AFTW282 (SEQ ID NO: 41), 284DRLLKA289 (SEQ ID NO: 42), 291KI292, 2 94DGVVTGVTTG303 (SEQ ID NO: 43), 305WQ306, 308LA309, 311EVEAMYLSGVAG322 (SEQ ID NO: 44), 324VALGI328 (SEQ ID NO: 45), 330T, 332MIS334, 337A, 341S, 343P, 346AV, 349ALTV352 (SEQ ID NO: 46), 354AVIGI358 (SEQ ID NO: 47), 360I, 362TSYI365 (SEQ ID NO: 48), 367AD368, 370AKALNNAV377 (SEQ ID NO: 49), 380LFK382; In the sequences, the number preceding the one-letter code of an amino acid residue indicates its position in SEQ ID NO: 23, and the number following the one-letter code of an amino acid residue indicates the position of the preceding amino acid residue in SEQ ID NO: 23 in the case of a stretch of two or more amino acid residues. Two or more amino acid residues separated by a " / " means that any one of the indicated residues is present at the position corresponding to the indicated position in SEQ ID NO: 23. The expression "amino acid residue or amino acid sequence stretch corresponding to ..." means that the position in a given protein may differ from the position in SEQ ID NO: 23. However, the corresponding position can be determined by aligning the amino acid sequence of the protein with those of SEQ ID NOs: 5-6 and 23 (as shown in Figure 10B) and counting the residues in the amino acid sequence of the protein starting from the first amino acid at the N-terminus to determine the corresponding position. SEQ ID NO: 23 is as shown at the end of this specification.

[0056] Klevicins with pore-forming activity, as defined with respect to SEQ ID NOs: 8 and 9, preferably have amino acid residues or amino acid sequence stretches corresponding to the following amino acid residues or amino acid sequence stretches: 1MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPD (SEQ ID NO: 50), 121K / R, 122TTTTENVGGGKNGHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAK (SEQ ID NO: 51), 23 8A, 239E / A, 240AKAKAEAEAKAKAEA254 (SEQ ID NO: 52), 255A / E, 256AKAKAEA262 (SEQ ID NO: 53), 263E / A, 264AKAKAEAEAKAKAEAEAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKINAQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWR405 (SEQ ID NO: 54), 406S / T, 407FFVKIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI466 (SEQ ID NO: 55); In the sequences, the number before the one-letter code of an amino acid residue indicates its position in SEQ ID NO: 24, and the number after the one-letter code of an amino acid residue indicates the position of the preceding amino acid residue in SEQ ID NO: 24 in the case of a stretch of two or more amino acid residues. Two or more amino acid residues separated by a " / " means that any one of the indicated residues is present at the position corresponding to the indicated position in SEQ ID NO: 24. The expression "amino acid residue or amino acid sequence stretch corresponding to ..." means that the position in a given protein may differ from the position in SEQ ID NO: 24. However, the corresponding position can be determined by aligning the amino acid sequence of the protein with those of SEQ ID NOs: 8-9 and 24 (as shown in Figure 10C) and counting the residues in the amino acid sequence of the protein starting from the first amino acid at the N-terminus to determine the corresponding position.

[0057] The definitions given above for consensus sequences can be combined with the definitions in the claims or with the embodiments described in the previous section. More specifically, the definition of a conserved residue may be combined with any of the definitions in items (Bi)-(B-iv), (Ci)-(C-iv), (Ei)-(E-iv), (Fi)-(F-iv), (bi)-(b-iv), and / or (ci)-(c-iv) above to define amino acid residues that should not be altered. Depending on the specific reference sequence used to define the protein of the present invention, the above indications of amino acid positions are replaced by the corresponding positions in the respective reference sequence. The corresponding positions in the reference sequence can be derived, for example, from the alignments shown in Figures 10A-C.

[0058] Krebicin Composition The compositions of the present invention comprise one or more proteins (klevicins) of the present invention as described above, and optionally any further components, such as a carrier. The compositions may comprise one or more different proteins (klevicins) as defined herein, for example, two, three, or four different proteins (klevicins) as defined herein. By "different," it is meant that the proteins differ in at least one amino acid residue. The compositions may comprise two, three, or more klevicins of the present invention from the same class as described in items (i) to (iv) above, or from any one of items (v) to (ix) above. Preferably, the compositions comprise at least two klevicins of the present invention from different classes, for example, at least one klevicin of the pore-forming type and at least one klevicin of the lipid II-cleaving type. The compositions may further comprise one or more Escherichia coli colicins or derivatives thereof, for example, as described in EP 3097783, for concomitant control of pathogenic E. coli (e.g., EHEC).

[0059] The present invention also provides compositions comprising one or more proteins of the present invention and one or more other bactericidal or bacteriostatic proteins. Such other bactericidal or bacteriostatic proteins may be Escherichia coli colicins or Salmonella colicins (salmosins). Escherichia coli colicins are known in the prior art and are described, in particular, in EP 3097783. Salmosins are known and are described in WO 2018 / 172065. Since the proteins of the present invention are preferably produced by expression in a plant or its cells, the composition may be plant material or an extract thereof, wherein the plant material is material derived from a plant expressing the protein (preferably a Nicotiana or edible plant expressing the protein). An extract of plant material is an aqueous solution containing water-soluble proteins, including the proteins of the present invention, present in or expressed in the plant material, or a dried version of such an aqueous solution. The extract is preferably obtained by removing water-insoluble components of the plant material, for example, by filtration or centrifugation. The plant material may be material derived from a plant selected from the group consisting of spinach, chard, beetroot, carrot, sugar beet, leafy beet, amaranth, and tobacco, and / or the plant material is one or more leaves, roots, tubers, or seeds, or crushed, ground, or comminuted leaves, roots, tubers, or seeds.

[0060] The composition or the extract from plant material may be a solid or liquid composition (e.g., a solution or dispersion) containing the klevicin of the present invention. The liquid composition may be aqueous, e.g., an aqueous solution. The concentration of the protein in the aqueous dispersion or solution may be 0.0001 to 1 mg / ml, preferably 0.001 to 0.1 mg / ml, and more preferably 0.005 to 0.05 mg / ml. When one or more klevicins capable of exerting a cytotoxic effect against Klebsiella are used, these concentrations refer to the total concentration of all such klevicins. The aqueous solution may contain a buffering agent in addition to one or more proteins of the present invention. The buffering agent may be an inorganic or organic acid or its salt. An example of an inorganic acid is phosphoric acid or its salt. Examples of organic acids include HEPES, acetic acid, succinic acid, tartaric acid, malic acid, benzoic acid, cinnamic acid, glycolic acid, lactic acid, citric acid, and ascorbic acid. Suitable organic acids are malic acid, lactic acid, citric acid, and ascorbic acid. The pH of the solution may generally be 4 to 8, preferably 5 to 8, and more preferably 6.0 to 7.5. When the object to which the composition is applied is a meat product, the pH of the solution may generally be 4 to 8, preferably 4.5 to 7, more preferably 5.0 to 6.5, and more preferably 5.0 to 6.0. Furthermore, the solution may contain an isotonic agent (e.g., glycerin or salt). A preferred salt used is sodium chloride. The aqueous solution containing one or more klebicins may be a buffered aqueous solution containing an additional solute such as a salt, for example, 50 to 400 mM NaCl, preferably 100 to 200 mM NaCl. The aqueous solution may further contain a sulfhydryl compound such as dithiothreitol (DTT), dithioerythritol, thioethanol, or glutathione, preferably DTT. The total concentration of the sulfhydryl compound in the aqueous solution may be 1 to 50 mM, preferably 2 to 20 mM, more preferably 4 to 10 mM.

[0061] When the composition of the present invention is a solid composition, it can be a powder, such as a solid composition after freeze-drying obtained by freeze-drying the above-mentioned extract or solution.The powder can contain additional solid components (such as those described above in the aqueous solution).It can be reconstituted with an appropriate liquid (such as water or a buffer solution) before use.The solid composition can contain the above-mentioned buffers, salts or other components so that the above-mentioned concentrations are achieved by reconstituting or dissolving the solid composition. Examples of carriers for the composition include solvents (e.g., water or aqueous buffer solution (as described above)), salts, sugars (e.g., monosaccharides and disaccharides), sugar alcohols, and other carriers (e.g., those known in pharmaceutical compositions). Examples of the latter include starch, cellulose, and other proteins (e.g., albumin). Examples of sugars include glucose, fructose, lactose, sucrose, and maltose.

[0062] The composition of the present invention may contain one or more klevicins of the present invention in an amount of at least 10, preferably at least 20, more preferably at least 30, even more preferably at least 50, and even more preferably at least 75% by mass, based on the total weight of protein in the composition. The content of klevicins in the composition can be measured by subjecting the composition to SDS-PAGE according to Reference Example 1, staining the resulting gel, and then analyzing the intensity of the bands on the gel. Thereby, the intensity of the bands due to klevicins can be measured relative to the sum of the intensities of the bands of all proteins in the composition. In one embodiment, the composition of the present invention is a pharmaceutical composition. The pharmaceutical composition may optionally contain, apart from one or more proteins of the present invention, an E. coli colicin. It may also contain one or more suitable pharmaceutically acceptable carriers and / or excipients, depending on whether it is liquid or solid and on the intended use. The excipients or carriers may be those described above.

[0063] The composition of the present invention as a pharmaceutical composition can be formulated for oral delivery to the small or large intestine. Accordingly, the present invention also provides an oral formulation comprising the protein of the present invention or the composition of the present invention, which can protect the protein from gastric conditions (e.g., acidic pH and / or proteases) and release the protein in the intestine. The ability to protect the protein from gastric conditions and release the protein in the intestine is preferably relevant for mammals, preferably human subjects. Components for delivering drugs to the intestine or preventing the degradation of drugs or active ingredients due to acidic or proteolytic gastric conditions are known to those skilled in the art. Solid compositions or formulations, such as tablets, can be coated with polymers that are resistant to gastric conditions but dissolve in the neutral intestinal conditions. Examples of commercially available products suitable for coating include Eudragit™ S100 from Evonik or enTRinsic™ drug delivery technology from Lonza. In another embodiment, the composition of the present invention as a pharmaceutical composition can be prepared for delivery to the lungs. Thus, the present invention also provides a pulmonary formulation comprising the protein of the present invention or the composition of the present invention. For a review of the localized delivery of protein therapeutics to the lungs, see, for example, Bodier-Montagutelli et al., EXPERT OPINION ON DRUG DELIVERY 2018, VOL. 15, NO. 8, 729-736; doi.org / 10.1080 / 17425247.2018.1503251. The formulation can be a dry powder for aerosol administration or a solution for nebulization therapy. Other usable embodiments of the formulation of the composition are as described below in the section on medical uses.

[0064] Application to objects The present invention provides a method for preventing or reducing Klebsiella contamination of an object, the method comprising contacting the object with one or more of the above-described proteins (klebicins) or the above-described composition. The object is an inanimate object. The object can be the surface of any non-organic or organic object (e.g., food). Klebsiella contamination of an object refers to the attachment of live Klebsiella cells to the object. Reducing Klebsiella contamination refers to reducing the number of live Klebsiella cells attached to the object. Measuring Klebsiella contamination of an object is part of common technical knowledge. For example, this can be performed by dilution plating of a homogenized food solution or dispersion, or dilution plating of a washing of another object, as performed in the examples, followed by counting bacterial colonies. Preferably, the object is food or animal feed. In order to treat or contact an object with the protein or composition of the present invention, in a broad sense, the object is contacted with a solution or liquid composition of the protein. For example, the object is sprayed with an aqueous solution of the composition of the present invention or immersed in the aqueous solution. The object can be immersed in the aqueous solution for at least 10 seconds, preferably at least 1 minute, preferably at least 5 minutes. Contacting the object with a liquid composition promotes the dispersion of the composition on the surface of the object. If a sufficiently uniform distribution can be achieved, it is also possible to contact the object with a solid composition according to the present invention.

[0065] Medical Use The present invention also provides a protein, composition, or pharmaceutical composition of the present invention for use in treating or preventing an infection caused by Klebsiella, particularly the aforementioned Klebsiella species, in a subject. The present invention also provides a method for treating or preventing an infection caused by Klebsiella (particularly the aforementioned Klebsiella species) in a subject, comprising administering to said subject one or more proteins (klevicins) or compositions of the present invention. The subject may be a human or a mammal (e.g., a livestock animal). Human subjects are preferred. The infection to be treated may be an infection caused by antibiotic-resistant Klebsiella. The resistance may be carbapenem resistance or multidrug resistance. The Klebsiella infection to be treated can be due to any of the Klebsiella species listed above. The klevicins KpneM (SEQ ID NO: 1) and KvarM (SEQ ID NO: 2), and the proteins defined herein using SEQ ID NO: 1 or SEQ ID NO: 2 as reference sequences, are suitable proteins for medical use due to their broad range of activity against a variety of Klebsiella isolates, as shown in the examples below. Therefore, these klevicins are preferably used for the treatment of infections by any Klebsiella, particularly Klebsiella pneumoniae (as well as for the prevention or reduction of infection, see above).

[0066] The Klebsiella infection to be treated may be an infection of the urinary tract, lower respiratory tract, bile duct, surgical wound, etc., or a syndrome (clinical syndrome) such as pneumonia, bacteremia, thrombophlebitis, cholecystitis, diarrhea, upper respiratory tract infection, osteomyelitis, and meningitis, preferably pneumonia, bacteremia, thrombophlebitis, urinary tract infection (UTI), diarrhea, upper respiratory tract infection, and wound infection. In a broad sense, a liquid or solid pharmaceutical composition comprising the above-mentioned klebicin and optionally other components is prepared for administration to a subject.The liquid composition can be an aqueous solution as described above.The solid composition can be a powder comprising at least one klebicin, for example, in a lyophilized form, or a tablet obtained from such a powder or a capsule filled with such a powder. The route of administration of the protein or pharmaceutical composition varies depending on the disease to be treated. In the case of treating diarrhea and upper respiratory tract infections, administration can be oral, for example in the form of a tablet or solution. In the case of treating diarrhea, the pharmaceutical preparation can be one that can pass through the stomach without being attacked by the acidic medium therein. The klebicin must then be released from the pharmaceutical composition in the intestine. Such formulations are known in the prior art. Examples are tablets and capsules that are resistant to the acidic medium of the stomach. It is also possible to orally administer biological material, such as E. coli or plant material, containing expressed klebicin to patients.

[0067] For the treatment of pneumonia, for example, when a subject suffers from a pulmonary infection caused by Klebsiella pneumoniae, the formulation can be administered to the subject as a pulmonary aerosol or powder. Methods for preparing proteins for pulmonary administration are known; for example, see Witt DM, Anderson L. Dornase alfa: a new option in the management of cystic fibrosis. Pharmacotherapy. 1996 Jan-Feb;16(1):40-8 for inhaled recombinant DNAse I or Dornase, and U.S. Patent Application Publication No. 2015 / 0024050 for dry powder formulations of DNAse I. See also the reviews by Depreter et al. 2013 and Bodier-Montagutelli et al. 2018. For the treatment of wound infections, the protein or pharmaceutical composition may be administered topically, for example, as an aqueous solution. For urinary tract infections (UTIs), the protein or pharmaceutical composition may be administered in the form of an aqueous solution using a catheter. For the treatment of cholecystitis or bile duct infections caused by Klebsiella, the protein or pharmaceutical composition may be administered in the form of an aqueous solution using a catheter.

[0068] Klevicin can be administered to adult humans in amounts of 1 mg to 1000 mg per day, preferably 10 mg to 250 mg per day for human patients. Such amounts may also be administered to animals. In a probiotic method, a patient can be treated by administering to the patient a genetically modified microorganism expressing at least one klevicin. The genetically modified microorganism can be a genetically modified non-pathogenic Escherichia coli or a lactic acid-producing microorganism commonly used in dairy fermentation. Examples of lactic acid-producing microorganisms include Lactobacillus bacteria, such as Lactobacillus lactis, and Bifidobacterium bacteria, such as Bifidobacterium bifidum or Bifidobacterium breve. Another route of administration is by infusion into the patient's bloodstream to prevent Klebsiella infection. For this purpose, klevicin can be dissolved in saline, and the solution can be sterilized.

[0069] Production of the proteins of the invention The klevicin or protein according to the present invention can be produced by known methods for expressing proteins in standard expression systems. When producing klevicin, the nucleotide sequence encoding it can be expressed in a suitable host organism. Methods that can be used to produce and purify the target protein have been described in the prior art, and any type of method can be used. For example, an E. coli expression system commonly known in the art can be used. When using a eukaryotic expression system, one or more introns can be inserted into the klevicin coding sequence to prevent toxicity to the bacterial organism used for cloning. Particularly efficient expression methods are plant expression systems, which are also known in the prior art. Plant expression systems that can be used to express klevicins according to the present invention are described in the Examples. A method that enables expression of a nucleotide sequence of interest in plants is the use of a self-replicating (viral) replicon containing a nucleotide sequence encoding a klevicin. The klevicin coding sequence can be codon-optimized for expression in plants or in the specific plant used as the expression host. Plant viral expression systems are described in many publications, such as WO 2012 / 019660, WO 2008 / 028661, WO 2006 / 003018, WO 2005 / 071090, WO 2005 / 049839, WO 2006 / 012906, WO 02 / 101006, WO 2007 / 137788, or WO 02 / 068664, and many more publications are cited in these publications. Various methods are known for introducing nucleic acid molecules (e.g., DNA molecules) into plants or plant parts, for example, for transient expression. Agrobacterium can be used to transfect plants with nucleic acid molecules (vectors) or nucleic acid constructs, for example, by agroinfiltration or spraying with an Agrobacterium suspension. For reference, see WO 2012 / 019660, WO 2014 / 187571, or WO 2013 / 149726. The nucleic acid molecule comprises a nucleotide sequence encoding the protein of the present invention.

[0070] In embodiments requiring strong expression of klevicin as a target protein, a nucleic acid molecule or nucleic acid construct containing a nucleotide sequence encoding klevicin may encode a viral vector capable of replicating in plant cells to form a viral vector replicon. For replication, viral vectors and replicons may contain an origin of replication that can be recognized by a nucleic acid polymerase present in plant cells (e.g., a viral polymerase expressed from the replicon). In the case of an RNA viral vector (referred to as an "RNA replicon"), the replicon can be formed by transcription under the control of a promoter active in plant cells after a DNA construct is introduced into the nucleus of the plant cell. In the case of a DNA replicon, the replicon can be formed by recombination between two recombination sites flanking the viral replicon-encoding sequence in the DNA construct, as described, for example, in International Publication Nos. 00 / 17365 and 99 / 22003. When the replicon is encoded by a DNA construct, an RNA replicon is preferred. The use of DNA and RNA viral vectors (DNA or RNA replicons) has been described in various publications for many years. Some examples are the following patent publications: WO 2008 / 028661, WO 2007 / 137788, WO 2006 / 003018, WO 2005 / 071090, WO 2005 / 049839, WO 02 / 097080, WO 02 / 088369, and WO 02 / 068664. Examples of DNA viral vectors are those based on geminiviruses. In the present invention, viral vectors or replicons based on plant RNA viruses, particularly those based on (+)-sense single-stranded RNA viruses, are preferably used. Therefore, the viral replicon can be a (+)-sense single-stranded RNA replicon. Examples of such viral vectors are those based on tobacco mosaic virus (TMV) and potexvirus X (PVX). By "based on" is meant that the viral vector uses a replication system (eg, replicase and / or other proteins involved in the replication of these viruses).Potexvirus-based viral vectors and expression systems are described in EP 2061890 or WO 2008 / 028661. As known from the cited references, an RNA replicon, such as a (+) sense single-stranded RNA replicon, can express a nucleotide sequence under the control of a subgenomic promoter located upstream of the nucleotide sequence. Through the action of a viral replicase that can be encoded by the same RNA replicon, the subgenomic RNA can be replicated in plant cells, and by including an (RNA) nucleotide sequence, a protein can be translated from the subgenomic RNA.

[0071] Krevicin can be expressed in multicellular plants or parts thereof, particularly higher plants or parts thereof. Monocotyledonous and dicotyledonous plants (crops) can be used. Common plants that can be used to express a protein of interest include Nicotiana benthamiana, Nicotiana tabacum, spinach, Brassica campestris, B. juncea, beet (Beta vulgaris), rapeseed, yellow bell pepper, mustard, strawberry, daisy (Chenopodium capitatum), lettuce, sunflower, cucumber, Chinese cabbage, cabbage, carrot, shallot, onion, radish, lettuce, snow pea, cauliflower, broccoli, burdock, turnip, tomato, eggplant, squash, watermelon, prince melon, and melon. Suitable plants are spinach, Swiss chard, beetroot, carrot, sugar beet, Nicotiana tabacum, and Nicotiana benthamiana. Expression in edible plants can be used to prevent Klebsiella contamination of the plant or foods produced therefrom. In one embodiment, plants that do not normally enter the human or animal food chain, such as Nicotiana species (e.g., N. tabacum and N. benthamiana), are used.

[0072] Generally, the target protein krebicin is expressed in the cytosol of the cells of a plant or plant part. In this case, a signal peptide that transports the target protein to a specific compartment is not added to the protein. Alternatively, the target protein can be expressed in or targeted to the chloroplast of a plant. In this latter case, an N-terminal signal peptide, commonly referred to as a plastid transit peptide or a chloroplast targeting peptide, is added to the N-terminus or C-terminus, preferably the N-terminus, of the target protein krebicin. The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a protein of the present invention, which may comprise (i) a transcriptional promoter active in plant cells, and (ii) a nucleic acid construct comprising the nucleotide sequence encoding the protein of the present invention for expressing the nucleotide sequence in plant cells under the control of the promoter.

[0073] The present invention also provides a nucleic acid molecule or nucleic acid construct encoding a protein of the present invention, said nucleic acid molecule or nucleic acid construct comprising (i) a transcriptional promoter, preferably active in a plant cell, and (ii) a nucleotide sequence encoding said protein for expressing the nucleotide sequence in a cell, preferably a plant cell, under the control of said promoter. The present invention further provides a nucleic acid molecule or nucleic acid construct encoding a protein of the present invention, wherein said nucleic acid molecule or nucleic acid construct is or encodes a viral (DNA or RNA) replicon comprising a nucleotide sequence encoding said protein for expressing said nucleotide sequence in a cell, preferably a plant cell, wherein said replicon may comprise a subgenomic promoter for expressing said nucleotide sequence in a plant cell or in a cell of a plant under the control of said subgenomic promoter. Since the protein of the present invention is preferably expressed in plants or plant cells, the present invention also provides a plant, plant tissue, or plant cell comprising the protein of the present invention. The present invention also provides a plant, plant tissue, or plant cell comprising the nucleotide sequence or nucleic acid molecule of the present invention. The plant may be any one of the above.

[0074] Production of the compositions of the present invention In a method for producing a composition comprising at least one klevicin, the first step is to express the klevicin in a plant (e.g., an edible plant) or plant cells. In a second step, plant material containing the expressed klevicin is recovered from the plant. For example, the plant material may be leaves, roots, tubers, or seeds, or may be crushed, ground, or pulverized leaves, roots, tubers, or seeds. In step (iii), the klevicin is extracted from the plant material using an aqueous buffer. Here, the plant material may be homogenized, and insoluble material may be removed by centrifugation or filtration. Soluble components, including the klevicin, are extracted into the aqueous buffer to prepare a solution of the klevicin in the aqueous buffer. The aqueous buffer may contain an inorganic or organic acid or a salt thereof and may have a pH as defined above for the aqueous solution of the composition of the present invention. Furthermore, the aqueous buffer may contain a salt and / or a sulfhydryl compound as described above for the aqueous solution of the composition of the present invention. If a relatively pure klebicin composition is desired, the klebicin solution in aqueous buffer can be further purified by removing unwanted components according to known protein purification methods.

[0075] The present invention therefore provides a method for producing a composition comprising a protein according to the invention, comprising the steps of: (i) expressing said protein in said plant, preferably an edible plant or Nicotiana; (ii) recovering plant material containing the expressed protein from said plant; (iii) extracting the protein from the plant material using an aqueous buffer to obtain a composition containing the protein; Optionally, removing undesirable contaminants from the composition. The present invention provides a method comprising: When klevicin is expressed in a plant, the plant or its tissue containing the expressed protein can be harvested, the tissue can be homogenized, and insoluble materials can be removed by centrifugation or filtration. If a relatively pure klevicin is desired, the klevicin can be further purified by commonly known protein purification methods, such as chromatography methods that can remove other host cell-derived proteins and plant metabolic products (e.g., alkaloids and polyphenols). The purified klevicin solution can be concentrated and / or lyophilized. When klebicins are expressed in food plants, crude protein extracts or semi-purified concentrates from the food plants can be used to prevent or reduce contamination of objects (eg, food products with Klebsiella). [Example]

[0076] (Reference Example 1) Soft agar overlay assay for evaluation of klebicin toxicity An overnight culture of Klebsiella quasipneumoniae subsp. similipneumoniae SB30 (DSM 28212) was grown in LB medium at OD 595 Equalize the mixture to 1.0 and dilute 100-fold with 0.8% top agar preheated in a 55°C water bath. Pour the mixed overlay components onto a plate containing solid agar (1.5% LB agar); hold the plate for a few minutes to allow the agar to harden. Place a sterile Whatman disk (6 mm diameter) on the soft agar, and apply a 20 μl aliquot of krebicin solution containing 10 μg of krebicin protein to the disk. Incubate the plate at 37°C for 16 hours. After 16 hours of incubation, measure the diameter of the krebicin inhibition zone.

[0077] Determination of klebicin concentration The concentration of klebicin in a liquid sample containing klebicin is determined by performing SDS-PAGE with Coomassie staining and reading the intensity of the klebicin band using a commercially available reader. This determined intensity is then compared with the band obtained by performing SDS-PAGE with Coomassie staining of serial dilutions of known concentrations of bovine serum albumin (BSA). A standard curve can be obtained from the band intensity of the BSA-stained SDS-PAGE gel. The concentration of BSA is determined using a Bradford protein assay (e.g., Bradford Reagent, B6916, Sigma-Aldrich, St. Louis, MO, USA).

[0078] (Example 1) Construction of klebicin expression vector KpneA (Klebsiella pneumoniae SAV78255.1), KaerA (Klebsiella aerogenes WP_063414841.1), KoxyY (Klebsiella oxytoca WP_024273778), KvarIa (Klebsiella variicola KDL88409), KpneIa (Klebsiella pneumoniae BAS34675), KpneM (Klebsiella pneumoniae EWD35590.1), KpneM2 (Klebsiella species WP_047066220), KvarM (Klebsiella variicola CTQ17225.1), KaerM (Klebsiella aerogenes The vector (WP_015367360.1) was optimized for expression in the host plant Nicotiana benthamiana and synthesized by Thermofisher Scientific (USA). It was inserted as a BsaI-BsaI fragment into the TMV-based magnICON® vector pICH29912 (Marillonnet et al., 2005) (Figure 1). The resulting plasmid was used to transform Agrobacterium tumefaciens GV3101.

[0079] (Example 2) Expression of klevicin in plants Nicotiana benthamiana plants were grown in a growth chamber at 25°C and 50% humidity under a 16-h light (1500 lux) and 8-h dark photoperiod. Four- to six-week-old plants were used for transfection of recombinant Agrobacterium tumefaciens. Agrobacterium tumefaciens 50mgL -1 of rifampicin and 50 mg L -1 The overnight Agrobacterium culture was sedimented at 3220 g for 5 minutes and grown at 30°C in LB medium containing 100 ml of kanamycin. 595 was resuspended in 1.5 ml tap water. Leaves from 4- to 6-week-old plants were infiltrated on the abaxial side using a needleless syringe containing a 1:1000 dilution of Agrobacterium tumefaciens strain containing the expression vector. Plant leaves were observed and harvested 4 to 7 dpi (days post-infiltration). SDS-PAGE and Coomassie staining analysis of soluble protein extracts from infiltrated plant leaves revealed that all nine klevicins were efficiently expressed in plants and were detected on the gel as very strong complementary bands (Figure 2). The weights of the polypeptides observed on electrophoresis corresponded closely to their expected theoretical molecular weights (KvarIa - 43.4 kDa, KpneIa - 48.5 kDa, KpneA - 40 kDa, Kaer A - 39 kDa, KoxyY - 48.7 kDa, KpneM - 30.3 kDa, KpneM2 - 29.7 kDa, KvarM - 29.8 kDa, and KaerM - 29 kDa). Expression levels of individual klevicins varied between 2.7 and 4.4 mg / g FW, with the highest expression levels achieved by two Klebsiella pneumoniae M-type klevicins, KpneM2 and KpneM (Table 1).

[0080] (Example 3) Purification of klevicin from plant biomass The KpneA, KaerA, KvarIa, KpneM, KpneM2, and KvarM bacteriocins were purified to homogeneity by protein chromatography. Highly pure KpneM, KpneM2, and KvarM proteins were obtained after a single-step hydrophobic interaction chromatography (HIC), but for best results, a second purification step by anion exchange chromatography was included. KpneA and KvarIa were also purified using hydrophobic interaction chromatography as the first step, but followed by a cation exchange chromatography column. KaerA was purified by two steps of ion exchange chromatography: a cation exchange column as the first step and an anion exchange column as the second step. Crude protein extracts were prepared as follows: A small portion of frozen leaf tissue was ground to a fine powder using a mortar and pestle with liquid nitrogen. The resulting powder was mixed with cold extraction buffer at a ratio of 1 g of plant material to 5 mL of buffer. The suspension was kept on ice for 15–20 min. Cellular debris was removed by centrifugation at 3220 g and 4°C for 20 min, and the supernatant was filtered through membrane filters (pore sizes of 5 μm and 0.22 μm). The resulting solution was used as total soluble protein and subjected to two-step chromatographic purification. The details of the purification protocol varied depending on the protein.

[0081] KpneM was purified using a combination of hydrophobic interaction chromatography (HIC) and anion exchange chromatography (AEXC) ( Figures 3A,B ). A small portion of the frozen leaf tissue was homogenized using a mortar and pestle chilled in liquid nitrogen. The resulting powder was mixed with cold extraction buffer (50 mM NaH2PO4 / Na2HPO4, 30 mM NaCl, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept at 20–25°C for 10–15 min. Cellular debris was removed by centrifugation at 3220 g and 4°C for 20 min. The pellet was discarded, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 0.70 M, and the pH of the solution was adjusted to 6. The precipitate formed was removed by centrifugation at 3220 g and 4°C for 5 min. The supernatant was used as total soluble protein and subjected to two-step purification.

[0082] In the first purification step, a chromatography column was packed with Phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, 0.70 M (NH4)2SO4, pH 6.0). The protein solution was loaded onto the column, and the Phenyl Sepharose-bound protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4 / Na2HPO4, 0.28 M (NH4)2SO4, pH 6.0). The collected protein fraction was transferred to a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 8-10-fold. The concentrate was diluted to the initial volume with a buffer containing 50 mM NaH2PO4 / Na2HPO4 (pH 8.0). The procedure was repeated until the conductivity decreased to less than 10 mS / cm, and the protein solution was subjected to a final purification step using Q Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and the Q Sepharose-unbound proteins were collected in the flow-through fraction. The KpneM was then lyophilized and subjected to analysis.

[0083] KpneM2 was purified using a combination of hydrophobic interaction chromatography (HIC) and anion exchange chromatography (AEXC) ( Figures 3C,D ). A small portion of frozen leaf tissue was homogenized using a mortar and pestle chilled in liquid nitrogen. The resulting powder was mixed with cold extraction buffer (50 mM NaH2PO4 / Na2HPO4, 30 mM NaCl, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept at 20–25°C for 10–15 min. Cellular debris was removed by centrifugation at 3220 g for 20 min at 4°C. The pellet was discarded, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 0.70 M, and the pH of the solution was adjusted to 6. The precipitate formed was removed by centrifugation at 3220 g for 5 min at 4°C. The supernatant was used as total soluble protein and subjected to two-step purification.

[0084] In the first purification step, a chromatography column was packed with Phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, 0.70 M (NH4)2SO4, pH 6.0). The protein solution was loaded onto the column, and the Phenyl Sepharose-bound protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4 / Na2HPO4, 0.42 M (NH4)2SO4, pH 6.0). The collected protein fraction was transferred to a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 8-10-fold. The concentrate was diluted to the initial volume with a buffer containing 50 mM NaH2PO4 / Na2HPO4 (pH 8.0). The procedure was repeated until the conductivity decreased to less than 10 mS / cm, and the protein solution was subjected to the final purification step using Q Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and the Q Sepharose-unbound proteins were collected in the flow-through fraction. KpneM2 was then lyophilized and subjected to analysis. KvarM was purified using a combination of hydrophobic interaction chromatography (HIC) and anion exchange chromatography (AEXC) ( Figures 3E,F ).

[0085] A small portion of the frozen leaf tissue was homogenized using a mortar and pestle chilled in liquid nitrogen. The resulting powder was mixed with cold extraction buffer (50 mM NaH2PO4 / Na2HPO4, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept at 20-25°C for 10-15 minutes. Cellular debris was removed by centrifugation at 3220 g for 20 minutes at 4°C. The pellet was discarded, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 0.95 M, and the pH of the solution was adjusted to 6. The precipitate formed was removed by centrifugation at 3220 g for 5 minutes at 4°C. The supernatant was used as total soluble protein and subjected to two-step purification.

[0086] In the first purification step, a chromatography column was packed with Phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, 0.95 M (NH4)2SO4, pH 6.0). The protein solution was loaded onto the column, and the Phenyl Sepharose-bound protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4 / Na2HPO4, 0.62 M (NH4)2SO4, pH 6.0). The collected protein fraction was placed in a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 8-10-fold. The concentrate was diluted to the initial volume with a buffer containing 50 mM NaH2PO4 / Na2HPO4 (pH 8.0). The procedure was repeated until the conductivity decreased to less than 10 mS / cm, and the protein solution was subjected to the final purification step using Q Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and the Q Sepharose-unbound proteins were collected in the flow-through fraction. The KvarM was then lyophilized and applied for analysis.

[0087] KpneA was purified using a combination of hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEXC) ( Figures 3G,H ). A small portion of frozen leaf tissue was homogenized using a mortar and pestle chilled in liquid nitrogen. The resulting powder was mixed with cold extraction buffer (20 mM NaH2PO4 / Na2HPO4, 30 mM NaCl, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept at 20–25°C for 10–15 min. Cellular debris was removed by centrifugation at 3220 g and 4°C for 20 min. The pellet was discarded, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 1.50 M, and the pH of the solution was adjusted to 6. The precipitate formed was removed by centrifugation at 3220 g and 4°C for 5 min. The supernatant was used as total soluble protein and subjected to two-step purification.

[0088] In the first purification step, a chromatography column was packed with Phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, 1.50 M (NH4)2SO4, pH 6.0). The protein solution was loaded onto the column, and the Phenyl Sepharose-bound protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4 / Na2HPO4, 0.90 M (NH4)2SO4, pH 6.0). The collected protein fraction was placed in a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 8-10-fold. The concentrate was diluted to the initial volume with a buffer containing 20 mM NaH2PO4 / Na2HPO4, 20 mM sodium citrate (pH 4.5). The procedure was repeated until the conductivity decreased to less than 9 mS / cm, and the protein solution was subjected to a final purification step using SP Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citric acid, pH 4.5). The protein solution was loaded onto the column, and the SP Sepharose-bound protein fraction was eluted with a linear gradient of cold wash buffer containing an additional 500 mM NaCl. KpneA was then lyophilized and subjected to analysis.

[0089] KaerA was purified using a combination of cation exchange chromatography (CEXC) and anion exchange chromatography (AEXC) ( Figures 3I,J ). A small portion of the frozen leaf tissue was homogenized using a mortar and pestle chilled in liquid nitrogen. The resulting powder was mixed with cold extraction buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citric acid, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept at 20–25°C for 10–15 min. Cellular debris was removed by centrifugation at 3220 g for 20 min at 4°C. The pellet was discarded, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.45 μm). The pH of the solution was adjusted to 4.5, and the precipitate formed was removed by centrifugation at 3220 g for 5 min at 4°C. The supernatant was used as total soluble protein and subjected to two-step purification.

[0090] In the first purification step, a chromatography column was packed with SP Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citric acid, pH 4.5). The protein solution was loaded onto the column, and the SP Sepharose-bound protein fraction was eluted with a linear gradient of cold wash buffer containing an additional 500 mM NaCl. The collected protein fraction was placed in a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 8-10-fold. The concentrate was diluted to its original volume with a buffer containing 20 mM NaH2PO4 / Na2HPO4 (pH 8.0). The procedure was repeated until the conductivity decreased to less than 8 mS / cm, and the protein solution was subjected to a final purification step using Q Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (20 mM NaHPO / NaHPO, pH 8.0). The protein solution was loaded onto the column, and unbound proteins were collected in the flow-through fraction. KaerA was then lyophilized and subjected to analysis. KvarIa was purified using a combination of hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEXC) (Fig. 3K,L).

[0091] A small portion of frozen leaf tissue was homogenized using a mortar and pestle chilled in liquid nitrogen. The resulting powder was mixed with cold extraction buffer (20 mM NaH2PO4 / Na2HPO4, 30 mM NaCl, pH 5.0) at a ratio of 1 g of plant material to 5 ml of buffer. The crude extract was kept at 20–25°C for 10–15 min. Cellular debris was removed by centrifugation at 3220 g and 4°C for 20 min. The pellet was discarded, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 1.35 M, and the pH of the solution was adjusted to 6. The precipitate formed was removed by centrifugation at 3220 g and 4°C for 5 min. The supernatant was used as total soluble protein and subjected to two-step purification.

[0092] In the first purification step, a chromatography column was packed with Phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibrated with cold buffer (50 mM NaH2PO4 / Na2HPO4, 1.35 M (NH4)2SO4, pH 6.0). The protein solution was loaded onto the column, and the Phenyl Sepharose-bound protein fraction was eluted by washing with elution buffer (50 mM NaH2PO4 / Na2HPO4, 0.81 M (NH4)2SO4, pH 6.0). The collected protein fraction was placed in a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution was reduced 8-10-fold. The concentrate was diluted to the initial volume with a buffer containing 20 mM NaH2PO4 / Na2HPO4, 20 mM sodium citrate (pH 4.5). The procedure was repeated until the conductivity decreased to less than 8 mS / cm, and the protein solution was subjected to a final purification step using SP Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibrated with cold buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citric acid, pH 4.5). The protein solution was loaded onto the column, and the SP Sepharose-bound protein fraction was eluted with a linear gradient of cold wash buffer containing an additional 500 mM NaCl. The KvarIa was then lyophilized and subjected to analysis.

[0093] The concentration of purified proteins was assessed by Bradford assay or by comparing band intensity with known BSA amounts run on the same SDS-PAGE gel. The results of klebicin purification are summarized in Table 1. Figure 4 shows purified KpneM, KpneM2, KvarM, KpneA, KaerA, and KvarIa klebicin proteins loaded on the same gel. As measured by capillary gel electrophoresis, all purified klebicins contained only 0.2–3.7% impurities. The yields of individual klebicins after purification ranged from 0.34–1.1 mg / g FW. Purifying klebicins at the highest expression levels resulted in the greatest final yield and also the highest quality purified protein. [Table 1]

[0094] (Example 4) Klevicin activity test in soft agar overlay assay The activity of the bacteriocin-expressing crude plant extracts was tested in a soft agar overlay assay using 12 Klebsiella strains belonging to different species (Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella oxytoca, Klebsiella variicola, and Klebsiella aerogenes). Klebsiella strains were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures and are listed in Table 2. Overnight Klebsiella cultures were grown in LB medium to reach OD 595 The mixture was equalized to a pH = 1.0 and diluted 100-fold with 0.8% top agar preheated in a 55°C water bath. The mixed overlay components were poured onto a plate containing solid agar (1.5% LB agar); the plate was kept at room temperature for several minutes to allow the agar to harden. Sterile Whatman disks (6 mm diameter) were placed on the soft agar, and the appropriate amount of krebicin (20 μl of crude extract or 10 μg of purified krebicin) was applied to the disk. The plate was incubated overnight at 37°C, and the diameter of the krebicin inhibition zone was observed. The results of this assay are summarized in Figure 5.

[0095] Two of the bacteriocins tested, KoxyY and KaerM, showed noticeable but narrow zones of inhibition in lawns of several tested strains, with KaerM forming a larger, more diffuse zone of inhibition only in lawns of Klebsiella aerogenes. Due to their weak activity, these two proteins were not included in further experiments. [Table 2]

[0096] All seven remaining bacteriocins formed large zones of inhibition in the lawns of several Klebsiella species and strains tested. All 12 strains tested were inhibited by not just one but several bacteriocins. The remaining three colM-like proteins exhibited the broadest spectrum of activity and similar activity patterns, targeting 11 of the 12 tested strains. However, KvarM formed a significantly larger zone of inhibition than both Klebsiella pneumoniae colM-like bacteriocins (KpneM and KpneM2). Two ColA-like proteins, KpneA and KaerA, also showed very similar activity patterns, although the diameter of the zones varied in some of the strains tested. Finally, both Colla-like proteins, KvarIa and KpneIa, showed very similar activity patterns (Figure 5). With the exception of KvarIa and KpneIa, all bacteriocins formed zones of inhibition in strains belonging to all five different Klebsiella species. The two colla-like proteins had little effect on any of the four Klebsiella pneumoniae strains tested.

[0097] (Example 5) Evaluation of klevicin activity against a panel of clinical Klebsiella isolates All six purified klebicins were then tested against a larger panel of Klebsiella strains: a total of 100 clinical Klebsiella isolates, including 89 Klebsiella pneumoniae and 11 Klebsiella oxytoca strains. The clinical Klebsiella strains used for the agar overlay assay were isolated at the Kaunas Clinic of the Lithuanian University of Health Sciences and are listed in Table 3. Purified, lyophilized klebicins were resuspended in deionized water and applied as 10 μl droplets (10 μg protein) to 6 mm Whatman disks placed on LB plates streaked with Klebsiella. After overnight incubation, the zones of inhibition were measured.

[0098] KvarM showed a surprisingly broad range of activity. 85% of strains were susceptible to this klebicin (Figure 6, Table 3). KpneM was not far behind, generally targeting 74% of the strains tested with a slightly smaller zone of inhibition. The specificities of the activity spectra of KvarM and KpneM largely overlapped, but KvarM targeted 11 more strains than KpneM, and only one KvarM-immune strain was susceptible to KpneM (Figure 6, Table 3). In contrast, a third M-type klebicin, KpneM2, was significantly less active, targeting only 20% of strains. The colA-like klebicins KpneA and KaerA both targeted 30% and 28% of strains, respectively, with partially overlapping profiles. Nine KaerA-immune strains were susceptible to KpneA, and seven KpneA-immune strains were susceptible to KaerA. KpneA also generally formed a larger zone of inhibition. KvarIa had the narrowest spectrum of activity, targeting only 10% of all strains (6 for Klebsiella oxytoca and 4 for Klebsiella pneumoniae) (Fig. 6, Table 3).

[0099] [Table 3] TIFF2026031930000004.tif222169 TIFF2026031930000005.tif222169 TIFF2026031930000006.tif145169

[0100] (Example 6) Evaluation of klevicin activity against Klebsiella strains in liquid culture and biofilms We next performed a more detailed analysis of klevicin activity in liquid medium and in young, 1-day-old biofilms with five representatives of different Klebsiella species: Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella oxytoca, Klebsiella variicola, and Klebsiella aerogenes.

[0101] To assess klebicin activity in liquid medium, overnight Klebsiella cultures were grown in iron-deficient casamino acid (CAA) medium (BD Bioscience) to an OD of up to 1.2 mL. 595 The lyophilized purified klebicins were resuspended in CAA medium, added to the diluted bacterial suspension, and incubated at 37°C with shaking (200 rpm) for 5.5–6.5 h. The antibacterial activity of klebicins was assessed by determining the cell count of bacterial test cultures. -1 , 10 -2 , 10 -3 , 10 -4 , and 10 -5 Serial dilutions of the culture were made, plated on LB agar plates, incubated overnight at 37°C, and CFU were calculated.

[0102] Biofilms were grown as described by Moskowitz et al. (2004) and Paskevicius et al. (2017) with some modifications. Briefly, Klebsiella quasipneumoniae, Klebsiella oxytoca, Klebsiella variicola, and Klebsiella aerogenes strains were grown overnight in LB and diluted to an OD of 0.1 in fresh CAA medium. 10 μl of bacterial culture was transferred to wells of a 96-well microtiter plate (Nalgene Nunc International, Rochester, NY) containing 90 μl of CAA medium. Bacterial biofilms were formed by immersing a peg of a modified polystyrene microtiter lid (Nunc TSP system) into the biofilm growth plate, followed by a 20-hour incubation at 30°C or 37°C thermostatically controlled. For krebicin treatment, peg lids were rinsed three times with sterile water and placed in microtiter plates containing 5 μg / mL krebicin diluted in 100 μl of CAA per well and incubated at 30°C or 37°C for 5 hours, depending on the strain. After incubation with krebicin, peg lids were again rinsed three times with sterile water, placed in a sterile microtiter plate containing CAA, and centrifuged at 810 g for 30 minutes. Six identically treated wells were pooled each time, serial dilutions were made, and bacteria were plated on LB plates for CFU counts.

[0103] For liquid culture assays, 5 μg mL -1One best-performing klebicin from each group (colM-like, colA-like, and cola-like) from Example 4 was tested at a concentration of 1000 kJ / mL. Klebicin KvarM inhibited growth of all five strains and reduced CFU counts to approximately the same extent. Klebsiella pneumoniae DSM 16231 was reduced by four orders of magnitude, and all remaining klebicins by approximately three orders of magnitude (Figure 7). KvarIa inhibited four strains and was the most efficient of all three klebicins, reducing CFU counts by four to nine orders of magnitude, depending on the strain (Figure 7). (As shown in Figure 5, Klebsiella pneumoniae DSM 16231 is insensitive to this Klebsiella). KpneA reduced CFU counts of the three strains by 4.6 to 5.7 logs (Figure 7). The biofilm assays used the same Klebsiella strains as in the liquid culture assays. We first tested the ability of these five strains to form biofilms. With the exception of Klebsiella pneumoniae DSM 16231, four of the tested strains formed biofilms under the conditions tested, so this strain was not used for further experiments. Biofilms from all four remaining strains were treated with two klebicins for 20 hours, which demonstrated the best results in the liquid culture assay. The results obtained with the biofilms closely mirrored those obtained in the liquid culture assays, with the exception of Klebsiella quasipneumoniae, whose biofilms were completely eradicated by KpneA and KvarIa (Figure 8). For all remaining strains, klebicin treatment reduced biofilm CFU counts to the same extent as in liquid culture, achieving only small Δlog variations (Figures 7 and 8).

[0104] (Example 7) Evaluation of klevicin antibacterial activity in vivo For the first demonstration of Klebsiella activity in vivo, a Klebsiella challenge assay was performed in a non-mammalian model, Galleria mellonella larvae. Galleria mellonella is the larger honey moth or wax moth, a moth in the family Pyralidae. Galleria mellonella is a convenient model organism for in vivo toxicology and pathogenicity testing and has been shown to replace the use of small mammals in such experiments (Harding et al. 2013; Paskevicius et al. 2017).

[0105] We selected KvarIa as one of the most active klevicins in this assay, and Klebsiella quasipneumoniae DSM 28212 as the KvarIa-susceptible challenge strain. Galleria mellonella challenge experiments were performed as described by Paskevicius et al. (2017) with some modifications. Overnight cultures of Klebsiella quasipneumoniae DSM 28212 were grown in CAA medium, diluted with 0.8% NaCl, and diluted to 1.2–3.2 × 10 in 10 μL of Klebsiella quasipneumoniae culture. 6 CFUmL -1 A concentration of 10 μL of klebicin solution was achieved by injecting 10 μL of the solution into the hemocoel of fifth-instar Galleria mellonella larvae (Livefood UK) near the left and / or right prolegs. Klebicin was injected 2 hours after infection with Klebsiella quasipneumoniae. Injected larvae were incubated at 37°C in 9 cm Petri dishes without food for up to 3 days. Caterpillars were considered dead when they showed no movement in response to mechanical stimulation of the head and produced a clear color change from cream to dark brown / black. 20 larvae were used for each treatment point. First, the minimum lethal dose (MLD) of the challenge strain sufficient to kill all larvae within 68 hours (the duration of the experiment) was 2.3 × 10 4 The present inventors determined that it was CFU.

[0106] Next, a challenge experiment was performed using the MLD, and two additional challenge doses were added, one inferior to the MLD and the other superior to the MLD by factors of 1.9 and 1.4, respectively. 15% of the larvae survived after 68 hours, resulting in a 1.2 × 10 challenge dose. 4 CFU was not sufficient to kill all larvae, but 2.3 and 3.2 × 10 4 Injection of KvarIa 2 hours after infection resulted in 1.2 and 2.3 × 10 CFU sufficient to kill all larvae at 44 hours. 4 All larvae infected with CFU were completely rescued. 4 CFU) were partially rescued, with 85% of the larvae surviving until the end of the experiment (Figure 9). In summary, activity assays demonstrate that KvarM has a very broad spectrum of activity, as it can target Klebsiella strains belonging to the species Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Klebsiella oxytoca, and Klebsiella aerogenes. It was also active against 85% of a panel of antibiotic-resistant clinical Klebsiella isolates. In liquid culture assays, this klebicin could reduce colony formation by 3–4 logs and by more than 2 logs in biofilm assays. Pore-forming klebicins are generally more efficient at reducing bacterial counts in liquid cultures or biofilms than peptidoglycan synthesis inhibitors, achieving 4–9 log reductions in CFU counts in liquid cultures and 2–nearly 6 log reductions in biofilms. Having demonstrated the highest efficacy in vitro, KvarIa was also tested in vivo in a Galleria mellonella larval challenge assay with very good results. However, its applicability to Klebsiella is currently undermined by the fact that it is not broadly active against Klebsiella pneumoniae, although it works well against the closely related Klebsiella quasipneumoniae.

[0107] (Example 8) Identification of the klebicin receptor / transposable element A universal feature of colicins is their domain organization, with each appearing to possess receptor-binding, translocation, and cytotoxicity domains, features conditioned by the need for these bacteriocins to cross the outer membrane of Gram-negative bacteria (Kleanthous, 2010). Alignment of pore-forming klevicin amino acid sequences with their Escherichia coli (E. coli) counterparts reveals that their killing domains show a significant degree of homology. However, as a rule, pore-forming klevicins are smaller than colicins. Their amino-terminal portions, which should contain the translocation and receptor-binding domains, are significantly shorter than the respective domains of colicins and share little or no sequence similarity. Therefore, we predicted that the translocation mechanism of pore-forming klevicins may differ from that of their E. coli counterparts. In contrast to some other bacteriocins (e.g., pyocins), which are strictly species-specific, klevicin activity is not restricted to the single species from which they were isolated, but rather to at least the genus. In this regard, it was important to investigate the players involved in the mechanism of klevicin reception and transposition. Klebsiella quasipneumoniae DSM 28212, a strain with a known genome sequence and susceptibility to all tested klevicins, was subjected to several rounds of transposon mutagenesis, and pooled mutants were tested for susceptibility to different klevicins.

[0108] Transposon mutagenesis of Klebsiella quasipneumoniae DSM 28212 was performed as described by Martinez-Garcia et al. (2011). Suicide delivery of the minitransposon contained in the pBAM1 plasmid was performed by triparental mating. This plasmid was mobilized from E. coli CC118λpir(pBAM1) donor cells into Klebsiella quasipneumoniae DSM 28212 cells with the help of the helper strain E. coli HB101(pRK600). The resulting kanamycin-resistant clones were confirmed for loss of ampicillin resistance, and their genomic DNA was used for PCR amplification of the transposon-flanking regions and subsequently sequenced as described by Martinez-Garcia et al. (2011). We isolated 29 independent mutant clones and successfully mapped the transposon insertions to 18 krebicin-resistant mutants. To confirm that the loss of krebicin sensitivity was due to the mapped mutations, we performed complementation assays by ectopic expression of the respective wild-type genes.

[0109] For the complementation assay, a Klebsiella genomic region containing the ExbB, ExbBD, OmpC, FhuA, TonB, and FimB gene ORFs, along with their 5' non-coding promoter regions, was PCR-amplified from Klebsiella quasipneumoniae DSM28212 genomic DNA using Phusion DNA polymerase (Thermofisher Scientific Baltics) and ligated into pJET1.2 (Thermofisher Scientific Baltics). After sequencing, the cloned fragments were excised with a pair of restriction endonucleases specific for each fragment, ligated into pACYC184 (NEB), and transformed into the respective Klebsiella quasipneumoniae mutants. The primer sequences and cloning strategy used are listed in Table 4.

[0110] [Table 4]

[0111] Summary results from the mutant klebicin susceptibility studies and complementation assays are shown in Table 5 . [Table 5]

[0112] Based on the results obtained, all klevicins except KvarIa resemble group B colicins and use the TonB-dependent translocation pathway. All three M-type klevicins require FhuA, TonB, and ExbB for receptor-translocation, as their E. coli homologs, colicins M.KpneA and KaerA, also rely on the TonB translocation pathway and additionally require a functional OmpC. Thus far, we have not been able to identify any other putative receptors for these two klevicins (Table 6). [Table 6]

[0113] KvarIa-resistant transposition mutants were extremely difficult to obtain, and only a few false-positive clones were isolated. Therefore, it was determined that only one protein, outer membrane protein C (OmpC), is involved in KvarIa reception and transposition. OmpC mutants were selected for resistance to KpneA and KaerA and appeared to be equally resistant to KvarIa. In summary, we have thus demonstrated that the three M-type klebicins, KpneA, KpneM2, and KvarM, all translocate by a mechanism similar to that of colicin M and require the FhuA receptor and TonB-associated translocation pathway to enter the periplasm and exert their activity.

[0114] Two klebicins, which we named KpneA and KaerA based on the similarity of their killing domains to colicin A, also appeared to depend on the TonB translocation pathway. This is in contrast to colicin A, which translocates via a TolA-dependent pathway. Colicin A also binds to BtuB, but we did not isolate BtuB mutants resistant to KpneA or KaerA. However, both KpneA and KaerA require functional OmpC, an analog of OmpF that is also involved in colA translocation (Kleanthous 2010). To date, no other putative receptors for these two klebicins have been identified.

[0115] KvarIa differs from all remaining klebicins because it appears to function in all TonB and ExbB mutants. Therefore, based on our results, KvarIa does not use the TonB-dependent transposition pathway. Considering that all described colicins use either TonB or TolA as transposable elements, this protein is expected to transpose via the Tol-dependent pathway. However, we did not isolate a single transposition mutant of a Tol-dependent pathway-related gene that was resistant to KvarIa. Obtaining KvarIa-resistant transposon mutants was extremely difficult, and only some false-positive clones were isolated; this may certainly be related to limitations of the method used. It is possible that the conditions used for selection did not yield mutations with a high fitness penalty. Therefore, so far, we have been able to identify the only protein involved in KvarIa receptor-transposition, outer membrane protein C (OmpC). OmpC mutants selected for resistance to KpneA and KaerA appeared to be equally resistant to KvarIa.

[0116] Further elucidation of the klevicin receptor and translocation element is also important for the practical application of these klevicins. Klevicins hold the greatest promise for use in combating antibiotic-resistant bacteria. However, 97.1% of carbapenem-resistant Klebsiella strains have been shown to not express or to express poorly expressed OmpC or OmpF (Ye et al., 2018). Although our study did not test carbapenem-resistant strains, it has been shown that carbapenem-resistant Klebsiella can be expected to be resistant to KpneA, KaerA, and KvarIa, since all of these klevicins require functional OmpC for their activity. The next step will be to attempt to alter the specificity of klevicins by engineering the proteins, for example, by swapping their receptor-translocation and killing domains. On the other hand, in the current state of research, we can conclude that there is a panel of six highly efficient plant-expressed klebsiella proteins that, together, can target approximately 91% of the clinical strains tested. Even without further manipulation and improvement, these proteins can be further developed for potential use in medicine as antibacterial agents against antibiotic-resistant Klebsiella.

[0117] (Example 10) MIC determination of klevicins KpneA, KaerA, KpneM, KpneM2, KvarM, and KvarIa against several Klebsiella species The minimum inhibitory concentration (MIC) was calculated as the lowest concentration of bacteriocin that prevented visible growth of the corresponding bacterial strain. To measure the MIC of individual klebicins, purified, lyophilized KpneA, KaerA, KpneM, KpneM2, and KvarM proteins were dissolved in sterile distilled water at a concentration of 5 μg / μl. For each individual bacteriocin, two-fold serial dilutions were prepared in MHB medium (Muller-Hinton Broth; Mueller & Hinton (1941) Experimental Biology and Medicine. 48(1):330-333). A 10 μl aliquot of each protein dilution was loaded into an empty well of a sterile 96-well microplate. Every evaluation point was repeated twice.

[0118] An overnight bacterial culture grown in MHB medium was measured at OD in 1 ml of MHB. 595 The bacterial suspension was diluted to a pH of 0.5 and then diluted 1000-fold with 10 ml of the same medium. A 90 μl aliquot of the diluted bacterial suspension was loaded into each well of a 96-well microplate already containing the protein dilutions using a multichannel pipette. An additional aliquot of the diluted bacterial suspension was plated on MHA medium (Mueller-Hinton agar; MHB containing 1.7% agar) for enumeration of CFU in the initial bacterial inoculum. For bacterial growth, the microplate and agar plate were incubated at 30°C or 37°C for 20 hours, depending on the optimal growth conditions for the Klebsiella strain. Klebsiella pneumoniae subsp. ozaene DSM 16358, Klebsiella variicola DSM 15968, and Klebsiella aerogenes DSM 30053 were incubated at 30°C, and Klebsiella quasipneumoniae subsp. similipneumoniae DSM 28212 and Klebsiella oxytoca DSM 5175 at 37°C.

[0119] MICs were determined by visual inspection of bacterial growth in microplate wells. If the difference between two replicates was at one protein dilution, the MIC was determined as the average of the two concentrations. Table 7 shows the MIC values ​​of six Klebsiella strains against five selected susceptible Klebsiella strains measured in μg protein / ml solution and in nM (μM). [Table 7]

[0120] In most cases, the measured klevicin MIC values ​​were less than 1-2 μg / ml. Nguen et al. (Scientific Reports (2018) 8:241) determined the MICs of 20 conventional antibiotics against 1,497 strains of Klebsiella. Typically, the MIC values ​​determined in this study were between 0.5 and 32 μg / ml. Therefore, klevicin is comparable to or superior to conventional antibiotics in terms of antibacterial activity calculated on a weight basis. Considering the difference in molecular weight (most antibiotics have a MW of less than 1 kDa), klevicin has significantly higher antibacterial activity calculated on a molar basis.

[0121] (Example 11) Stability of klebicins KpneA, KaerA, KpneM, KpneM2, KvarM, and KvarIa during storage To evaluate stability, purified lyophilized klevicin protein samples were stored at -20°C, 5°C, and room temperature (approximately 23°C). Protein stability was assessed based on antibacterial activity at the following time points: day 0, 1 week, 2 weeks, 3 weeks, 5 weeks, 3 months, 6 months, 10 months, and 12 months of storage. Protein activity against susceptible bacteria was assessed by liquid culture or radial diffusion assay. Klevicin was tested against the following bacterial strains: KpneM and KpneM2 were tested against Klebsiella pneumoniae DSM 16358, KvarIa was tested against Klebsiella oxytoca DSM 5175, and KpneA, KaerA, and KvarM were tested against Klebsiella quasipneumoniae DSM 28212.

[0122] Lyophilized protein samples were resuspended in distilled water (0.2–0.4 mg / ml). The soluble protein concentration of each sample was measured using a Bradford assay. For stability evaluation in liquid culture, 5 μg of bacteriocin solution was incubated at OD in CAA medium. 600 A 1000mg / mL suspension of a susceptible bacterial strain with a logarithm of 0.3 was added to 1 ml of the bacteriocin (time point "0"). The bacteria mixed with the bacteriocin were incubated on a shaker for 4.5 hours. Klebsiella oxytoca DSM 5175 and Klebsiella quasipneumoniae DSM 28212 were incubated at 37°C, and Klebsiella pneumoniae DSM 16358 was incubated at 30°C. Serial dilutions were made in LB medium. Bacteria were plated on LB agar plates and incubated overnight at 30°C or 37°C, after which CFU were calculated. Antibacterial activity was expressed as CFU / mL Δlog with respect to the untreated sample. 10 was evaluated as. For the radial diffusion assay, serial 1:2 dilutions of solubilized protein in PBS buffer were made. Five microliters of the protein dilutions (1–1.8 μg of undiluted protein) were spotted onto soft agar plates containing susceptible bacterial strains. Residual activity of klebicin was assessed after overnight incubation of the plates. Antibacterial activity was assessed as specific radioactivity units (AU), i.e., the highest dilution that accounts for the difference in the area of ​​unaffected bacterial growth determined by visual inspection of the plate for bacterial growth inhibition by holding the plate in front of a light source. The highest dilution with growth inhibition was recorded as the activity in AU / μg bacteriocin. All experiments were performed in triplicate.

[0123] At −20°C, the activities of all klebicins KpneM, KpneM2, KvarM, KpneA, KaerA, and KvarIa remained stable throughout the year (Fig. 11A,D), despite a slight decrease in the concentration of soluble proteins (Fig. 11G). When stored at 5°C, five of the six klebicins remained active for 1 year; the activity of KpneA decreased (Fig. S11E). The concentrations of KvarM and KpneM2 in solution decreased significantly, suggesting some loss of solubility upon storage (Fig. S11H). In general, klevicins were less stable during storage at room temperature. Nevertheless, the activity of klevicins KvarIa and KpneM remained stable for one year. The activity of KpneA, KvarM, and KpneM2 was dramatically reduced (Figure ​(Figure11C). 11C). This decrease in activity correlated with the concentration of the protein in solution, suggesting a decrease in solubility during storage (Figure 11I).

[0124] (Example 12) Evaluation of bacteriocin KvarIa activity against Klebsiella quasipneumoniae in a mouse gastrointestinal model Klebsiella pneumoniae is a normal component of the human intestinal microbiota. Gastrointestinal carriage has been considered a major reservoir of Klebsiella pneumoniae infection, especially in intensive care unit patients (Gorrie et al., 2017). A 1971 prospective study showed that 18.5% of patients who became colonized with multidrug-resistant Klebsiella pneumoniae after hospitalization had a higher risk of developing a subsequent infection caused by the same bacterium within 21 days compared with patients who did not become intestinal carriage (45% vs. 11%) (Martin and Bachman, 2018). Orally administered klebicin has the potential to be an efficient tool for eradicating asymptomatic, multidrug-resistant Klebsiella pneumoniae from the intestines of hospitalized patients. Klebicin is rapidly inactivated by gastrointestinal enzymes when administered orally and must be formulated for gastroprotection and release in the small and large intestines. In this example, klebicin KvarIa was formulated with Eudragit S100 for delivery to the ileum and colon (release of klebicin at a pH above 7) and administered by oral gavage to mice colonized with Klebsiella quasipneumoniae.

[0125] KvarIA coating A 5% Eudragit S100 solution was prepared by dissolving 0.5 g of Eudragit S100 (Evonik) in 10 ml of miliQ HO and sonicating in an ultrasonic bath for 30 min at 25 °C. 250 μg of KvarIa was dissolved in 200 μg of 5% Eudragit S100. The resulting solution was lyophilized at -51 °C for 24 h. Activity evaluation by simulated gastric digestion and radial diffusion assay. To investigate whether Eudragit-S100-coated KvarIa was resistant to pepsin digestion, a simulated gastric digestion experiment was performed. Protein exposure to simulated gastric fluid (SGF, a commercially available acidic pepsin extract) was performed using a low enzyme-to-substrate ratio. The method was adapted from Moreno et al. (2005), Mandalari et al. (2009), and Eiwegger et al. (2006). Briefly, plant-produced KvarIa and Eudragit-S100-coated KvarIa were mixed with the recommended concentration of SGF and incubated at 37°C for up to 60 min. Every few minutes, samples of the digestion mixture were taken for analysis. Protein digestion into fragments was assessed using SDS-PAGE. In parallel, residual antibacterial activity was assessed using a radial diffusion assay. Coomassie staining on the gel was used to visualize proteolysis and estimate the MW of the peptide products, but this method was only used for uncoated KvarIa because Eudragit S100 distorted the migration of the protein on SDS-PAGE gels.

[0126] Protein samples were incubated at 37°C with rotation at 200 rpm for 10 minutes. Pepsin (0.15 M NaCl, 5 mg / ml) was added to obtain 80-113 U of pepsin per mg of protein (pepsin:protein ratio 1:40) in a final digestion mixture containing 1 mg of protein and 0.025 mg of pepsin. Samples were placed on a shaker (200 rpm, 37°C). Aliquots (50 μl) of the reaction were removed at various time points (0.5, 5, 10, 20, 30, and 60 minutes). Digestion was stopped by adding 0.5 M ammonium bicarbonate (10 μl of NH4HCO3) to inactivate the pepsin and raise the pH to 6.5. Eudragit-coated KvarIa samples were adjusted to pH 8 to dissolve the Eudragit coating. All samples were then diluted 1:2 with distilled water, and 5 μL aliquots of the diluted samples were spotted onto MHA plates with Klebsiella quasipneumoniae DSM28212 lawns for the soft agar overlay assay.

[0127] Under the conditions used (pepsin:protein ratio 1:40), protein coating with Eudragit S100 appears to provide transient resistance to pepsin digestion. While coated KvarIa still exhibited detectable activity in an agar diffusion assay after 20 min of in vitro gastric digestion, uncoated KvarIa was inactivated very rapidly in simulated gastric fluid, completely losing its activity already after 0.5 min of digestion (Figure 12A). The SDS-PAGE profile of the uncoated KvarIa digestion product reveals that uncoated KvarIa is digested very rapidly by pepsin, with no full-length protein detected on the gel after 5 min of digestion (Figure 12B).

[0128] Colonization of the mouse intestine by treatment with Klebsiella quasipneumoniae DSM28212 and KvarIa Prior to the experiment, BALB / c mice (n = 12) were acclimatized in individual cages for 3 days. To eradicate the intestinal microbiota, the mice were given ampicillin (2000 U / ml) and streptomycin (2 mg / ml) in their drinking water for 3 consecutive days. Ampicillin in the drinking water was continued until the end of the experiment. On days 4–6 and 11–13 of the experiment, oral gavage of Klebsiella quasipneumoniae DSM28212 was administered. 9 Mice were administered 100 μg of KvarIa (cfu) once daily. Five days after the last Klebsiella quasipneumoniae gavage (day 18), mice were divided into four groups (n = 3): group 1 received PBS gavage, group 2 received 100 μg of KvarIa, group 3 received 100 μg of Eudragit-S100-coated KvarIa, and group 4 received 1000 μg of Eudragit-S100-coated KvarIa. Gavage continued once daily from days 18 to 21 (4 days). Fecal samples were collected before Klebsiella quasipneumoniae inoculation and then daily from day 18 (just before the start of KvarIa treatment) through day 22 of the experiment. Cages were changed daily during treatment. During all experiments, mice had free access to normal diet, food, and water, but animals were fasted for 6 h before oral gavage. Feces collected on day 18 of the experiment (just before the start of KvarIa treatment) and day 22 of the experiment (1 day after the last KvarIa treatment) were used to quantify the amount of Klebsiella quasipneumoniae DNA by real-time PCR.

[0129] Real-time PCR DNA was extracted from 50 mg of feces using the QIAamp Fast DNA Stool Mini Kit (Qiagen). The Klebsiella hemolysin gene (khe) marker was used for amplification. The khe gene amplification primers used were: forward: 5-GATGAAACGACCTGATTGCATTC-3 (SEQ ID NO: 56), reverse: 5-CCGGGCTGTCGGGATAAG-3 (SEQ ID NO: 57), and probe: 5-6FAM-CGCGAACTGGAAGGGCCCG-TAMRA-3 (SEQ ID NO: 58). TAqMan Universal Master Mix II with UNG and TaqMan probe (Applied Biosystems, JAV) were used. 14 ng of DNA was used for each PCR reaction. The following controls were used in real-time PCR: Klebsiella quasipneumoniae DSM28212 DNA (Klebsiella hemolysin gene khe amplification at cycle 13), Escherichia coli (E. coli) DNA (no khe amplification), and blank (no khe amplification).

[0130] [Table 8] The real-time PCR was validated by setting up a standard curve for detecting Klebsiella quasipneumoniae. According to this curve, CT38 was found to be 10 times more effective than Klebsiella quasipneumoniae. 3 CFU, CT24 is equivalent to 10 6 CFU, and CT17 is 10 8 This corresponds to CFU (Figure 13).

[0131] Colonization of the mouse intestine by Klebsiella quasipneumoniae DSM28212 before and after oral gavage of klebicin Real-time PCR results confirmed that all mice showed the presence of Klebsiella quasipneumoniae DNA in their feces on day 18 of the experiment (before the start of Klebsiella treatment). On day 18 of the experiment, the median C values ​​for each group of mice ranged from 18.3 to 20.5 (Figure 14 and Table 9). Real-time PCR results showed that the number of Klebsiella quasipneumoniae bacteria continued to increase in the feces of mice treated with PBS and uncoated KvarIa. On day 22, the day after the last KvarIa treatment, the median Ct values ​​in the PBS group decreased from 19.97 to 17.23, and in the KvarIa-treated group they decreased from 20.05 to 18.36.

[0132] In contrast to mice treated with PBS and uncoated KvarIa, the amount of Klebsiella quasipneumoniae DNA decreased sharply in the feces of mice treated with Eudragit-S100-coated KvarIa (both concentrations). The median C values ​​increased by 8–8.5 cycles (from 18.5 to 26.3 in the Eudragit_S100-KvarIa 100 μg group and from 18.86 to 27.43 in the Eudragit_S100-KvarIa 1 mg group) (Figure 14 and Table 9).

[0133] [Table 9]

[0134] Thus, real-time PCR results showed that Eudragit-S100-coated KvarIa significantly reduced the amount of Klebsiella quasipneumoniae DNA in mouse feces. The reduction obtained was similar for both doses used (100 μg and 1 mg). In contrast, the amount of Klebsiella quasipneumoniae DNA was slightly increased in uncoated KvarIa- and PBS-treated mice. In conclusion, Eudragit-S100 coated KvarIa showed high activity in reducing Klebsiella quasipneumoniae DNA levels in the intestine of mice, indicating a reduction in the population of this bacterium.

[0135] Nucleic acid and amino acid sequences SEQ ID NO: 1 (called KpneM) (Klebsiella pneumoniae EWD35590.1) MSETMVVATPTGFEPAGYGGGLFSPSTPNHSPQGQIFLQVTLPYYQSTKFCQDSMAWLAQYVKTHGAQDPLTIQVVANNIRYFLNADTNLCHNPKQNVWEAFHSEMTHSGPPPAKYDYHSMSLKQMSGN VVTPAAA FGHYLWGNGEARYVNLPDVGLKITPQMIPELMNIVNSGVTGHIPVDIKFVHDTSVSGGIVPAAYLGHITLRTEGTLDIQSGGAWTYNGVARAF NDTYDFNLGDFRGPIAESMTFLGSQFTGKQYEISMPGQINISGSGRR SEQ ID NO:2 (called KvarM) (Klebsiella varicola CTQ17225.1) MSDTMIVVATPTPGFSYASGLTYGGGAFAGAPANGPSEGQIFFQTVLPAYQSPNLCIGQLAWMTDYINKNGVGNPKTWEVISQNVLIFCSADTALVLNPRIAVYDGFHKTKWAPAKFNFKTQSQEKFSGNVTTPIAAFG HYLWGEGKPRTVDLSSVGLKIQANQIDPVMIAVKNNAAGTYQISGNFNRNTFIDGDIPGLYLGNITMKTEGTLKIDAKGNWNYNGVVRAFNDTYDANPSTHRSKSAEDLTTLLRLTQGTPYEIRIPGELKVSGSGKK

[0136] SEQ ID NO: 3 (called KpneM2) (Klebsiella sp. WP_047066220) MSETLVVVAPAPSAPSMTYGGGLIYSSIPSGPNEGQIFFQTVLPAYSSPNFCTDRLRWMVKFINENGVGNPDTWKTLADVIRYYASADTAISKNPKTNPYDAWHKCPWPPASFDVKTMSVEKFSGSVNTPIVAFGHYL WGEGKPRSVDLSTVGLKVQANQIDPVMIAVKSYGAGTYQINGNFNRNTFDDGVIPGLYLGNITLKTEGTLKIEKNGSWNYNGVIRAFNDTYDANPSN HRSQAAEDLTTLLRITQGTPYEIRIPGEIKVSGSGKK SEQ ID NO: 4 (called KaerM) (Klebsiella aerogenes WP_015367360.1) MTDTLTVTATIPNGSSFNFQFEGMGNYYAAGSSTWDDPAMADAAHLYNAIQSMEDGSFTKALFADWLQFNAKGRENIPMINARFATMETMRFNDPGKAYFQFAQYNEYEGHTPGNNFTSGAFAPFLGLWHYIS GNGVETSLDITTIGLTFNQSNLTPVNDALKSQPPGNYPISSNFGKSVAEDNLYVAALLGRISMKTEGTLSIGESGEWSYNGVVRAYNDTYDANFDPSRGVIAQASTTVLSWFNGKPYPIALPGEIPVQLSGHR

[0137] SEQ ID NO: 5 (called KpneA) (Klebsiella pneumoniae SAV78255.1) MPEETLTVVGGGNNSCNVSWGGGNGNNGGAGYSGKYGGTSYEGATSMLKLNDRVLIQLYLCNPLNPDYIGAPWGSDKDAESIIRANRDKPGKFKANIQNWKTSGTGSLGSPVVGKSYSSGDVDTYSVSFGKEKYNVLYNRKKDSFTTAYVDGGANKPEHSMKDQAIAVVKLYLLNE SQASVIDTTSGIITDSGKTLSGKLGDKYNTLAREAADNIKNFQGKKLRSFNDAMASINELANNPKMKLSQADKTVVSNALKQMDLSALADRFKGLEKAFTWGDRLLKAEKIRDGVVTGVTTGDWQKLAFEVEAMYLSGVAGAVALGITTAMISTVAVALSLPSVAVSALTVVAVIGI SILTSYIDADKAKALNNAVLGLFK

[0138] SEQ ID NO: 6 (called KaerA) (Klebsiella aerogenes WP_063414841.1) MANEDSMTVNGNAGSGVHWGGGSGNGNNGGAGSNGGANVALGGTMEVELGNGFTMIVDGTHPINPGIGGAPWSDDKSNKSAVDALNANKSKPAKFKANIQNYKSGTQGSLNSPAVNKSSSSGDVDTYAVSFGKEKYNV MYNRKKDSFTSGYVDGGATKPEHSMKDQAIAVVQLYLLNEKEKDVITTAAEIISSSGETISGKLGEKYKGLAQGVANDIRNFQGKKIRSFKDAMSSLEQFTKNPNMKLNQADKAALVNALNQVNLSTLADRFKGLERA FTWADRLLKAQKIKDGVVTGVTTGNWQPLALEVEAMYLSGVAGSVALGIVTGMISGLAALISIPALAVTAL TVTAVIGIAIATSYINADTAKALNNAVADLFK

[0139] SEQ ID NO: 7 (called Koxy) (Klebsiella oxytoca WP_024273778) MAGFSYGGFGDGTTWSKERGTGPLPGGGSSGNSGNHSNTTPAEQKQINAIRADKNVRARLSNLIKAARKLNPSVKITVHAISPEGTMAISMEGLTATQARQAGLTGLVMGITVPGYIGSVGDFETGHKYNLKNPEKLNS IGVGTPLDGFNGGENIDTTPKKYRNWRATDEKSFYYVGTTVPMRLLHHLTVSRNKETDTYTMYFKAKDIKALYKIEVKNGDLDNMKLTTLAQGHPLFTAEFAKDIVRNFASVKNESDKEVLDKTSGVIISVGDKAGALLGEKY KALSREVASNIQNFQGKQIRTYDQAMASMNKLMTNPNMKIKAADKTAVINAWKAFNVEDMGNKFTALGRAFKVADYVTKGNNVREKSITGYETGNWGPLMREVESWTVSGLTSSVALAVFSATLGAMLVAAGVST AVVGIIGIIIAGLIGALIDDKFIDKLNNEIIRPAY

[0140] SEQ ID NO:8 (called KpneIa) (Klebsiella pneumoniae BAS34675) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPDKTTTTENVGGGKN GHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAKAEAEAAKAKAEAEAKAKAKAEAAAKAKAEAEAKAKAEAEAKAKAEAAAKAKAEAEAKAKAEAEAKAKAEAAAKAKAEAEAKAKAE AEAKAKAEAEAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKINAQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWRSFFV KIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI

[0141] SEQ ID NO: 9 (referred to as KvarIa) (Klebsiella variicola KDL88409) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPDRTTTTENVGGGKNGH NVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAKAAAKAKAEAEAKAKAEAEAKAKAEAAAKAKAEAEAAKAKAEAAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLAT QAKGKNIR NIDDALKAYEKHKTNINKKINAQDRAAIAKALESVDVKEAAKNFAKFSKGL GYVGPTMDVVDLVLELRKAIKEDNWRTFFVKIEAIAISFGATQLAALAFASLLGAPVGLLG YALIMAGIGALVSDDVVDAANKIIGI SEQ ID NO: 10: ExbB Eco88I fwd AAACTCGGGTTGATGAACCTGTTTTTATACGTCT SEQ ID NO: 11 ExbB Eco81I rev AAACCTGAGGTCAACCTACCCGTAATTTCTGCG

[0142] SEQ ID NO: 12 ExbB Eco88I fwd AAACTCGGGTTGATGAACCTGTTTTTATACGTCT SEQ ID NO: 13 ExbD Eco81I rev AAACCTGAGGTTATTTGGCTTTGACGGTCTC SEQ ID NO: 14 FhuA Eco81I fwd AAACCTCAGGTTTAAGCCCTAAGACCAGACCC SEQ ID NO: 15 FhuA Eco 81I rev AAACCTGAGGTTAGAAAACGGAAGGTGGGCGGTG SEQ ID NO: 16 FimB Eco88I fwd AAACTCGGGGCTCCCGTAGCAAATAAAAACG SEQ ID NO: 17 FimB Eco81I rev AAACCTGAGGGTTACTGAAGCAGCGACAGGCG SEQ ID NO: 18 OmpC Eco88I fwd AAACTCGGGCTTGTGGCTGAACGACTCATCA SEQ ID NO: 19 OmpC Eco81I rev AAACCTGAGGTTAGAACTGGTAAACCAGGCCC

[0143] SEQ ID NO:20 TonB PsyI fwd AAAGACCGGGTCGGCAAAGCTCCTTATCAATAAACA SEQ ID NO: 21 TonB BseSI rev AAAGTGCCCTCAGTTAATCTCGACGCCGTTG SEQ ID NO: 22 Consensus sequence M (KpneM2, KvarM, KpneM, KaerM) MSXTXVVVATPXXXXXXXXTYGGGLFYXXXPXGPSEGQIFFQTVLPAYQSPNFCXDXLAWMADYINXNGVGNPXTWEVIAXNIRYFASADTALXXNPKXXVYDAFHKXXWPPAKXDXXTMSXEKFSGNVXTPIAAFG HYLWGXGKPRSVDLSTVGLKIQANQIDPVMIAVKSXXAGTYXISGNFNRNTFXDGXIPXXYLGNITXKTEGTLKIXXXGXWNYNGVVRAFNDTYDANPSXHRXXIAEDLTTLLXXXQGXPYEIRIPGEIKVSGSGKX

[0144] SEQ ID NO: 23 Consensus sequence A (KaerA, KpneA) MXXEXXXXVXGXNXXXXVXWGGXXGNGNNGGAGXXGXXGXXXXXGXTXXXXLXBXXXXXXXXXXPJNPXXXGAPWXXXXSBKXAXXXJXANXXKP XKFKANIQNXKXXXXGSLXSPXVXKSXSSGDVDTYXVSFGKEKYNVXYNRKKDSFTXXYVDGGAXKPEHSMKDQAIAVVXLYLLNEXZXXVIXTXX XIIXXSGXTJSGKLGXKYXXLAXXXABBIXNFQGKKJRSFXDAMXSJXZXXXNPXMKLXQADKXXXXNALXQXBLSXLADRFKGLEXAFTWXDRL LKAZKIXDGVVTGVTTGBWQXLAXEVEAMYLSGVAGXVALGIXTXMISXXAXXJSJPXXAVXALTVXAVIGIXIXTSYIBADXAKALNNAVXXLFK

[0145] SEQ ID NO: 24 Consensus sequence Ia (KpneIa, KvarIa) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHD AMPDXTTTTENVGGGKNGHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAKXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXAXAKAKAEEAAKAKAEAXAKAKAEAXAKAKAEEAAKAKAEEAAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKIN AQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWRXFFVKIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI

[0146] SEQ ID NO: 25 (sequence stretch conserved in SEQ ID NO: 22) TEGTL SEQ ID NO: 26 (sequence stretch conserved in SEQ ID NO: 22) YNGV SEQ ID NO: 27 (sequence stretch conserved in SEQ ID NO: 22) RAFNDTYD SEQ ID NO: 28 (sequence stretch conserved in SEQ ID NO: 23) GNGNNGGAG SEQ ID NO: 29 (sequence stretch conserved in SEQ ID NO: 23) GAPW SEQ ID NO: 30 (sequence stretch conserved in SEQ ID NO: 23) KFKANIQN

[0147] SEQ ID NO: 31 (sequence stretch conserved in SEQ ID NO: 23) SSGDVDTY SEQ ID NO: 32 (sequence stretch conserved in SEQ ID NO: 23) VSFGKEKYNV SEQ ID NO: 33 (sequence stretch conserved in SEQ ID NO: 23) YNRKKDSFT SEQ ID NO: 34 (sequence stretch conserved in SEQ ID NO: 23) YVDGGA SEQ ID NO: 35 (sequence stretch conserved in SEQ ID NO: 23) KPEHSMKDQAIAVV SEQ ID NO: 36 (sequence stretch conserved in SEQ ID NO: 23) LYLLNE

[0148] SEQ ID NO: 37 (sequence stretch conserved in SEQ ID NO: 23) SGKLG SEQ ID NO: 38 (sequence stretch conserved in SEQ ID NO: 23) NFQGKK SEQ ID NO: 39 (sequence stretch conserved in SEQ ID NO: 23) QADK SEQ ID NO: 40 (sequence stretch conserved in SEQ ID NO: 23) LADRFKGL SEQ ID NO: 41 (sequence stretch conserved in SEQ ID NO: 23) AFTW SEQ ID NO: 42 (sequence stretch conserved in SEQ ID NO: 23) DRLLKA SEQ ID NO: 43 (sequence stretch conserved in SEQ ID NO: 23) DGVVTGVTTG SEQ ID NO: 44 (sequence stretch conserved in SEQ ID NO: 23) EVEAMYLSGVAG

[0149] SEQ ID NO: 45 (sequence stretch conserved in SEQ ID NO: 23) VALGI SEQ ID NO: 46 (sequence stretch conserved in SEQ ID NO: 23) ALTV SEQ ID NO: 47 (sequence stretch conserved in SEQ ID NO: 23) AVIGI SEQ ID NO: 48 (sequence stretch conserved in SEQ ID NO: 23) TSYI SEQ ID NO: 49 (sequence stretch conserved in SEQ ID NO: 23) AKALNNAV SEQ ID NO: 50 (sequence stretch conserved in SEQ ID NO: 24) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPD

[0150] SEQ ID NO: 51 (sequence stretch conserved in SEQ ID NO: 24) TTTTENVGGGKNGHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAK SEQ ID NO: 52 (sequence stretch conserved in SEQ ID NO: 24) AKAKAEAEAKAKAEA SEQ ID NO: 53 (sequence stretch conserved in SEQ ID NO: 24) AKAKAEA SEQ ID NO: 54 (sequence stretch conserved in SEQ ID NO: 24) AKAKAEAEAKAKAEAEAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKINAQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWR

[0151] SEQ ID NO: 55 (sequence stretch conserved in SEQ ID NO: 24) FFVKIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI SEQ ID NO: 56 khe gene amplification forward primer GATGAAACGACCTGATTGCATTC SEQ ID NO: 57 khe gene amplification reverse primer CCGGGCTGTCGGGATAAG SEQ ID NO: 58 khe gene amplification probe sequence (labeled with 6FAM at the 5' end and TAMRA at the 3' end) CGCGAACTGGAAGGGCCCG

[0152] References TIFF2026031930000013.tif220170 TIFF2026031930000014.tif194170The contents of European Patent Application No. 19178 676.3, filed June 6, 2019, are incorporated herein by reference, including the description, claims, figures, and sequence listing.

Claims

1. A protein having cytotoxic activity against Klebsiella or a composition comprising said protein for use in therapy.

2. 2. The protein or composition for use according to claim 1, wherein the protein has lipid II cleavage activity or pore-forming activity in the cell membrane of Klebsiella cells.

3. 3. A protein or composition for use according to claim 1 or 2 for use in a method of treating an infection in a subject caused by Klebsiella, such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella variicola, wherein the Klebsiella may be antibiotic resistant, such as carbapenem resistant.

4. 1. A protein or a composition comprising said protein for use in a method of treating an infection in a subject caused by Klebsiella, e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella variicola, wherein said Klebsiella may be antibiotic-resistant, e.g., carbapenem-resistant.

5. 1. A method of treating an infection with Klebsiella in a subject in need thereof, comprising administering to the subject a protein having cytotoxic activity against Klebsiella or a composition comprising said protein.

6. 1. A method for preventing or reducing infection or contamination of an object with one or more Klebsiella species, the method comprising contacting the object with a protein having cytotoxic activity against Klebsiella or a composition comprising said protein.

7. 7. The protein, composition or method of any one of claims 1 to 6, wherein the protein comprises or consists of a first amino acid sequence segment and a second amino acid sequence segment, the first segment preferably being an N-terminal segment of the protein and the second segment being a C-terminal segment of the protein.

8. (A) a first segment; (A-ii) amino acid residues 1 to 127 of SEQ ID NO: 2 (KvarM); (Ai) amino acid residues 1 to 128 of SEQ ID NO: 1 (KpneM); (A-iii) amino acid residues 1 to 123 of SEQ ID NO: 3 (KpneM2); (A-iv) amino acid residues 1 to 118 of SEQ ID NO: 4 (KaerM); (A-v) amino acid residues 1 to 170 of SEQ ID NO: 5 (KpneA); (A-vi) amino acid residues 1 to 172 of SEQ ID NO: 6 (KaerA); (A-vii) amino acid residues 1 to 255 of SEQ ID NO: 7 (Koxy); (A-viii) amino acid residues 1 to 288 of SEQ ID NO: 8 (KpneIa); or (A-ix) Amino acid residues 1 to 236 of SEQ ID NO: 9 (KvarIa) comprising or consisting of the amino acid sequence of or (B) the first segment is (B-ii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (Bi) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (B-iii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (B-iv) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (Bv) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO:5 (B-vi) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (B-vii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (B-viii) has at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8; or (B-ix) having at least 70% sequence identity with the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9 Contains an amino acid sequence; or (C) the first segment is (C-ii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2; (C-i) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1; (C-iii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3; (C-iv) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4; (C-v) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5; (C-vi) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6; (C-vii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7; (C-viii) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 288 of SEQ ID NO: 8; or (C-ix) having 1 to 40 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO:

9.

8. The protein, composition or method of claim 7, comprising an amino acid sequence.

9. In item (B), any one of the sequence identities is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%; and / or 9. The protein, composition or method according to claim 8, wherein in item (C), the number of amino acid substitutions, additions, insertions and / or deletions is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably at least 1 to 5 compared to any one of the amino acid sequences.

10. (D) the second segment is (D-ii) amino acid residues 128 to 276 of SEQ ID NO: 2 (KvarM); (Di) amino acid residues 129 to 278 of SEQ ID NO: 1 (KpneM); (D-iii) amino acid residues 124 to 272 of SEQ ID NO: 3 (KpneM2); (D-iv) amino acid residues 119 to 266 of SEQ ID NO: 4 (KaerM); (D-v) amino acid residues 171 to 377 of SEQ ID NO: 5 (KpneA); (D-vi) amino acid residues 173 to 379 of SEQ ID NO: 6 (KaerA); (D-vii) amino acid residues 256 to 452 of SEQ ID NO: 7 (Koxy); (D-viii) amino acid residues 289 to 466 of SEQ ID NO: 8 (KpneIa), or (D-ix) Amino acid residues 237 to 414 of SEQ ID NO: 9 (KvarIa) comprising or consisting of the amino acid sequence of or (E) the second segment is (E-ii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 128 to 276 of SEQ ID NO: 2; (E-i) having at least 70% sequence identity with the amino acid sequence of amino acid residues 129 to 278 of SEQ ID NO: 1; (E-iii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 124 to 272 of SEQ ID NO: 3; (E-iv) having at least 70% sequence identity with the amino acid sequence of amino acid residues 119 to 266 of SEQ ID NO: 4; (E-v) having at least 70% sequence identity with the amino acid sequence of amino acid residues 171 to 377 of SEQ ID NO: 5; (E-vi) having at least 70% sequence identity with the amino acid sequence of amino acid residues 173 to 379 of SEQ ID NO: 6; (E-vii) having at least 70% sequence identity with the amino acid sequence of amino acid residues 256 to 452 of SEQ ID NO: 7; (E-viii) has at least 70% sequence identity with the amino acid sequence of amino acid residues 289 to 466 of SEQ ID NO: 8; or (E-ix) having at least 70% sequence identity with the amino acid sequence of amino acid residues 237 to 414 of SEQ ID NO: 9 Contains an amino acid sequence; or (F) the second segment is (F-ii) having 1 to 30 amino acid substitutions, additions, insertions or deletions compared to the amino acid sequence of amino acid residues 128 to 276 of SEQ ID NO: 2; (Fi) having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 129 to 278 of SEQ ID NO: 1; (F-iii) having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 124 to 272 of SEQ ID NO: 3; (F-iv) having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 119 to 266 of SEQ ID NO: 4; (F-v) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 171 to 377 of SEQ ID NO: 5; (F-vi) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 173 to 379 of SEQ ID NO: 6; (F-vii) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 256 to 452 of SEQ ID NO: 7; (F-viii) having 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 289 to 466 of SEQ ID NO: 8; (F-ix) has 1 to 35 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 237 to 414 of SEQ ID NO:

9.

10. The protein, composition or method of any one of claims 7, 8 and 9, comprising an amino acid sequence.

11. 11. The protein, composition or method of claim 9 or 10, wherein the first segment is any one of items Ai to A-iv, Bi to B-iv or Ci to C-iv, and the second segment is any one of items Di to D-iv, Ei to E-iv or Fi to F-iv.

12. 12. The protein, composition or method of claim 11, wherein the first segment is any one or more of items Ai to A-iv and the second segment is any one of items Di to D-iv, respectively; or the first segment is any one of items Bi to B-iv and the second segment is any one of items E-i to E-iv, respectively; or the first segment is any one of items Ci to C-iv and the second segment is any one of items F-i to F-iv, respectively.

13. The protein is (a) (a-ii) SEQ ID NO: 2 (KvarM); (ai) SEQ ID NO: 1 (KpneM); (a-iii) SEQ ID NO: 3 (KpneM2); (a-iv) SEQ ID NO: 4 (KaerM); (a-v) SEQ ID NO: 5 (KpneA); (a-vi) SEQ ID NO: 6 (KaerA); (a-vii) SEQ ID NO: 7 (Koxy); (a-viii) SEQ ID NO: 8 (KpneIa), or (a-ix) SEQ ID NO: 9 (KvarIa) the amino acid sequence of or (b)(b-ii) has at least 70% sequence identity with the amino acid sequence of SEQ ID NO:2; (b-i) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1; (b-iii) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 3; (b-iv) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 4; (b-v) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 5; (b-vi) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 6; (b-vii) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 7; (b-viii) has at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 8; or (b-ix) having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 9 amino acid sequence; or (c) (c-ii) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 2; (ci) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 1; (c-iii) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 3; (c-iv) having 1 to 80 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 4; (c-v) having 1 to 110 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 5; (c-vi) having 1 to 110 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 6; (c-vii) having 1 to 130 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 7; (c-viii) has 1 to 130 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 8; or (c-ix) having 1 to 120 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of SEQ ID NO: 9 Amino acid sequence 7. The protein, composition or method of any one of claims 1 to 6, comprising or consisting of an amino acid sequence comprising or consisting of:

14. In item (b), any one of the sequence identities is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%; and / or 14. The protein, composition or method according to claim 13, wherein in item (c), the number of amino acid substitutions, additions, insertions and / or deletions is 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably at least 1 to 5 compared to any one of the amino acid sequences.

15. 15. The protein, composition or method of any one of claims 1 to 14, having cytotoxic activity against Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella granulomatis, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella variicola.

16. the protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24, wherein each X represents any one of the 20 standard amino acid residues or the absence of an amino acid residue, and each J represents either L (leucine) or I (isoleucine); or 16. The protein, composition or method of any one of claims 1 to 15, wherein the protein is any one defined in SEQ ID NOs: 1 to 4, 7, 8 or 22, preferably any one defined in SEQ ID NOs: 1 to 4 or 22.

17. The cytotoxic activity of the protein is - said protein, and - Comparison protein of the amino acid sequence of SEQ ID NO: 1 is such that spotting 20 microliters of each of a solution of said protein and a comparative protein onto a lawn of a susceptible Klebsiella strain on an agar plate, followed by incubation of the agar plate at 37°C for 16 hours, produces a spot of at least the same diameter that is free of viable bacteria of susceptible Klebsiella quasipneumoniae subsp. similipneumoniae SB30 (DSM 28212), 17. The protein, composition or method of any one of claims 1 to 16, wherein the concentration of the protein in solution is up to 5 times the concentration of a solution of a comparison protein.

18. A protein having cytotoxic activity against Klebsiella, which has lipid II cleavage activity or pore-forming ability in the cell membrane of Klebsiella cells, and preferably comprises or consists of a first amino acid sequence segment and a second amino acid sequence segment.

19. 19. The protein of claim 18, further defined in any one of claims 7 to 17.

20. A composition comprising one or more proteins as defined in any one of claims 7 to 12, 13 to 17 and 18 to 19, and a carrier.

21. A pharmaceutical composition comprising one or more proteins as defined in any one of claims 7 to 12, 13 to 17 and 18 to 19.

22. A composition, preferably a pharmaceutical composition, comprising one or more proteins, wherein said one or more proteins comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24, wherein each X represents any one of the 20 standard amino acid residues or the absence of an amino acid residue, and each J represents either L (leucine) or I (isoleucine); or the protein is any one of SEQ ID NOs: 1-4, 7, 8 or 22, preferably any one of SEQ ID NOs: 1-4 or 22; The composition preferably comprises a carrier.

23. 23. The composition according to any one of claims 20 to 22, which is plant material or an extract thereof, wherein the plant material may be material from a plant expressing said one or more proteins, preferably an edible plant expressing said one or more proteins.

24. 24. The composition of any one of claims 20 to 23, wherein the one or more proteins are formulated for oral delivery to the small intestine or large intestine.

25. 25. An oral formulation comprising a protein as defined in any one of claims 1 to 17, 18 and 19 or a composition according to any one of claims 20 to 24, which is capable of protecting the protein from gastric conditions and releasing the protein in the intestine.

26. 20. A nucleic acid molecule or nucleic acid construct encoding a protein as defined in any one of claims 1 to 17, 18 or 19, preferably a protein as defined in any one of claims 7 to 17, said nucleic acid molecule or nucleic acid construct comprising a transcription promoter that is preferably active in plant cells and a nucleotide sequence encoding said protein for expressing said nucleotide sequence under the control of said promoter in a cell, preferably a plant cell.

27. A nucleic acid molecule or nucleic acid construct encoding a protein as defined in any one of claims 1 to 17, 18 or 19, preferably a protein as defined in any one of claims 7 to 17, which is or encodes a viral (DNA or RNA) replicon comprising a nucleotide sequence encoding said protein for expressing said nucleotide sequence in a cell, preferably a plant cell.

28. A plant, plant tissue or plant cell comprising a protein as defined in any one of claims 1 to 17, 18 and 19, or comprising a nucleic acid molecule or nucleic acid construct as defined in claim 26 or 27.

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