Klebsiella control

Abstract

The present invention provides a protein having cytotoxic activity against Klebsiella, wherein the protein has lipid II cleavage activity or pore-forming ability in the Klebsiella cell membrane.

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 said proteins. The present invention further provides proteins having cytotoxic activity against Klebsiella and compositions comprising the said proteins for therapeutic use. The present invention also provides proteins or compositions comprising the said proteins for use in methods of treating infections of a subject caused by Klebsiella. Furthermore, it provides oral enteric-coated formulations for delivering proteins or compositions to the small or large intestine. Furthermore, it provides pulmonary formulations for delivering proteins or compositions to the lungs. The present invention also provides methods for preventing or reducing infection or contamination of objects by Klebsiella, methods for treating Klebsiella infections in a subject or patient requiring such treatment, and methods for producing compositions comprising the said 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 said proteins, and plants, plant tissues or plants comprising the said nucleic acid molecules. [Background technology]

[0002] Klebsiella are non-motile, rod-shaped, Gram-negative bacteria encased in a capsular polysaccharide that confers resistance to many host defense mechanisms. Klebsiella are opportunistic pathogens found in the environment and on the mucous membranes of mammals. The following three species of the genus Klebsiella are commonly associated with human diseases: K. pneumoniae, K. oxytoca, and K. granulomatis. Recently, two more Klebsiella species, K. variicola and K. quasipneumoniae, have also been found to cause fatal infections (Long et al. 2017). The primary sources of infection with the pathogen are the patient's gastrointestinal tract and the hands of hospital staff. Outside of hospitals, Klebsiella infection typically occurs in the lungs. The disease typically affects middle-aged and older men with debilitating conditions such as alcoholism, diabetes, or chronic bronchopulmonary spirochetosis (Chan et al., 2009). This patient population is thought to have impaired host defense mechanisms in the respiratory tract. The organism enters the host after the host inhales colonizing oropharyngeal microorganisms into 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 hospital-acquired infections include the urinary tract, lower respiratory tract, bile duct, and surgical wound sites. The range of clinical syndromes includes pneumonia, bacteremia, thrombophlebitis, urinary tract infections (UTIs), cholecystitis, diarrhea, upper respiratory tract infections, wound infections, osteomyelitis, and meningitis (Miftode et al., 2008). The presence of invasive devices, contamination of respiratory support devices, use of urinary catheters, and use of antibiotics are factors that increase the likelihood of hospital-acquired infections by Klebsiella species (Weisenberg et al., 2009). K. pneumoniae is one of the six pathogens that make up hospital-acquired ESKAPE infections (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter) that rapidly develop antibiotic resistance. In 2016, an outbreak of hospital-acquired pneumonia was reported in China, resulting in the deaths of five surgical patients due to infection with the highly virulent carbapenem-resistant K. pneumoniae (CRPK) ST11 strain (Gu et al, 2017). This strain is highly virulent and highly resistant, and can therefore be referred to as a "superbug." The ST11 CR HvKP strain infects relatively healthy populations with normal immunity. These are mucoid strains and adhere to all surfaces in the intensive care unit. Colistin is a last-resort antibiotic against carbapenem-resistant enterobacteria, but it is largely ineffective against this strain. For the time being, ceftazidime / avibactam can be used to treat these infections, but resistance to these antibiotics may soon be acquired.

[0004] The increasing drug resistance of pathogens observed to date is an international challenge, and the development of a new generation of antimicrobial agents is urgently needed. Bacteria produce toxic proteins called bacteriocins when competing for ecological 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, inhibition of DNase and RNase activity, and inhibition of protein synthesis or DNA replication. Bacteriocins produced by Gram-positive bacteria are usually called bacteriocins of a predetermined class according to their characteristics, while bacteriocins produced by Gram-negative bacterial 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 produced solely by bacteria, but also by various organisms when faced with bacterial infection. Antimicrobial peptides are used in pharmaceuticals as peptide antibiotics, such as colistin (polymyxin derived from Paenibacillus polymyxa) and vancomycin (derived from Amycolatopsis orientalis). However, such antibiotics are mostly used for topical administration or as a last resort. Other challenges with peptides include the inefficiency and high cost of purification from natural sources. While this can be overcome by chemical synthesis, it remains expensive. The production of recombinant peptides in heterologous hosts is also difficult due to their toxicity to host cells (Li, 2011).

[0005] To date, no colicin-like antibiotics have been registered. However, there are studies in the scientific literature on the potential use of colicin-like bacteriocins as antimicrobial agents against Gram-negative pathogens. Colicin is the most studied bacteriocin, and several research groups have studied pyocin (Grinter et al., 2013; Ghequire, de Mot, 2014). During this time, bacteriocins belonging to the class of nucleases derived from Klebsiella have received little attention, and only a few research results have been published (James et al, 1987; Riley et al, 2001; Chavan et al, 2005). Detailed research results on the expression, purification, and activity testing of klebicin do not exist. Regardless of prior art, the object of the present invention is to provide a drug that is active against Klebsiella. Another object of the present invention is to provide a drug or composition that can be used to treat a target Klebsiella infection (particularly an infection caused by antibiotic-resistant Klebsiella). Furthermore, an object of the present invention is to provide a method for preventing or reducing contamination of an object (for example, food by one or more Klebsiella species). [Overview of the project]

[0006] The inventors have discovered a novel bacteriocin that is active against Klebsiella. Therefore, the present invention provides the following: 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 can bind to components of Klebsiella cells and the second amino acid sequence segment has lipid II cleavage activity or pore-forming ability in the cell membrane of Klebsiella cells. 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 1-128 of sequence number 1 (KpneM), (A-ii) Amino acid residues 1-127 of sequence number 2 (KvarM), (A-iii) Amino acid residues 1-123 of sequence number 3 (KpneM2), (A-iv) Amino acid residues 1-118 of sequence number 4 (KaerM), (Av) Amino acid residues 1-170 of sequence number 5 (KpneA), (A-vi) Amino acid residues 1-172 of sequence number 6 (KaerA), (A-vii) Amino acid residues 1-255 of sequence number 7 (Koxy), (A-viii) Amino acid residues 1-288 of sequence number 8 (KpneIa), or (A-ix) Amino acid residues 1-236 of sequence number 9 (KvarIa) Containing or consisting of the amino acid sequence; or (B) The first segment is (Bi) The amino acid sequence of amino acid residues 1-128 of sequence number 1 has at least 70% sequence identity. (B-ii) Having at least 70% sequence identity with amino acid residues 1-127 of SEQ ID NO: 2 (B-iii) Having at least 70% sequence identity with amino acid residues 1-123 of SEQ ID NO: 3, (B-iv) Having at least 70% sequence identity with amino acid residues 1-118 of sequence number 4, (Bv) The amino acid sequence of amino acid residues 1-170 of sequence number 5 has at least 70% sequence identity. (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) having 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 comprising an amino acid sequence; or (C) wherein the first segment (C-i) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1, (C-ii) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO: 2, (C-iii) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 123 of SEQ ID NO: 3, (C-iv) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 118 of SEQ ID NO: 4, (C-v) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 170 of SEQ ID NO: 5, (C-vi) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 172 of SEQ ID NO: 6, (C-vii) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with the amino acid sequence of amino acid residues 1 to 255 of SEQ ID NO: 7, (C-viii) has 1 to 40 amino acid substitutions, additions, insertions and / or deletions as compared with 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 as compared with the amino acid sequence of amino acid residues 1 to 236 of SEQ ID NO: 9 The protein according to any one of items 2 or 3, comprising an amino acid sequence.

[0009] 5) (A) The amino acid sequence of the first segment is (A-i) 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); (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) is the amino acid sequence of; or (B) The amino acid sequence of the first segment is (B-i) 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; (B-v) 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 amino acid residues 1-172 of sequence number 6, (B-vii) At least 70% sequence identity with amino acid residues 1-255 of sequence number 7, (B-viii) At least 70% sequence identity with amino acid residues 1-288 of sequence number 8, or (B-ix) At least 70% sequence identity with amino acid residues 1-236 of sequence number 9. Is it sharp? or (C) The amino acid sequence of the first segment is (Ci) Compared to the amino acid sequence of amino acid residues 1-128 of SEQ ID NO: 1, 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-ii) Compared with the amino acid sequence of amino acid residues 1-127 of SEQ ID NO: 2, 1-40 amino acid substitutions, additions, insertions and / or deletions, (C-iii) Compared to the amino acid sequence of amino acid residues 1-123 of SEQ ID NO: 3, 1-40 amino acid substitutions, additions, insertions and / or deletions, (C-iv) Compared with the amino acid sequence of amino acid residues 1-118 of SEQ ID NO: 4, 1-40 amino acid substitutions, additions, insertions and / or deletions. (Cv) Compared to the amino acid sequence of amino acid residues 1-170 of SEQ ID NO: 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-vi) Compared with the amino acid sequence of amino acid residues 1-172 of SEQ ID NO: 6, 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-vii) Compared to the amino acid sequence of amino acid residues 1-255 of SEQ ID NO: 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-viii) Compared to the amino acid sequence of amino acid residues 1-288 of SEQ ID NO: 8, 1-40 amino acid substitutions, additions, insertions and / or deletions, (C-ix) Compared to the amino acid sequence of amino acid residues 1-236 of SEQ ID NO: 9, 1-40 amino acid substitutions, additions, insertions and / or deletions. A protein having the properties described in either item 2 or 3.

[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 The protein according to item 4 or 5, wherein in item (C), the number of substitutions, additions, insertions and / or deletions of the amino acids 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.

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

[0012] 8)(D) The amino acid sequence of the second segment is (Di) Amino acid residues 129-278 of sequence number 1 (KpneM), (D-ii) Amino acid residues 128-276 of sequence number 2 (KvarM), (D-iii) Amino acid residues 124-272 of sequence number 3 (KpneM2), (D-iv) Amino acid residues 119-266 of sequence number 4 (KaerM), (Dv) Amino acid residues 171-377 of sequence number 5 (KpneA), (D-vi) Amino acid residues 173-379 of sequence number 6 (KaerA), (D-vii) Amino acid residues 256-452 of sequence number 7 (Koxy), (D-viii) Amino acid residues 289-466 of sequence number 8 (KpneIa), or (D-ix) From amino acid residues 237-414 of sequence number 9 (KvarIa) It contains or consists of the following amino acid sequences: or (E) The amino acid sequence of the second segment is (Ei) At least 80% sequence identity with amino acid residues 129-278 of sequence number 1, (E-ii) At least 80% sequence identity with amino acid residues 128-276 of sequence number 2, (E-iii) At least 80% sequence identity with amino acid residues 124-272 of sequence number 3, (E-iv) At least 80% sequence identity with amino acid residues 119-266 of sequence number 4, (Ev) At least 80% sequence identity with amino acid residues 171-377 of sequence number 5, (E-vi) At least 80% sequence identity with amino acid residues 173-379 of sequence number 6, (E-vii) At least 80% sequence identity with amino acid residues 256-452 of sequence number 7, (E-viii) At least 80% sequence identity with amino acid residues 289-466 of sequence number 8, or (E-ix) At least 80% sequence identity with amino acid residues 237-414 of sequence number 9. Do you have it? or (F) The amino acid sequence of the second segment is (Fi) Compared to the amino acid sequence of amino acid residues 129-278 of SEQ ID NO: 1, 1-30 amino acid substitutions, additions, insertions and / or deletions. (F-ii) Compared with the amino acid sequence of amino acid residues 128-276 of SEQ ID NO: 2, 1-30 amino acid substitutions, additions, insertions and / or deletions, (F-iii) Compared to the amino acid sequence of amino acid residues 124-272 of SEQ ID NO: 3, 1-30 amino acid substitutions, additions, insertions and / or deletions, (F-iv) Compared to the amino acid sequence of amino acid residues 119-266 of SEQ ID NO: 4, 1-30 amino acid substitutions, additions, insertions and / or deletions. (Fv) Compared to the amino acid sequence of amino acid residues 171-377 of SEQ ID NO: 1-35 amino acid substitutions, additions, insertions and / or deletions. (F-vi) Compared to the amino acid sequence of amino acid residues 173-379 of SEQ ID NO: 6, 1-35 amino acid substitutions, additions, insertions and / or deletions. (E-vii) Compared to the amino acid sequence of amino acid residues 256-452 of SEQ ID NO: 1-35 amino acid substitutions, additions, insertions and / or deletions. (F-viii) Compared to the amino acid sequence of amino acid residues 289-466 of SEQ ID NO: 8, 1-35 amino acid substitutions, additions, insertions and / or deletions, (F-ix) Compared to the amino acid sequence of amino acid residues 237-414 of SEQ ID NO: 9, 1-35 amino acid substitutions, additions, insertions and / or deletions. A protein having any one of the items 2 to 7.

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

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

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

[0016] 14) The cytotoxic activity of the protein is such that, when 20 microliters each of the protein and the comparative protein of the amino acid sequence of Sequence ID No. 1 are spotted onto a colony of susceptible Klebsiella strains on an agar plate, and the agar plate is incubated at 37°C for 16 hours, spots free of viable Klebsiella quasipneumoniae subspecies similipneumoniae SB30 (DSM 28212) are produced, and the concentration of the protein in the solution is up to 5 times the concentration of the respective comparative protein solution, as described in any one of items 4 to 13.

[0017] 15)(a)(ai) Sequence ID 1(KpneM), (a-ii) Sequence ID 2 (KvarM), (a-iii) Sequence ID 3 (KpneM2), (a-iv) Sequence ID 4 (KaerM), (av) Sequence ID 5 (KpneA), (a-vi) Sequence ID 6 (KaerA), (a-vii) Sequence ID 7 (Koxy), (a-viii) Sequence ID 8 (KpneIa), or (a-ix) Sequence ID 9 (KvarIa) The amino acid sequence; or (b)(bi) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, (b-ii) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 2, (b-iii) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 3, (b-iv) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 4, (bv) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 5, (b-vi) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 6, (b-vii) Having at least 70% sequence identity with the amino acid sequence of sequence number 7, (b-viii) Having at least 70% sequence identity with the amino acid sequence of sequence number 8, or (b-ix) Has at least 70% sequence identity with the amino acid sequence of Sequence ID No. 9. amino acid sequence; or (c)(ci) Compared to the amino acid sequence of SEQ ID NO: 1, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (c-ii) Compared to the amino acid sequence of SEQ ID NO: 2, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (c-iii) Compared to the amino acid sequence of SEQ ID NO: 3, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (c-iv) Compared to the amino acid sequence of SEQ ID NO: 4, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (cv) Compared to the amino acid sequence of SEQ ID NO: 5, it has 1 to 110 amino acid substitutions, additions, insertions and / or deletions. (c-vi) Compared to the amino acid sequence of SEQ ID NO: 6, there are 1 to 110 amino acid substitutions, additions, insertions and / or deletions. (c-vii) Compared to the amino acid sequence of SEQ ID NO: 7, it has 1 to 130 amino acid substitutions, additions, insertions and / or deletions. (c-viii) Compared to the amino acid sequence of SEQ ID NO: 8, it has 1 to 130 amino acid substitutions, additions, insertions and / or deletions, or (c-ix) Compared to the amino acid sequence of SEQ ID NO: 9, it has 1 to 120 amino acid substitutions, additions, insertions, and / or deletions. amino acid sequence A protein as described in any one of items 1 to 14, which contains, or comprises an amino acid sequence consisting thereof.

[0018] 16) (a) The amino acid sequence of the protein is (ai) Sequence ID 1 (KpneM), (a-ii) Sequence ID 2 (KvarM), (a-iii) Sequence ID 3 (KpneM2), (a-iv) Sequence ID 4 (KaerM), (av) Sequence ID 5 (KpneA), (a-vi) Sequence ID 6 (KaerA), (a-vii) Sequence ID 7 (Koxy), (a-viii) Sequence ID 8 (KpneIa), or (a-ix) Sequence ID 9 (KvarIa) Is it the amino acid sequence? or (b) The amino acid sequence of the protein is (bi) At least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, (b-ii) At least 70% sequence identity with the amino acid sequence of Sequence ID No. 2, (b-iii) At least 70% sequence identity with the amino acid sequence of Sequence ID No. 3, (b-iv) At least 70% sequence identity with the amino acid sequence of Sequence ID No. 4, (bv) Amino acid sequence of sequence number 5 has at least 70% sequence identity, (b-vi) At least 70% sequence identity with the amino acid sequence of sequence number 6, (b-vii) At least 70% sequence identity with the amino acid sequence of sequence number 7, (b-viii) At least 70% sequence identity with the amino acid sequence of sequence number 8. ,or (b-ix) At least 70% sequence identity with the amino acid sequence of Sequence ID No. 9 Is it sharp? or (c) The amino acid sequence of the protein is (ci) Compared to the amino acid sequence of SEQ ID NO: 1, 1 to 80 amino acid substitutions, additions, insertions and / or deletions, (c-ii) Compared to the amino acid sequence of SEQ ID NO: 2, 1 to 80 amino acid substitutions, additions, insertions and / or deletions, (c-iii) Compared to the amino acid sequence of SEQ ID NO: 3, 1 to 80 amino acid substitutions, additions, insertions and / or deletions, (c-iv) Compared to the amino acid sequence of SEQ ID NO: 4, 1 to 80 amino acid substitutions, additions, insertions and / or deletions, (cv) Compared to the amino acid sequence of SEQ ID NO: 5, 1 to 110 amino acid substitutions, additions, insertions and / or deletions. (c-vi) Compared to the amino acid sequence of SEQ ID NO: 6, 1 to 110 amino acid substitutions, additions, insertions and / or deletions. (c-vii) Compared to the amino acid sequence of SEQ ID NO: 7, 1 to 130 amino acid substitutions, additions, insertions and / or deletions. (c-viii) Compared to the amino acid sequence of SEQ ID NO: 8, 1 to 130 amino acid substitutions, additions, insertions and / or deletions, (c-ix) Compared to the amino acid sequence of SEQ ID NO: 9, 1 to 120 amino acid substitutions, additions, insertions and / or deletions. A protein having the properties described in item 1 or 15.

[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 The protein according to either item 15 or 16, wherein in item (c), the number of substitutions, additions, insertions and / or deletions of the amino acids 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.

[0020] 18) The cytotoxic activity of any one of the proteins in items (bi)~(b-ix) or (ci)~(c-ix) is such that, when 20 microliters each of the solutions of the protein and the comparison protein of the amino acid sequence numbers in items (bi)~(b-ix) or (ci)~(c-ix) are spotted onto a colony of susceptible Klebsiella strains on an agar plate, and the agar plate is incubated at 37°C for 16 hours, spots free of viable Klebsiella quassinneumoniae subspecies simili pneumoniae SB30 (DSM 28212) are produced, according to any one of items 15, 16, or 17, wherein the concentration of the protein in the solution is up to 5 times the concentration of the respective comparison protein solution.

[0021] 19) A 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 to undecaprenyl phosphate. 20) A protein described in any one of items 2 to 17, wherein the first segment includes a transposition and receptor-binding domain. 21) A protein according to any one of items 1 to 20, having bactericidal or bacteriostatic activity against Klebsiella pneumoniae, Klebsiella oxytoka, Klebsiella granulomatis, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella variicola. 22) A protein having cytotoxic activity against Klebsiella, optionally one of the proteins described in item 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) A protein according to any one of items 1 to 22, wherein the reference sequence is any one defined with respect to sequence numbers 1 to 4, 7, 8, or 22, preferably any one defined with respect to sequence numbers 1 to 4 or 22. 24) The cytotoxic activity of the protein is such that, when 20 microliters each of the protein and the comparative protein of the amino acid sequence of Sequence ID No. 1 are spotted onto a colony of susceptible Klebsiella strains on an agar plate, and the agar plate is incubated at 37°C for 16 hours, spots free of viable Klebsiella quasinneumoniae subspecies simili pneumoniae SB30 (DSM 28212) are produced of at least the same diameter, and the concentration of the protein in the solution is up to 5 times the concentration of the comparative protein solution, as described in item 1 or 23. 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 the comparative composition containing the comparative protein of the amino acid sequence of SEQ ID NO: 1 is such that when 20 microliters each of the solution of the composition and the comparative solution of the comparative composition are spotted onto a colony of susceptible Klebsiella strains on an agar plate, and the agar plate is incubated at 37°C for 16 hours, spots free of viable Klebsiella quasinneumoniae subspecies simili pneumoniae SB30 (DSM 28212) are produced, and the concentration of the protein in the solution of the composition is up to 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 salmonsine. 28) A pharmaceutical composition, as described in any one of items 25 to 27. 29) A composition according to any one of items 25 to 28, wherein the plant material is a plant expressing the one or more proteins, preferably a material derived from an edible plant expressing the one or more proteins.

[0024] 30) The composition according to item 29, wherein the plant material is a plant-derived material selected from the group consisting of spinach, chard, beet root, carrot, sugar beet, beet leaf, amaranth, tobacco, preferably Nicotiana benthamiana, and / or the plant material is one or more leaves, roots, tubers or seeds, or a product obtained by crushing, pulverizing or grinding the 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 a dispersed form, preferably in a dissolved form. 32) Proteins or compositions according to any one of items 1 to 31, for use in treatment, preferably in methods for treating infections of the subject caused by Klebsiella, e.g., Klebsiella pneumoniae, Klebsiella oxytoka, Klebsiella quasinneumoniae, Klebsiella aerogenes and / or Klebsiella barriicola. 33) The Klebsiella protein for use as described in item 32, wherein the Klebsiella is antibiotic resistant, for example, carbapenem resistant. 34) A protein for use as described in item 32 or 33, which is defined as any one of sequence numbers 1-4, 7, 8, or 22 as a reference sequence, preferably any one of sequence numbers 1-4 or 22 as a reference sequence.

[0025] 35) A method for preventing or reducing infection or contamination of an object by one or more Klebsiella species, comprising bringing the object into contact 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 an infection caused by Klebsiella in a subject requiring such treatment, comprising administering to the subject 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. 37) The method according to item 35 or 36, wherein the Klebsiella includes Klebsiella pneumoniae, Klebsiella oxytoka, Klebsiella quasinneumoniae, Klebsiella erogenes and / or Klebsiella variicola. 38) A method for producing a composition comprising a protein as defined in any one of items 1 to 24, (i) A step of expressing the protein in a plant, preferably an edible plant or tobacco, (ii) A step of recovering plant material containing the protein expressed from the plant, (iii) A step of extracting the protein from the plant material using an aqueous buffer to obtain a composition containing the protein, (iv) Optionally, the step of removing undesirable impurities from the composition. Methods that include...

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

[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 a viral (DNA or RNA) replicon containing the nucleotide sequence encoding the protein for expressing the nucleotide sequence in a cell, preferably a plant cell; the replicon may contain the subgenome promoter for expressing the nucleotide sequence in a plant cell or a plant cell under the control of a subgenome promoter. 44) A plant, plant tissue, or plant cell containing a protein as defined in any one of items 1 through 24. 45) Plants, plant tissues, or plant cells containing nucleic acid molecules or nucleic acid constructs as defined in item 42 or 43. [Brief explanation of the drawing]

[0028] [Figure 1]Figure 1 schematically shows the T-DNA region of a TMV-based vector for clebicin expression. RB: Right T-DNA boundary, Act2: Arabidopsis thaliana actin promoter, RdRp: RNA-dependent RNA polymerase, 3'NTR: 3' untranslated region, T: nos terminator, LB: Left T-DNA boundary, KpneM-cat1: Coding sequence of clevicin KpneM (K. pneumoniae EWD35590.1) containing the first intron of the catalase gene (cat-1) derived from castor bean (Ricinus communis), KpneM2: Coding sequence of clevicin KpneM2 (Klebsiella sp.WP_047066220), KvarM: Coding sequence of clevicin KvarM (K. barricola CTQ17225.1), KaerM: Coding sequence of clevicin KaerM (K. erogenes WP_015367360.1), KpneA: clevicin The coding sequence for KpneA (K. pneumoniae SAV78255.1), KaerA: the coding sequence for KaerA (K. erogenes WP_063414841.1), KoxyY: the coding sequence for KoxyY (K. oxytoka WP_024273778), KvarIa: the coding sequence for KvarIa (K. barriicola KDL88409), and KpneIa: the coding sequence for KpneIa (K. pneumoniae BAS34675). [Figure 2]Figure 2 shows the SDS-PAGE analysis of crebicin expression in leaves of *N. benthamiana*. Plant material (50 mg) (3 leaf samples pooled, 4 dps of crebicin KaerA, 5 dps of the remaining crebicin) was collected 5 or 7 days (dps) after spraying, pulverized with liquid nitrogen, and 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 minutes. The solution containing 5 μg of protein was separated on a 12% polyacrylamide gel and stained with Coomassi stain. M: PageRulerPrestained Protein Ladder (ThermoFisher Scientific Baltics), WT: Crude extract of unsprayed tobacco leaves, KvarIa, KpneIa, KpneA, KaerA, KoxyY, KpneM, KpneM2, KvarA, KaerM: Extract of tobacco leaves sprayed with crebicin expression constructs. Bands corresponding to recombinant crebicin are indicated by asterisks. [Figure 3A]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3B]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3C]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3D]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3E]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3F]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3G]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3H]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3I]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3J]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3K]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 3L]Figure 3 illustrates the purification of crebicin from the biomass of tobacco leaves. A, C, E, G, I, K: Purification schemes 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 stain. B: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneM Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on Q Sepharose, Lane 9 - KpneM Flow-through of Q Sepharose, Lane 10 - Impurity Eluten (after Q Sepharose), D: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 3 - Flow-through of Phenyl Sepharose, Lane 4 - KpneM2 Eluten (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: Lane 1 and 7 - PageRuler™ Prestained protein ladder, Lane 2 - Crude extract, Lane 3 - Total soluble protein loaded onto Phenyl Sepharose, Lane 4 - Flow-through from Phenyl Sepharose, 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: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KpneA Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded on SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KpneA Eluten (after SP Sepharose), J: Lane 1 and 6 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded on SP Sepharose, Lane 4 - Flow-through of SP Sepharose, Lane 5 - KaerA Eluten (after SP Sepharose), Lane 7 - Protein Loaded on Q Sepharose, Lane 8 - Flow-through of KaerA from Q Sepharose. L: Lane 1 and 7 - PageRuler™ Prestained Protein Ladder, Lane 2 - Crude Extract, Lane 3 - Total Soluble Protein Loaded onto Phenyl Sepharose, Lane 4 - Flow-through of Phenyl Sepharose, Lane 5 - KvarIa Eluten (after Phenyl Sepharose), Lane 6 - Impurity Eluten (after Phenyl Sepharose), Lane 8 - Protein Loaded onto SP Sepharose, Lane 9 - Flow-through of SP Sepharose, Lane 10 - KvarIa Eluten (after SP Sepharose). Arrows indicate recombinant proteins. [Figure 4] Figure 4 shows purified crebicin on a single gel. 0.5 μg of purified crebicin was separated on a 12% SDS-PAGE gel and stained with Coomassi stain. M: PageRuler Unstained Protein Ladder (ThermoFisher Scientific Baltics). [Figure 5]Figure 5 illustrates the evaluation of crebicin activity against Klebsiella strains in a soft agar overlay assay. Bacterial cultures were grown overnight in CAA medium, homogenized to OD595=1.0, diluted 100-fold with molten top CAA agar, and poured onto a CAA agar plate. 20 μL drops of crude protein extract were added to a 6 mm Whatman disc, and the Petri plate was incubated overnight at 30°C or 37°C. [Figure 6] Figure 6 shows the susceptibility of Klebsiella clinical isolates to clebicin expressed in six plants. 100 Klebsiella clinical isolates (89 K. pneumoniae and 11 K. oxytoka) were tested using a plate-drop assay. Strains susceptible to each clebicin were classified according to the size of the inhibition zone. [Figure 7] Figure 7 shows the cytotoxicity assay of crebicin in liquid culture. Klebsiella overnight cultures were diluted with CAA medium to OD600 = 0.3, treated with 5 μg mL-1 of any of the crebicin solutions, and the bacteria were cultured for a further 5 hours with shaking (200 rpm). The antimicrobial activity of crebicin was evaluated by counting the colony-forming units of the tested cultures. The bars represent the standard deviation. [Figure 8] Figure 8 shows the activity of crebicin against biofilms. Biofilms of K. quasinneumoniae, K. oxytoka, K. variikola, and K. aerogenes, grown in CAA medium for 1 day, were treated with 5 μg mL-1 of any of the crebicin concentrations. The antimicrobial activity of crebicin was evaluated by counting the colony-forming units in the tested culture media. The bar represents the standard deviation. [Figure 9]Figure 9 shows the impact of KvarIa treatment on the survival of Galleria mellonella larvae after challenge with K. quassineumonier DSM 28212. G. mellonella larvae were infected with 12,000–32,000 CFU of K. quassineumonier DSM 28212 and treated with 10 μg of KvarIa 2 hours after infection. The larvae were incubated in Petri dishes at 37°C for up to 68 hours. Twenty larvae were used at each treatment stage. [Figure 10A] Figure 10 shows the estimated consensus sequences for sequence numbers 22-24. "Single-letter codes" refer to 20 standard amino acids, "X" indicates insufficient conservation for estimating the consensus amino acid at each position, and "-" indicates an amino acid omitted in the consensus sequence estimation 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 sequence estimation was performed using the Geneious Prime Clustal W software with standard settings. Figure 10A shows the estimation for sequence number 22. Figure 10B shows the estimation for sequence number 23. Figure 10C shows the estimation for sequence number 24. [Figure 10B] Figure 10 shows the estimated consensus sequences for sequence numbers 22-24. "Single-letter codes" refer to 20 standard amino acids, "X" indicates insufficient conservation for estimating the consensus amino acid at each position, and "-" indicates an amino acid omitted in the consensus sequence estimation 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 sequence estimation was performed using the Geneious Prime Clustal W software with standard settings. Figure 10A shows the estimation for sequence number 22. Figure 10B shows the estimation for sequence number 23. Figure 10C shows the estimation for sequence number 24. [Figure 11A]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11B]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11C]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11D]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11E]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11F]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11G]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11H]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 11I]Figure 11 shows the activity and concentration evaluation of crebicin KpneM, KpneM2, KvarIa, KpneA, KaerA, and KvarM when stored as lyophilized purified proteins. The activity of crebicin against susceptible bacteria was evaluated in liquid culture or by radial diffusion assay. Antimicrobial activity was expressed as CFU / mL Δlog10 when evaluated in liquid culture, or as specific activity units (AU) when using radial diffusion assay for evaluation. Crebicin concentration was measured by Bradford assay. Data are mean ± SD of three independent experiments. (A) Activity of KvarIa after storage at -20°C with crebicin KpneM and KpneM2. (B) Activity of KvarIa after storage at 5°C with crebicin KpneM and KpneM2. (C) Activity of KvarIa after storage at room temperature with crebicin KpneM and KpneM2. (D) Activity of crebicin KpneA, KaerA, and KvarM after storage at -20°C. (E) Activity of crebicin KpneA, KaerA, and KvarM after storage at 5°C. (F) Activity of crebicin KpneA, KaerA, and KvarM after storage at room temperature. (G) Trend line of crebicin concentration after storage at -20°C. (H) Trend line of crebicin concentration after storage at 5°C. (I) Trend line of crebicin concentration after storage at room temperature. [Figure 12A]Figure 12 shows the residual activity of KvarIa and KvarIa coated with Eudragit S100 after in vitro gastric digestion in a soft agar overlay assay, as well as the evaluation of the fragments of KvarIa after pepsin digestion by SDS-PAGE. (A) Evaluation of residual activity of KvarIa and KvarIa coated with Eudragit S100 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 for all samples were prepared with distilled water in a 1:2 ratio, and aliquots of 5 μL of diluted samples were dropped onto MHA plates containing the K. quasinneumoniae DSM28212 flora. (B) Tricin SDS-PAGE assay of KvarIa digestion. Coomassi staining was used to visualize protein degradation and estimate the MW of peptide products. The presence or absence of pepsin and Kvar Ia is indicated. Time is shown in minutes, and these correspond to (A). [Figure 12B]Figure 12 shows the residual activity of KvarIa and KvarIa coated with Eudragit S100 after in vitro gastric digestion in a soft agar overlay assay, as well as the evaluation of the fragments of KvarIa after pepsin digestion by SDS-PAGE. (A) Evaluation of residual activity of KvarIa and KvarIa coated with Eudragit S100 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 for all samples were prepared with distilled water in a 1:2 ratio, and aliquots of 5 μL of diluted samples were dropped onto MHA plates containing the K. quasinneumoniae DSM28212 flora. (B) Tricin SDS-PAGE assay of KvarIa digestion. Coomassi staining was used to visualize protein degradation and estimate the MW of peptide products. The presence or absence of pepsin and Kvar Ia is indicated. Time is shown in minutes, and these correspond to (A). [Figure 13] Figure 13 shows a standard curve for the detection of K. quasinneumoniae, obtained by real-time PCR based on amplification of the khe gene. [Figure 14] The real-time PCR results in Figure 14 show that K. quasinneumoniae colonized mouse fecal samples before and after treatment with crebicin. Fecal samples from three mice were used at each time point in the experiment. 18d: Fecal samples were collected on day 18 of the experiment, before the start of crebicin treatment; 22d: Fecal samples were collected on day 22 of the experiment (the day after the last oral intake of crebicin). [Modes for carrying out the invention]

[0029] The inventors of the present invention have identified a protein that has bactericidal or bacteriostatic activity against Klebsiella. Such a protein is referred to herein as "Clevicin." The protein of the present invention or Clevicin preferably has lipid II cleavage activity or pore-forming ability in the bacterial cell membrane. The protein or clebicin of the present invention broadly comprises at least two amino acid sequence segments (sometimes abbreviated as "segments" herein). In this specification, an amino acid sequence segment means a number of adjacent amino acid residues in the primary structure of a protein or polypeptide, wherein the protein or polypeptide has more amino acid residues in its primary structure than the number of segments. The protein of the present invention broadly comprises or comprises a first segment and a second segment. The first segment broadly provides a protein having the ability to bind to components of Klebsiella cells (e.g., to receptors), and / or it provides a protein having the ability to be introduced or taken up (translocated intracellularly) by Klebsiella cells. The second segment may have lipid II cleavage activity or pore-forming ability in bacterial cell membranes. 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. Therefore, the protein of the present invention may include, or consist of, a first segment at the N-terminus and a second segment at the C-terminus. 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. Therefore, the protein of the present invention may include, or consist of, a second segment at the N-terminus and a first segment at the C-terminus.

[0030] The first segment of the protein of the present invention The protein of the present invention may include a first segment containing 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 crebicin identified by the inventors. Preferably, the amino acid sequence of the first segment is the amino acid sequence of items (Ai) to (A-ix) defined above.

[0031] However, the present invention is not limited to crebicin having a specific crebicin first segment identified by the inventors. Instead of the amino acid sequences of items (Ai) to (A-ix), the first segment may each contain any one of the amino acid sequences of items (Bi) to (B-ix) as defined above. The expression "the first segment contains an amino acid sequence having at least 70% sequence identity with the amino acid sequence from amino acid residues x to y of SEQ ID NO: Z" (where x and y refer to the start and end sites, and Z refers to the SEQ ID NO: number) 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 residues x to y of SEQ ID NO: Z, and has the sequence identity described above over at least the entire length from residues x to y of SEQ ID NO: Z. This principle applies to items (Bi) to (B-ix) and all other sequence identities as defined in this specification. In this specification, sequence identity determination is performed using Clustal Omega (CLUSTAL O 1.2.4) and based on standard parameters. Preferably, the amino acid sequence of the first segment is that of any one of items (Bi) to (B-ix). The expression "the amino acid sequence of the first segment has at least 70% sequence identity with the amino acid sequence from amino acid residues 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 residues x to y of SEQ ID NO: Z, and has the sequence identity described above over the entire length from residues x to y of SEQ ID NO: Z. This applies to items (Bi) to (B-ix) and all other sequence identities as defined in this specification.

[0032] In another embodiment, the first segment comprises one amino acid sequence of any of the items (Ci) to (C-ix) defined above. Preferably, the amino acid sequence of the first segment is defined as one of the items (Ci) to (C-ix) defined above. The definition of items (Ci) to (C-ix) means that the amino acid sequence is that of the indicated amino residue range of the indicated sequence number, except for the indicated number of substitutions, additions, insertions and / or deletions. Where a protein is defined in this specification by a predetermined 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 predetermined number or range refers to the total of all amino acid substitutions, additions, insertions, and deletions. Of 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., an addition to the C-terminus or N-terminus). The term "addition" means an addition to the C-terminus or N-terminus of the amino acid sequence of a reference sequence. A deletion may be a deletion of an amino acid residue at the terminal or within the reference sequence. In this specification, the term "reference sequence" refers to the amino acid sequence in the sequence listing in which the amino acid sequence of the protein of the present invention is defined. 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 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 amino acid sequences.

[0034] Therefore, items (i) to (ix) below in 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 is preferably one of the following amino acid sequences: (Bi) The amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1 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. (B-ii) Having sequence identity of at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% with the amino acid sequence of amino acid residues 1-127 of SEQ ID NO: 2, (B-iii) Having sequence identity of at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% with the amino acid sequence of amino acid residues 1-123 of SEQ ID NO: 3, (B-iv) Having sequence identity of at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% with the amino acid sequence of amino acid residues 1-118 of SEQ ID NO: 4, (Bv) The amino acid sequence of amino acid residues 1-170 of SEQ ID NO: 5 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. (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 amino acid residues 1-172 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 1-255 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 1-288 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 1-236 of SEQ ID NO: 9, or, (Ci) Compared to the amino acid sequence of amino acids 1-128 of SEQ ID NO: 1, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (C-ii) Compared to the amino acid sequence of amino acids 1-127 of SEQ ID NO: 2, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (C-iii) Compared to the amino acid sequence of amino acids 1-123 of SEQ ID NO: 3, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (C-iv) Compared to the amino acid sequence of amino acids 1-118 of SEQ ID NO: 4, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (Cv) Compared to the amino acid sequence of amino acids 1-170 of SEQ ID NO: 5, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (C-vi) Compared to the amino acid sequence of amino acids 1-172 of SEQ ID NO: 6, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (C-vii) Compared to the amino acid sequence of amino acids 1-255 of SEQ ID NO: 7, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions. (C-viii) Compared to the amino acid sequence of amino acids 1-288 of SEQ ID NO: 8, it has 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, 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 other embodiments, the amino acid sequence of the first segment of the protein of the present invention preferably has the following: (Bi) The amino acid sequence of amino acid residues 1 to 128 of SEQ ID NO: 1 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. (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 amino acid residues 1-127 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 amino acid residues 1-123 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 amino acid residues 1-118 of SEQ ID NO: 4. (Bv) The amino acid sequence of amino acid residues 1-170 of SEQ ID NO: 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 1-170 of SEQ ID NO: (B-vi) The amino acid sequence of amino acid residues 1-172 of SEQ ID NO: 6 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. (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 amino acid residues 1-255 of sequence number 7. (B-viii) The amino acid sequence of amino acid residues 1-288 of sequence number 8 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, or (B-ix) The amino acid sequence of amino acid residues 1-236 of sequence number 9 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. or, (Ci) Compared to the amino acid sequence of amino acid residues 1-128 of SEQ ID NO: 1, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (C-ii) Compared to the amino acid sequence of amino acid residues 1-127 of SEQ ID NO: 2, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (C-iii) Compared to the amino acid sequence of amino acid residues 1-123 of SEQ ID NO: 3, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (C-iv) Compared to the amino acid sequence of amino acid residues 1-118 of SEQ ID NO: 4, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (Cv) Compared to the amino acid sequence of amino acid residues 1-170 of SEQ ID NO: 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (C-vi) Compared to the amino acid sequence of amino acid residues 1-172 of SEQ ID NO: 6, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (C-vii) Compared to the amino acid sequence of amino acid residues 1-255 of SEQ ID NO: 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (C-viii) Compared to the amino acid sequence of amino acid residues 1-288 of SEQ ID NO: 8, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (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 present invention Since the protein (Clevicin) of the present invention has both a first and a second segment, the protein can be defined by both its first and second segments. Accordingly, the protein can be defined by a combination of any one of the first segments described above and the second segment as defined herein. The second segment of the protein of the present 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., colisin from E. coli).

[0037] The second segment may contain 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 one of the amino acid sequences 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 are combined based on the same sequence number as a reference sequence. 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 crebicin having a second segment of a specific crebicin identified by the inventors. Instead of the amino acid sequences of items (Di) to (D-ix), the second segment may include or consist of any one of the amino acid sequences 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 include or consist of any one of the amino acid sequences of items (Fi) to (F-ix) defined above. Preferably, the amino acid sequence of the first segment is any one of the amino acid sequences of items (Fi) to (F-ix) defined above.

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

[0040] In other embodiments, the amino acid sequence of the second segment of the protein of the present invention preferably has the following: (Ei) For the segment from amino acid residues 129-278 of SEQ ID NO: 1, 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, (E-ii) For the segment from amino acid residues 128-276 of SEQ ID NO: 2, 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, (E-iii) For the segment from amino acid residues 124-272 of SEQ ID NO: 3, 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, (E-iv) For the segment from amino acid residues 119-266 of SEQ ID NO: 4, 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, (Ev) For the segment from amino acid residues 171-377 of SEQ ID NO: 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, (E-vi) For the segment from amino acid residues 173-379 of SEQ ID NO: 6, 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, (E-vii) For the segment from amino acid residues 256-452 of SEQ ID NO: 7, 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, (E-viii) For the segment from amino acid residues 289-466 of sequence number 8, 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, (E-ix) For the segment from amino acid residues 237-414 of sequence number 9, 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, or, (Fi) Compared to the amino acid sequence of amino acids 129-278 of SEQ ID NO: 1, 1-20, preferably 1-15, more preferably 1-10 amino acids are substituted, added, inserted, and / or deleted. (F-ii) Substitution, addition, insertion and / or deletion of 1 to 20 amino acids, preferably 1 to 15, more preferably 1 to 10, 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 amino acids, preferably 1 to 15, more preferably 1 to 10, compared to the amino acid sequence of amino acids 124 to 272 of SEQ ID NO: 3, (F-iv) Compared to the amino acid sequence of amino acids 119-266 of SEQ ID NO: 1-20, preferably 1-15, more preferably 1-10 amino acids have been substituted, added, inserted, and / or deleted. (Fv) Compared to the amino acid sequence of amino acids 171-377 of SEQ ID NO: 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (F-vi) Compared to the amino acid sequence of amino acids 173-379 of SEQ ID NO: 6, 1 to 30, preferably 1 to 20, more preferably 1 to 10 amino acid substitutions, additions, insertions and / or deletions, (F-vii) Compared to the amino acid sequence of amino acids 256-452 of SEQ ID NO: 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (F-viii) Compared to the amino acid sequence of amino acids 289-466 of SEQ ID NO: 8, 1-30, preferably 1-20, more preferably 1-10 amino acid substitutions, additions, insertions and / or deletions, (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 aforementioned first segments can be combined with any of the second segments to form the protein of the present invention. In one embodiment, the protein of the present invention includes one first segment from items (i) to (iv) and one second segment from 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, the protein of the present invention includes one first segment from items (Ci) to (C-iv) and one second segment from items (Fi) to (F-iv). In another embodiment, the protein of the present invention comprises one first segment from items (v) to (ix) and one second segment from 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 present invention comprises one first segment from items (Cv) to (C-ix) and one second segment from items (Fv) to (F-ix).

[0042] Other embodiments are as follows: In one embodiment, the protein of the present invention may have, in the broadest and most preferred embodiment, a first segment comprising or consisting of any amino acids of the categories (Ai)~(A-iv), (Bi)~(B-iv), or (Ci)~(C-iv), and a second segment comprising or consisting of any one amino acid sequence of the categories (Di)~(D-iv), (Ei)~(Eiv), or (Fi)~(F-iv). Preferably, the first segment comprises or consists of any one amino acid sequence of the categories (Ai)~(A-iii), (Bi)~(B-iii), or (Ci)~(C-iii), and the second segment comprises any one amino acid of (Di)~(D-iii), (Ei)~(Eiii), or (Fi)~(F-iii). More preferably, the first segment comprises one amino acid sequence of (A-ii), (B-ii), or (C-ii), and the second segment comprises one amino acid sequence 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 includes one amino acid sequence of items (Av)~(A-ix), (Bv)~(B-ix), or (Cv)~(C-ix), and the second segment includes one amino acid sequence of items (Dv)~(D-ix), (Ev)~(Eix), or (Fv)~(F-ix). In a preferred embodiment, the first segment includes one amino acid sequence of items (Av), (A-vi), (Bv), (B-vi), (Cv), and (C-vi), and the second segment includes one amino acid sequence 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 further alternative embodiments, the first segment comprises one amino acid sequence of items (A-viii), (A-ix), (B-viii), (B-ix), (C-viii), or (C-ix), and the second segment comprises one amino acid sequence 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 protein of the present invention are as defined in items (ai) to (a-ix), (bi) to (b-ix), and (ci) to (c-ix) above. In item (b), "an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: Z" (where Z is the SEQ ID NO: number) 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 sequence identity shown at least over the entire length of SEQ ID NO: Z. This applies to all items (bi) to (b-ix) and to the corresponding sequence identity as defined in this specification. The definitions in items (ci) to (c-ix) mean that the amino acid sequence is that of all amino sequences 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 may be defined as follows: (b') In one embodiment, the amino acid sequence of the protein of the present invention is: (b'-i) The amino acid sequence is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to that of SEQ ID NO: 1. (b'-ii) The amino acid sequence is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to that of SEQ ID NO: 2. (b'-iii) The amino acid sequence is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to that of SEQ ID NO: 3. (b'-iv) The amino acid sequence is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to that of SEQ ID NO: 4. (b'-v) The amino acid sequence of SEQ ID NO: 5 is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical. (b'-vi) The amino acid sequence of SEQ ID NO: 6 is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical. (b'-vii) The amino acid sequence of SEQ ID NO: 7 is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical. (b'-viii) The amino acid sequence of (b'-viii) is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical to that of SEQ ID NO: 8, or (b'-ix) The amino acid sequence of SEQ ID NO: 9 is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% identical. (b'') In one embodiment, the amino acid sequence of the protein of the present invention is: (b''-i) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of SEQ ID NO: 1, (b''-ii) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of Sequence ID No. 2, (b''-iii) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of Sequence ID No. 3, (b''-iv) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of Sequence ID No. 4, (b''-v) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of SEQ ID NO: 5 (b''-vi) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of SEQ ID NO: 6, (b''-vii) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of sequence number 7, (b''-viii) Sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% with the amino acid sequence of sequence number 8, or (b''-ix) shares at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence identity with the amino acid sequence of sequence number 9.

[0047] Cytotoxic activity of the protein of the present invention The clebicin of the present invention can exert cytotoxic effects against Klebsiella bacteria (e.g., K. pneumoniae, K. granulomatis, K. oxytoka, K. aerogenes, K. quasipneumoniae, and K. variicola). Preferably, the clebicin of the present invention is active against K. pneumoniae and K. oxytoka. K. pneumoniae is the most preferred. The cytotoxic effect may be bacteriostatic or bactericidal. Whether the protein has a cytotoxic effect can be experimentally tested, for example, using the assay in 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 one of embodiments (i) to (iv) defined above. The proteins selected from items (i) to (iv), (vii), and (viii) are proteins defined using SEQ ID NOs: 1 to 4, 7, or 8 as reference sequences.

[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 sequence numbers 5 to 9, as well as the derivatives of items (bv) to (b-ix) and (cv) to (c-ix), respectively, have pore-forming activity. It is assumed that this 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 assumed to possess lipid II cleavage activity, by analogy with the activity of E. coli coli colisin M. The inventors assume that this class of proteins possess 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 coli colisin M (Gross and Braun, Mol. Gen. Genet. 251 (1996) 388-396; Barreteau et al., Microbial Drug Resistance 18 (2012), 222-229). The released C55 polyisoprenol no longer allows the MurNAc-pentapeptide-GlcNAc transposition across the cell membrane. Their clevisins are therefore assumed to exert toxicity against Klebsiella cells after they are taken up into the periplasm through the outer cell wall. This property of the proteins of the present invention can be analyzed according to a standard assay for cholinin 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 (bi) to (b-iv) and (ci) to (c-iv), possess peptidoglycanase or lipid II cleavage activity. This activity is attributed to the presence of a second amino acid sequence segment in these proteins.

[0050] The cytotoxic activity of the protein of the present invention is preferably such that the protein and the comparative protein of the amino acid sequence of Sequence ID No. 1 produce spots of at least the same diameter that do not contain viable bacteria of the susceptible Klebsiella strain after 16 hours of spotting 20 μL each of the solutions of the protein and the comparative protein onto a colony of susceptible Klebsiella pneumoniae subspecies similipneumoniae SB30 (DSM 28212) on an agar plate, and the agar plate is incubated at 37°C, with the concentration of the protein in the solution being up to 5 times the concentration of the comparative protein solution. The solution is an aqueous solution. This test can be carried out 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 the above items (bi)~(b-ix) or (ci)~(c-ix), and the protein and a comparison protein of the amino acid sequence of the sequence numbers of items (bi)~(b-ix) or (ci)~(c-ix) have cytotoxic activity such that when 20 μL of each solution of the protein and the comparison protein are spotted onto a colony of susceptible Klebsiella pneumoniae subsp. similis pneumoniae SB30 (DSM 28212) on an agar plate, and the agar plate is incubated at 37°C for 16 hours, spots without viable bacteria of the susceptible Klebsiella strain are produced, and the concentration of the protein in the solution is up to 5 times the concentration of the comparison protein solution. The solution is an aqueous solution. This test can be carried out 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 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 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, when 20 μL of each solution of the protein and the comparative protein of the amino acid sequence of SEQ ID NO: 1 is spotted onto a colony of susceptible Klebsiella pneumoniae subsp. simili pneumoniae SB30 (DSM 28212) on an agar plate, and the agar plate is incubated at 37°C for 16 hours, spots free of viable bacteria of the susceptible Klebsiella strain are produced, and the concentration of the protein in the solution is up to 5 times the concentration of the comparative protein solution. The assay for cytotoxic activity is performed as described in Reference Example 1. In the 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. Conserved positional relationships and / or amino acid sequence stretches are 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 related to the function of binding to and transposition to the receptor. In the second amino acid sequence segment, the conserved positional relationships or stretches are usually related to cytotoxicity against Klebsiella. Crebicin KpneM2, KvarM, KpneM, and KaerM share the consensus sequence of SEQ ID NO: 22. Crebicin KaerA and KpneA share the consensus sequence of SEQ ID NO: 23. Crebicin KpneIa and KvarIa share the consensus sequence of SEQ ID NO: 24. In sequence numbers 22, 23, and 24, X represents 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, crebicins having lipid II cleavage activity (for example, 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 (Sequence ID 25), 212I, 216G, 218W, 220YNGV223 (Sequence ID 26), 225RAFNDTYD232 (Sequence ID 27), 234N, 239R, 243A, 247T, 255G, 258Y, 260I, 262P, 262G, 271S, 272G; In the sequence, the number before the single-letter code of an amino acid residue indicates its position in SEQ ID NO: 22, and the number after the single-letter code 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 " / " mean that any one of the indicated residues is located at the position corresponding to the indicated position in SEQ ID NO: 22. The expression "corresponding amino acid residue or amino acid sequence stretch..." means that the position of 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 counting the residues of the amino acid sequence of the protein starting from the first amino acid sequence at the N-terminus to determine the corresponding position.

[0055] Crebicin having pore-forming activity, as defined with respect to SEQ ID NOs. 5 and 6, preferably has 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 (Sequence ID 37), 208KY209, 212LA213, 217A, 220I, 222NFQGKK227 (Sequence ID 38), 229RSF231, 233DAM235, 237S, 244NP245, 247MKL249, 251QADK254 (Sequence ID 39), 259NAL261, 263Q, 266LS267, 269LADRFKGL277 (Sequence ID 40), 279AFTW282 (Sequence ID 41), 284DRLLKA289 (Sequence ID 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 sequence, the number before the single-letter code of an amino acid residue indicates its position in SEQ ID NO: 23, and the number after the single-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 " / " mean that any one of the indicated residues is present at the position corresponding to the indicated position in SEQ ID NO: 23. The expression "corresponding amino acid residue or amino acid sequence stretch..." means that the position of 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 of the amino acid sequence of the protein starting from the first amino acid sequence at the N-terminus to determine the corresponding position. SEQ ID NO: 23 is shown at the end of this specification.

[0056] Crebicin having pore-forming activity, as defined with respect to SEQ ID NOs: 8 and 9, preferably has amino acid residues or amino acid sequence stretches corresponding to the following amino acid residues or amino acid sequence stretches: 1MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPD120 (SEQ ID NOs: 50), 121K / R, 122TTTTENVGGGKNGHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAK185 (SEQ ID NOs: 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 sequence, the number before the single-letter code of an amino acid residue indicates its position in SEQ ID NO: 24, and the number after the single-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 " / " mean that any one of the indicated residues is located at the position corresponding to the indicated position in SEQ ID NO: 24. The expression "corresponding amino acid residue or amino acid sequence stretch..." means that the position of 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 of the amino acid sequence of the protein starting from the first amino acid sequence at the N-terminus to determine the corresponding position.

[0057] The definitions of consensus arrays described above may be combined with the definitions in the claims or the embodiments described in the previous section. More specifically, the definition of conserved residues may be combined with any of the above definitions of (Bi)~(B-iv), (Ci)~(C-iv), (Ei)~(Eiv), (Fi)~(F-iv), (bi)~(b-iv), and / or (ci)~(c-iv) 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 sequences. The corresponding positions in the reference sequences can be derived, for example, from the alignments shown in Figures 10A~C.

[0058] Crebicin composition The compositions of the present invention comprise one or more proteins (crebicins) of the present invention as described above, and optionally any further components such as carriers. The compositions may comprise one or more different proteins (crebicins) as defined herein, for example, two, three, or four different proteins (crebicins) as defined herein. "Different" means that the proteins differ in at least one amino acid residue. The compositions may comprise two, three, or more crebicins of the present invention from the same class represented by items (i) to (iv) above, or by any one of items (v) to (ix) above. Preferably, the compositions comprise at least two crebicins of the present invention from different classes, for example, at least one crebicin of the pore-forming type and at least one crebicin of the lipid II-cleaving type. The compositions may further comprise one or more E. coli colicins or derivatives thereof, for example, in conjunction with the control of pathogenic E. coli (e.g., EHEC), as described in, for example, European Patent Application Publication No. 3097783.

[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 colicin or Salmonella colisin (salmocins). Escherichia coli colicin is known in the prior art and is described in particular in European Patent Application Publication No. 3097783. Salmocins are known and are described in International Publication No. 2018 / 172065. Since the protein of the present invention is preferably produced by expression in plants or their cells, the composition may be a plant material or an extract thereof, wherein the plant material is a material derived from a plant expressing the protein (preferably a Nicotiana or edible plant expressing the protein). The extract of the plant material is an aqueous solution containing a water-soluble protein, including the protein of the present invention present in or expressed in the plant material, or a dried product of such aqueous solution. The extract is preferably obtained by removing the water-insoluble components of the plant material, for example, by filtration or centrifugation. The plant material may be a material derived from a plant selected from the group consisting of spinach, chard, beet root, 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 pulverized leaves, roots, tubers or seeds.

[0060] The composition or extract from the plant material may be a solid or liquid composition (e.g., a solution or dispersion) containing the crebicin of the present invention. The liquid composition may be aqueous, for example, 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. If one or more crebicins capable of exerting a cytotoxic effect against Klebsiella are used, these concentrations relate to the total concentration of all such crebicins. The aqueous solution may contain a buffer in addition to one or more proteins of the present invention. The buffer may be an inorganic or organic acid or a salt thereof. An example of an inorganic acid is phosphoric acid or a salt thereof. Examples of organic acids are hepe, acetic acid, succinic acid, tartaric acid, malic acid, benzoic acid, cinnamic acid, glycolic acid, lactic acid, citric acid, and ascorbic acid. Preferred organic acids are malic acid, lactic acid, citric acid, and ascorbic acid. The pH of the solution may be typically 4 to 8, preferably 5 to 8, and more preferably 6.0 to 7.5. If the object to which the composition is applied is a meat product, the pH of the solution may be typically 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 a salt). A preferred salt used is sodium chloride. An aqueous solution containing one or more crebicins may be a buffered aqueous solution containing a further solute, such as a salt, for example, 50-400 mM NaCl, preferably 100-200 mM NaCl. The aqueous solution may further contain a sulfhydryl compound, preferably DTT, such as dithiothreitol (DTT), dithioerythritol, thioethanol, or glutathione. The total concentration of the sulfhydryl compound in the aqueous solution may be 1-50 mM, preferably 2-20 mM, more preferably 4-10 mM.

[0061] If the composition of the present invention is a solid composition, it may be a powder, such as a lyophilized solid composition obtained by lyophilizing the above-mentioned extract or solution. The powder may contain further solid components (e.g., those described above in aqueous solutions). It may be reformatted with a suitable liquid (e.g., water or buffer solution) before use. The solid composition may contain the above-mentioned buffer, salt or other components so as to achieve the above-mentioned concentration by reformatting or dissolving the solid composition. Examples of carriers for compositions include solvents (e.g., water or aqueous buffers (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 compositions of the present invention may contain one or more of the crebicins 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 of the total weight of the proteins in the composition. The crebicin content in the composition can be measured by performing an analysis according to Reference Example 1, in which the composition is subjected to SDS-PAGE, the resulting gel is stained, and the intensity of the bands on the gel is measured. Thereafter, the intensity of the band due to crebicin can be measured relative to the sum of the band intensities of all the proteins in the composition. In one embodiment, the composition of the present invention is a pharmaceutical composition. The pharmaceutical composition may optionally contain Escherichia coli colicin, apart from one or more proteins of the present invention. It also comprises one or more suitable pharmaceutically acceptable carriers and / or excipients, depending on whether they are liquid or solid and depending on the intended use. The excipients or carriers may be those described above.

[0063] The compositions of the present invention, as pharmaceutical compositions, can be formulated for oral delivery to the small or large intestine. Accordingly, the present invention also provides oral formulations comprising the protein or compositions of the present invention that 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 relates preferably to mammals, preferably to human subjects. Components for drug delivery to the intestine, or for avoiding the degradation of a drug or active ingredient due to acidic gastric conditions or proteolytic conditions in the stomach, 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 under neutral intestinal conditions. Examples of commercially available coatings suitable for this purpose 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, may be prepared for delivery to the lungs. Therefore, the present invention also provides a lung formulation comprising the protein of the present invention or the composition of the present invention. For an overview of the topical 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 may be a dry powder for aerosol administration or a solution for spray therapy. Other possible embodiments of the formulation of the composition are described in the section on medical uses below.

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

[0065] Medical use The present invention also provides proteins, compositions, or pharmaceutical compositions for use in treating or preventing infections of a target by Klebsiella, particularly by the aforementioned Klebsiella species. The present invention also provides a method for treating or preventing an infection of a target by Klebsiella (particularly the aforementioned Klebsiella species), comprising administering one or more of the present invention proteins (clebicin) or compositions to the target. The target may be a human or a mammal (e.g., livestock). A human target is preferred. The infection to be treated may be an infection by antibiotic-resistant Klebsiella. The resistance may be resistance to carbapenems or multidrug resistance. The Klebsiella infection being treated may be caused by any of the Klebsiella species described above. The proteins KpneM (SEQ ID NO: 1) and KvarM (SEQ ID NO: 2), as defined in this specification using SEQ ID NO: 1 or SEQ ID NO: 2 as the reference sequence, are suitable for medical use because of their broad activity against a variety of Klebsiella isolates, as shown in the examples below. Therefore, these Klebsiella proteins are preferably used, particularly for treating infections caused by any Klebsiella, including Klebsiella pneumoniae (and for the prevention or reduction of contamination, see above).

[0066] The Klebsiella infections treated may be infections of the urinary tract, lower respiratory tract, bile ducts, surgical wounds, or syndromes (clinical syndromes) such as pneumonia, bacteremia, thrombophlebitis, cholecystitis, diarrhea, upper respiratory tract infections, osteomyelitis, and meningitis, preferably pneumonia, bacteremia, thrombophlebitis, urinary tract infections (UTIs), diarrhea, upper respiratory tract infections, and wound infections. In a broad sense, liquid or solid pharmaceutical compositions containing the above-mentioned crebicin and optionally further components are prepared for administration to a subject. The liquid composition may be an aqueous solution as described above. The solid composition may be, for example, a powder containing at least one crebicin in a lyophilized form, or a tablet obtained from such powder or a capsule filled with such powder. The route of administration of proteins or pharmaceutical compositions varies depending on the disease being treated. For the treatment of diarrhea and upper respiratory tract infections, administration may be orally, for example, in the form of tablets or solutions. For the treatment of diarrhea, the pharmaceutical preparation may be able to pass through the stomach without being attacked by acidic solvents. Crebicin must then be released from the pharmaceutical composition in the intestines. Such formulations are known in the prior art. Examples include tablets and capsules that exhibit resistance to acidic solvents in the stomach. It is also possible to orally administer biological materials, such as E. coli or plant materials containing expressed crebicin, to the patient.

[0067] In the treatment of pneumonia, for example, if the subject suffers from a lung infection caused by Klebsiella pneumoniae, the formulation may be administered to the subject as an aerosol or powder to the lungs. Methods for preparing proteins for intrapulmonary administration are known; for example, for recombinant DNAse I or Dornase for inhalation, see Witt DM, Anderson L. Dornase alfa: a new option in the management of cystic fibrosis. Pharmacotherapy. 1996 Jan-Feb;16(1):40-8, and for a dried powder formulation of Dnase I, see U.S. Patent Application Publication 2015 / 0024050. 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 aqueous solution form using a catheter. For the treatment of cholecystitis or bile duct infections caused by Klebsiella, the protein or pharmaceutical composition may be administered in aqueous solution form using a catheter.

[0068] Klebsiella can be administered to adult humans in doses of 1 mg to 1000 mg per day, preferably 10 mg to 250 mg per day to human patients. Such doses may also be administered to animals. In a probiotic manner, patients may be treated by administering a genetically modified microorganism expressing at least one molecule of Klebsiella. The genetically modified microorganism may be a genetically modified non-pathogenic Escherichia coli or lactic acid-producing microorganism commonly used in dairy fermentation. Examples of such 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 infection with Klebsiella. For this purpose, Klebsiella can be dissolved in saline solution and the solution may be sterilized.

[0069] Production of the protein of the present invention The crebicin or protein according to the present invention can be produced by known methods for expressing proteins in a standard expression system. When producing crebicin, the nucleotide sequence encoding it can be expressed in a suitable host organism. Methods available for producing and purifying the target protein are described in the prior art, and any type of method can be used. For example, an E. coli expression system, which is generally known in the art, can be used. When using an expression system in eukaryotic cells, toxicity to the bacterial organism used for cloning can be prevented by inserting one or more introns into the coding sequence of crebicin. A particularly efficient expression method is a plant expression system, which is also known in the prior art. A plant expression system that can be used to express crebicin according to the present invention is described in the examples. A method that enables the expression of the target nucleotide sequence in a plant is the use of a self-replicating (viral) replicon containing the nucleotide sequence encoding crebicin. The coding sequence of crebicin can be codon-optimized for expression in a plant or in a specific plant used as an expression host. Plant virus expression systems are described in many publications, including International Publication Nos. 2012 / 019660, 2008 / 028661, 2006 / 003018, 2005 / 071090, 2005 / 049839, 2006 / 012906, 02 / 101006, 2007 / 137788 or 02 / 068664, and many more publications are cited in these documents. Various methods are known for introducing nucleic acid molecules (e.g., DNA molecules) into plants or parts of plants, for example, for transient expression. Plants can be transfected with nucleic acid molecules (vectors) or nucleic acid constructs using Agrobacterium, for example, by agroinfiltration or spraying with an Agrobacterium suspension. See International Publication No. 2012 / 019660, No. 2014 / 187571 or Brochure No. 2013 / 149726 for reference. The nucleic acid molecule comprises a nucleotide sequence encoding the protein of the present invention.

[0070] In embodiments where potent expression of crebicin as the target protein is required, a nucleic acid molecule or nucleic acid construct containing a nucleotide sequence encoding crebicin may encode a viral vector that can replicate in plant cells to form a replicon of a viral vector. For replication, the viral vector and replicon may contain replication origins that can be recognized by nucleic acid polymerases present in plant cells (e.g., viral polymerase expressed from the replicon). In the case of RNA viral vectors (referred to as "RNA replicons"), the replicon may be formed by transcription under the control of a promoter active in the plant cell, after the DNA construct has been introduced into the nucleus of a plant cell. In the case of DNA replicons, the replicon may be formed by recombination between two recombination sites adjacent to the sequence encoding the viral replicon 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, RNA replicons are preferred. The use of DNA and RNA viral vectors (DNA or RNA replicons) has been documented in various literatures over many years. Some examples are the following patent publications: International Publication Nos. 2008 / 028661, 2007 / 137788, 2006 / 003018, 2005 / 071090, 2005 / 049839, 02 / 097080, 02 / 088369, and 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, in particular those based on (+) sense single-stranded RNA viruses, may be preferably used. Thus, the viral replicon may be a (+) sense single-stranded RNA replicon. Examples of such viral vectors are those based on tobacco mosaic virus (TMV) and potexvirus X (PVX). "Based on" means that the viral vector uses a replication system (e.g., replicases and / or other proteins involved in the replication of these viruses).Viral vectors and expression systems based on Potexvirus are described in European Patent No. 2061890 or International Publication No. 2008 / 028661. As is known from the references, RNA replicons, e.g., (+) sense single-stranded RNA replicons, can express nucleotide sequences under the control of a subgenome promoter located upstream of the nucleotide sequence. Subgenomic RNA can be replicated in plant cells by the action of a viral replicase that may be encoded by the same RNA replicon, and by containing (RNA) nucleotide sequences, proteins can be translated from the subgenomic RNA.

[0071] Crebicin can be expressed in multicellular plants or parts thereof, particularly in higher plants or parts thereof. Monocots and dicots (crops) can be used. Common plants that can be used to express the target protein include Nicotiana benthamiana, Nicotiana tabacum, spinach, rapeseed (Brassica campestris), mustard (B. juncea), beet (Beta vulgaris), rapeseed, yellow radish, mustard, strawberry, daisy (Chenopodium capitatum), lettuce, sunflower, cucumber, Chinese cabbage, cabbage, carrot, shallot, onion, radish, snow pea, cauliflower, broccoli, burdock, turnip, tomato, eggplant, squash, watermelon, cantaloupe, and melon. Suitable plants include spinach, Swiss chard, beet root, carrots, sugar beets, Nicotiana tabacum, and tobacco benthamiana. By using expression in edible plants, contamination of plants or foods made therefrom by Klebsiella can be prevented. In one embodiment, plants that do not normally enter the human or animal food chain (e.g., Nicotiana species (e.g., N. tabacum and N. benthamiana)) are used.

[0072] Generally, the target protein, crebicin, is expressed in the cytosol of plant or plant part cells. In this case, the signal peptide that transports the target protein to a specific compartment is not attached to the protein. Alternatively, the target protein can be expressed in or targeted in plant chloroplasts, in which case an N-terminal signal peptide, commonly referred to as a plastid transport peptide or chloroplast target peptide, is attached to the N-terminus or C-terminus, preferably the N-terminus, of the target protein, crebicin. The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the protein of the present invention. The nucleic acid molecule may comprise (i) a transcription 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 the protein of the present invention, the nucleic acid molecule or nucleic acid construct comprising (i) a transcription promoter which is preferably active in plant cells, and (ii) a nucleotide sequence encoding the protein for expressing the nucleotide sequence in cells, preferably in plant cells, under the control of the promoter. Furthermore, the present invention provides a nucleic acid molecule or nucleic acid construct encoding the protein of the present invention, wherein the nucleic acid molecule or nucleic acid construct is a viral (DNA or RNA) replicon comprising a nucleotide sequence encoding the protein for expressing the nucleotide sequence in a cell, preferably a plant cell, wherein the replicon may include a subgenome promoter for expressing the nucleotide sequence in a plant cell or in a plant cell under the control of a subgenome promoter. Since the proteins of the present invention are preferably expressed in plants or plant cells, the present invention also provides plants, plant tissues or plant cells comprising the proteins of the present invention. The present invention also provides plants, plant tissues or plant cells comprising the nucleotide sequence or nucleic acid molecule of the present invention. The plant may be any one of the foregoing.

[0074] Production of the composition of the present invention In a method for producing a composition containing at least one crebicin, the first step involves expressing crebicin in a plant (e.g., an edible plant) or plant cells. In the next step, plant material containing the expressed crebicin is recovered from the plant expressing crebicin. For example, the plant material may be leaves, roots, tubers, or seeds, or it may be crushed, ground, or pulverized leaves, roots, tubers, or seeds. In step (iii), crebicin is extracted from the plant material using an aqueous buffer. The plant material may be homogenized, and insoluble substances may be removed by centrifugation or filtration. The soluble components containing crebicin are extracted into an aqueous buffer to prepare a crebicin solution in the aqueous buffer. The aqueous buffer may contain inorganic or organic acids or salts thereof, and may have the pH defined above with respect to the aqueous solution of the composition of the present invention. Furthermore, the aqueous buffer may contain the above-mentioned salts and / or sulfhydryl compounds with respect to the aqueous solution of the composition of the present invention. If a relatively high-purity crebicin composition is desired, the crebicin solution in aqueous buffer can be further purified by removing unwanted components according to known protein purification methods.

[0075] Therefore, the present invention relates to a method for producing a composition comprising the protein according to the present invention, (i) A step of expressing the protein in the above-mentioned plants, preferably edible plants or tobacco plants, (ii) A step of recovering plant material containing the protein expressed from the plant, (iii) A step of extracting the protein from the plant material using an aqueous buffer to obtain a composition containing the protein, Optionally, a step of removing undesirable impurities from the composition. This provides a method that includes [something]. If crebicin is expressed in plants, the plants or tissues containing the expressed protein can be recovered, homogenized, and insoluble substances can be removed by centrifugation or filtration. If relatively pure crebicin is desired, it can be further purified by generally known protein purification methods, such as chromatography, which can remove other host cell-derived proteins and plant metabolites (e.g., alkaloids and polyphenols). The purified crebicin solution can be concentrated and / or freeze-dried. When clebicin is expressed in edible plants, crude protein extracts or semi-purified concentrates from these plants can be used to prevent or reduce contamination of objects (e.g., food by Klebsiella). [Examples]

[0076] (See Example 1) Soft agar overlay assay for evaluating clebicin toxicity The overnight culture of Klebsiella quassineumonier subspecies simili pneumonier SB30 (DSM 28212) was mixed with LB medium and OD. 595 Equalize to 1.0 and dilute 100-fold in 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 several minutes to allow the agar to harden. Place a sterile Whatman disc (6 mm in diameter) on the soft agar and spread a 20 μl aliquot of crebisin solution containing 10 μg of crebisin protein onto the disc. Incubate the plate at 37°C for 16 hours. After 16 hours of incubation, measure the diameter of the crebisin inhibition zone.

[0077] Determination of Klebicin concentration The crebicin concentration in a liquid sample containing crebicin is determined by performing SDS-PAGE with Coomersie staining and reading the intensity of the crebicin band using a commercially available reader, and by comparing this determined intensity with the bands obtained by performing SDS-PAGE with Coomersie staining of serial dilutions of known concentrations of bovine serum albumin (BSA). A calibration curve can be obtained from the intensity of the bands on a BSA-stained SDS-PAGE gel. The concentration of BSA is determined using the Bradford protein assay (e.g., Bradford reagent, B6916, Sigma-Aldrich, St. Louis, Missouri, USA).

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

[0079] (Example 2) Expression of crebicin in plants Nicotiana benthamiana plants were grown in a growth chamber at 25°C and 50% humidity with a photoperiod of 16 hours of light (1500 lux) and 8 hours of darkness. Plants 4-6 weeks old were used for transfection with recombinant Agrobacterium tumefaciens. Agrobacterium tumefaciens, 50 mgL -1 Rifampicin and 50 mg L -1 The cultures were incubated overnight at 30°C in LB medium containing kanamycin. The overnight cultures of Agrobacterium were allowed to settle at 3220g for 5 minutes, and OD was used. 595 It was resuspended in 1.5 parts tap water. Leaves from 4-6 week-old plants were infused abaxially with a 1:1000 dilution of Agrobacterium tumefaciens strain containing the expression vector using a needleless syringe. The plant leaves were observed and collected at 4-7 dpi (days post infiltration). SDS-PAGE and Coomassi staining analysis of soluble protein extracts from infiltrated plant leaves revealed that all nine crebicins were efficiently expressed in the plants and detected as very strong supplemental bands on the gel (Figure 2). The polypeptide weights observed by electrophoresis closely corresponded to the expected theoretical molecular weights (KvarIa-43.4kDa, KpneIa-48.5kDa, KpneA-40kDa, KaerA-39kDa, KoxyY-48.7kDa, KpneM-30.3kDa, KpneM2-29.7kDa, KvarM-29.8kDa, and KaerM-29kDa). The expression levels of individual clebicins varied in the range of 2.7–4.4 mg / g FW, which is the highest expression level achieved by the two M-type clebicins of Klebsiella pneumoniae, KpneM2 and KpneM (Table 1).

[0080] (Example 3) Purification of crebicin from plant biomass KpneA, KaerA, KvarIa, KpneM, KpneM2, and KvarM bacteriocins were homogeneously purified by protein chromatography. Very pure KpneM, KpneM2, and KvarM proteins were obtained after a single-step hydrophobic interaction chromatography (HIC), but a second purification step by anion chromatography was included for best results. KpneA and KvarIa were also purified using hydrophobic interaction chromatography as the first step, but subsequently using 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 into a fine powder using a mortar and pestle with liquid nitrogen. The prepared powder was mixed with cold extraction buffer at a ratio of 1 g of plant material per 5 mL of buffer. The suspension was maintained on ice for 15–20 minutes. Cell debris was removed by centrifugation at 3220 g at 4°C for 20 minutes, and the supernatant was filtered through membrane filters (pore sizes 5 μm and 0.22 μm). The resulting solution was taken as total soluble protein and subjected to two-step chromatography for 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) (Figure 3A, B). A small portion of the frozen leaf tissue was homogenized using a mortar and pestle cooled in liquid nitrogen. The prepared 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 held at 20–25°C for 10–15 minutes. Cell debris was removed by centrifugation at 3220 g at 4°C for 20 minutes. The pellet was discarded, and the supernatant was filtered by passing the solution through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 0.70 M to adjust the pH of the solution to 6. The formed precipitate was removed by centrifugation at 3220 g at 4°C for 5 minutes. The supernatant was taken as total soluble protein and subjected to two-step purification.

[0082] In the initial purification step, a chromatography column was packed with phenyl Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) and pre-equilibriumized 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 replaced with a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution decreased 8 to 10 times. The concentrate was diluted to primary 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-equilibrium with cold buffer (50 mM NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and Q Sepharose-unbound proteins were collected in the efflux fraction. KpneM was then lyophilized and applied to the analysis.

[0083] KpneM2 was purified using a combination of hydrophobic interaction chromatography (HIC) and anion exchange chromatography (AEXC) (Figure 3C, D). A small portion of the frozen leaf tissue was homogenized using a mortar and pestle cooled in liquid nitrogen. The prepared 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 held at 20–25°C for 10–15 minutes. Cell debris was removed by centrifugation at 3220 g at 4°C for 20 minutes. The pellet was discarded, and the supernatant was filtered by passing the solution through membrane filters (pore sizes 5 μm and 0.45 μm). Ammonium sulfate was added to 0.70 M to adjust the pH of the solution to 6. The formed precipitate was removed by centrifugation at 3220 g at 4°C for 5 minutes. The supernatant was taken as total soluble protein and subjected to two-step purification.

[0084] In the initial 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 replaced with a diafiltration concentrator (10 kDa) and centrifuged at 3220 g until the volume of the protein solution decreased 8 to 10 times. The concentrate was diluted to primary volume with 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-equilibrium with cold buffer (50 mM NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and Q Sepharose-unbound proteins were collected in the flow-through fraction. KpneM2 was then lyophilized and applied to the analysis. KvarM was purified using a combination of hydrophobic interaction chromatography (HIC) and anion exchange chromatography (AEXC) (Figure 3E, F).

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

[0086] In the initial 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 to 10 times. The concentrate was diluted to primary 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-equilibrium with cold buffer (50 mM NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and Q Sepharose-unbound proteins were collected in the efflux fraction. KvarM was then lyophilized and applied to the analysis.

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

[0088] In the initial 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 to 10 times. The concentrate was diluted to primary volume with a buffer containing 20 mM NaH2PO4 / Na2HPO4 and 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 the final purification step using SP Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibriumized with cold buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citrate, pH 4.5). The protein solution was loaded onto the column, and the SP Sepharose-bound protein fraction was eluted by a linear gradient of cold wash buffer containing an additional 500 mM NaCl. KpneA was then lyophilized and applied to the analysis.

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

[0090] In the initial purification step, SP Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden) was packed into a chromatography column and pre-equilibrated with cold buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citrate, pH 4.5). The protein solution was loaded onto the column, and the SP Sepharose-bound protein fraction was eluted by 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 times. The concentrate was diluted to primary volume with 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 the 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 NaH2PO4 / Na2HPO4, pH 8.0). The protein solution was loaded onto the column, and Q Sepharose-unbound proteins were collected in the efflux fraction. KaerA was then lyophilized and applied to the analysis. KvarIa was purified using a combination of hydrophobic interaction chromatography (HIC) and cation exchange chromatography (CEXC) (Figure 3K, L).

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

[0092] In the initial 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 to 10 times. The concentrate was diluted to primary volume with a buffer containing 20 mM NaH2PO4 / Na2HPO4 and 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 the final purification step using SP Sepharose FF resin (GE Healthcare Life Sciences, Uppsala, Sweden). The chromatography medium was pre-equilibriumized with cold buffer (20 mM NaH2PO4 / Na2HPO4, 20 mM citrate, pH 4.5). The protein solution was loaded onto the column, and the SP Sepharose-bound protein fraction was eluted by a linear gradient of cold wash buffer containing an additional 500 mM NaCl. KvarIa was then lyophilized and applied to the analysis.

[0093] The concentration of purified protein was assessed by the Bradford assay or by comparing the band intensity performed on the same SDS-PAGE gel with the known BSA level. The results of crebicin purification are summarized in Table 1. Figure 4 shows purified KpneM, KpneM2, KvarM, KpneA, KaerA, and KvarIa crebicin proteins loaded onto the same gel. Capillary gel electrophoresis revealed that all purified crebicins contained only 0.2–3.7% impurities. The yield of individual crebicins after purification ranged from 0.34–1.1 mg / gFW. Purifying crebicins at the maximum expression level yields the maximum final yield and also the highest quality purified protein. [Table 1]

[0094] (Example 4) Clevicin activity testing in soft agar overlay assay The activity of bacteriocin-expressing crude plant extracts was tested using a soft agar overlay assay with 12 Klebsiella strains belonging to different species (Klebsiella pneumoniae, Klebsiella quasii pneumoniae, Klebsiella oxytoka, Klebsiella valicola, and Klebsiella aerogenes). The Klebsiella strains were purchased from the Leibniz Institute DSMZ - German Microbial and Cell Culture Collection and are listed in Table 2. Klebsiella cultures left overnight in LB medium (OD) 595 The mixture was equalized to 1.0 and diluted 100-fold in 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. A sterile Whatman disc (6 mm in diameter) was placed on the soft agar, and the respective amounts of crebicin (20 μl crude extract or 10 μg purified crebicin) were applied to the disc. The plate was incubated overnight at 37°C, and the diameter of the crebicin inhibition zone was observed. The results of this assay are summarized in Figure 5.

[0095] Two of the bacteriocins tested, KoxyY and KaerM, showed a narrow, perceptible inhibition zone in the colonies of several tested strains, while KaerM formed a larger, ambiguous inhibition zone only in the colonies of Klebsiella aerogenes. Due to their weak activity, these two proteins were not included in subsequent experiments. [Table 2]

[0096] All of the remaining seven bacteriocins formed large inhibitory zones in the colonies of several Klebsiella species and strains tested. All 12 strains tested were inhibited by several bacteriocins, not just one. The remaining three colM-like proteins showed the largest activity spectra and similar activity patterns, targeting 11 of the 12 tested strains. However, KvarM formed a significantly larger inhibitory zone than both of the Klebsiella pneumoniae colM-like bacteriocins (KpneM and KpneM2). The two ColA-like proteins KpneA and KaerA also showed very similar activity patterns, although the zone diameters differed in some of the tested strains. 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 inhibitory zones 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 clebicin activity in a panel of clinical Klebsiella isolates. Next, all six purified crebicin were tested against a larger panel of Klebsiella strains: a total of 100 clinical Klebsiella isolates, including 89 Klebsiella pneumoniae and 11 Klebsiella oxytoka strains. The clinical Klebsiella strains used for the agar overlay assay were isolated at the Kaunas Clinic of the University of Health Sciences, Lithuania, and are listed in Table 3. The purified lyophilized crebicin was resuspended in deionized water and inoculated as 10 μl droplets (10 μg protein) onto 6 mm Whatman discs placed on LB plates streaked with Klebsiella. After overnight incubation, the inhibition zone was measured.

[0098] KvarM exhibited remarkably broad activity. 85% of strains were susceptible to this crebicin (Figure 6, Table 3). KpneM lagged less, generally with a slightly smaller inhibition zone, targeting 74% of the strains tested. While the specificity of the activity spectra of KvarM and KpneM largely overlapped, KvarM targeted 11 more strains than KpneM, and only one strain immunized to KvarM was susceptible to KpneM (Figure 6, Table 3). In contrast, the third M-type crebicin, KpneM2, was considerably less active, targeting only 20% of strains. Both colA-like crebicins, KpneA and KaerA, targeted 30% and 28% of strains, respectively, with partially overlapping profiles. Nine strains immunized to KaerA were susceptible to KpneA, and seven strains immunized to KpneA were susceptible to KaerA. KpneA also generally formed a larger inhibition zone. The activity spectrum of KvarIa was the narrowest, targeting only 10% of all strains (6 in Klebsiella oxytoka and 4 in Klebsiella pneumoniae) (Figure 6, Table 3).

[0099] [Table 3] TIFF0007856871000004.tif208157 TIFF0007856871000005.tif213161 TIFF0007856871000006.tif135157

[0100] (Example 6) Evaluation of clebicin activity against Klebsiella strains in liquid cultures and biofilms. Next, the inventors performed a more detailed analysis of klebicin activity with five representative examples of different Klebsiella species, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella oxytoca, Klebsiella variicola, and Klebsiella aerogenes, in a liquid medium and young 1-day-old biofilms.

[0101] To evaluate klebicin activity in a liquid medium, an overnight Klebsiella culture was diluted to an OD 595 = 0.3 in iron-depleted casamino acid (CAA) medium (BD Bioscience) up to a maximum of 1.2 mL. Lyophilized purified klebicin was resuspended in CAA medium, added to the diluted bacterial suspension, and incubated at 37°C for 5.5 - 6.5 hours with shaking (200 rpm). The antibacterial activity of klebicin was evaluated by determining the cell number of the bacterial test culture. Serial dilutions of 10, 10 -1 、10 -2 、10 -3 、10 -4 、and 10 -5 were performed, plated on LB agar plates, incubated overnight at 37°C, and CFUs were calculated.

[0102] Biofilms were grown with some modifications as described in Moskowitz et al., (2004) and Paskevicius et al., (2017). Briefly, Klebsiella quasinneumoniae, Klebsiella oxytoka, Klebsiella barriicola, and Klebsiella aerogenes strains were grown overnight in LB and diluted to OD=0.1 in fresh CAA medium. 10 μl of bacterial culture was transferred to the wells of a 96-well microtiter plate (Nalgene Nunc International, Rochester, New York) containing 90 μl of CAA medium. Bacterial biofilms were formed by immersing pegs of modified polystyrene microtiter lids (Nunc TSP system) in the biofilm growth plate and subsequently incubating for 20 hours at a thermostat-controlled 30°C or 37°C. To treat with crebicin, the lids of the pegs were rinsed three times with sterile water and placed in microtiter plates containing 5 μg / mL crebicin diluted in 100 μl of CAA per well. Depending on the strain, the plates were incubated at 30°C or 37°C for 5 hours. After incubation with crebicin, the lids of the pegs were again rinsed three times with sterile water and placed in CAA on sterile microtiter plates, and centrifuged at 810 g for 30 minutes. Six wells, each treated in the same manner, were pooled, serially diluted, and the bacteria were plated onto LB plates for CFU counting.

[0103] For liquid culture assays, 5 μg mL -1At the specified concentrations, one of the most effective klebicin compounds from each group (colM-like, colA-like, and cola-like) from Example 4 was tested. Klebicin KvarM inhibited the growth of all five strains and reduced the CFU count to approximately the same extent. It reduced the CFU count by four orders of magnitude in Klebsiella pneumoniae DSM16231 and by approximately three orders of magnitude in all other klebicin compounds (Figure 7). KvarIa inhibited four strains and was the most efficient of all three klebicin compounds, reducing the CFU count by four to nine orders of magnitude depending on the strain (Figure 7) (as shown in Figure 5, Klebsiella pneumoniae DSM16231 is insensitive to this Klebsiella). KpneA reduced the CFU count of three strains by 4.6 to 5.7 logarithmically (Figure 7). The biofilm assay used the same Klebsiella strains as the liquid culture assay. The inventors 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 tested conditions and were therefore not used in further experiments. The remaining four stained biofilms were each treated with two crebicin solutions for 20 hours, which demonstrated the best results obtained in the liquid culture assay. The results obtained in the biofilm assays very well reflected the results obtained in the liquid culture assays, with the exception of Klebsiella quasi pneumoniae, whose biofilms were completely eradicated by KpneA and KvarIa (Figure 8). For all remaining strains, crebicin treatment reduced the number of CFUs in the biofilms to a similar degree as in the liquid culture assays, achieving only slight variations in Δlog (Figures 7 and 8).

[0104] (Example 7) Evaluation of the antibacterial activity of Crebisin in vivo For the first demonstration of Klebsiella activity in vivo, a Klebsiella challenge assay was performed in the larvae of the non-mammalian model Galleria mellonella (honeycomb moth). Galleria mellonella is a larger honeycomb moth or honeycomb moth and is a moth of the Pyralidae family. 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] In this assay, the inventors selected KvarIa as one of the most active crebicins and Klebsiella quasinensis DSM28212 as the KvarIa-sensitive challenge strain. The Galleria mellonella challenge experiment was performed as described by Paskevicius et al., (2017), with some modifications. Overnight cultures of Klebsiella quasinensis DSM 28212 were grown in CAA medium, diluted with 0.8% NaCl, and then mixed with 10 μL of Klebsiella quasinensis culture medium for a concentration of 1.2–3.2 × 10⁶. 6 CFUmL -1 A concentration of crebicin was achieved, and 10 μL of crebicin solution was injected into the hemocoel of 5th instar wax moth (Galleria melonella) larvae (Livefood UK) near the left and / or right prolegs. Crebicin was injected 2 hours after infection with Klebsiella quassine pneumoniae. The injected larvae were incubated at 37°C in a 9 cm petri dish without food for up to 3 days. Caterpillars were considered dead if they were motionless in response to mechanical stimulation of the head and showed a clear color change from cream to dark brown / black. Twenty larvae were used per treatment point. First, the minimum lethal dose (MLD) of the challenge strain sufficient to kill all larvae within 68 hours (the experimental period) is 2.3 × 10⁻⁶. 4 The inventors determined that it was a CFU.

[0106] Next, a challenge experiment was performed using MLD, with two additional challenge doses added, one of which was inferior to MLD by coefficients of 1.9 and 1.4 respectively, and the other was superior to MLD. Since 15% of the larvae survived after 68 hours, 1.2 × 10⁻⁶ 4 CFU was not sufficient to kill all the larvae. However, 2.3 and 3.2 × 10 4 CFU was sufficient to kill all larvae within 44 hours. Injection of KvarIa 2 hours after infection resulted in 1.2 and 2.3 × 10⁶ larvae. 4 All larvae infected with CFU were completely rescued. The highest amount of bacteria (3.2 × 10) 4 Larvae infected with CFU were partially rescued, and 85% of the larvae survived until the end of the experiment (Figure 9). In summary, activity assays demonstrate that KvarM possesses a very broad activity spectrum, as it can target Klebsiella strains belonging to the species Klebsiella pneumoniae, Klebsiella quasii pneumoniae, Klebsiella variicola, Klebsiella oxytoka, and Klebsiella aerogenes. It was also active against 85% of the antibiotic-resistant clinical Klebsiella isolates in a panel. In liquid culture assays, this crebicin can reduce colony formation by 3–4 logarithms, and in biofilm assays by more than 2 logarithms. Pore-forming crebicins are generally more efficient than peptidoglycan synthesis inhibitors in reducing bacterial counts in liquid culture or biofilms, achieving reductions of 4–9 logarithms in CFU counts in liquid culture and 2–approximately 6 logarithms in CFU counts in biofilms. KvarIa, which showed the highest efficiency in vitro, was also tested in vivo in a challenge assay against wax moth (Galleria melonella) larvae and yielded very good results. However, its applicability to Klebsiella is currently limited by the fact that, while it works well against the closely related Klebsiella quasinneumonier, it lacks broad activity against Klebsiella pneumonier.

[0107] (Example 8) Identification of clebicin receptors / transition factors A universal characteristic of cholicins is their domain configuration, with each cholicin appearing to possess receptor-binding, translocation, and cytotoxic domains, a feature conditioned by the need for these bacteriocins to cross the outer membrane of Gram-negative bacteria (Kleanthous, 2010). Amino acid sequence alignment of pore-forming crebicins with their E. coli counterparts has revealed that their killing domains exhibit a significant degree of homology. However, as a rule, pore-forming crebicins are smaller than cholicins. Their amino-terminal portions, which should contain the translocation and receptor-binding domains, are considerably shorter than the respective domains of cholicins, resulting in little to no sequence similarity. Therefore, we anticipated that the translocation mechanism of pore-forming crebicins may differ from that of their E. coli counterparts. In contrast to several other bacteriocins that are strictly species-specific (e.g., pyosin), clebisin activity is not limited to the single species from which they are isolated, but at least to the genus. In this regard, it was important to investigate the players involved in the mechanisms of clebisin reception and transposition. Klebsiella quasinneumoniae DSM 28212 is a strain with a known genome sequence and susceptibility to all clebisins tested, and after undergoing several transposon mutagenesis, pooled mutants were tested for susceptibility to different clebisins.

[0108] Transposon mutagenesis of Klebsiella quasipneumoniae DSM 28212 was performed as described in Martinez-Garcia et al., (2011). Suicide delivery of a minitransposon localized to the pBAM1 plasmid was performed by triplicate mating. This plasmid was recruited from E. coli CC118λpir(pBAM1) donor cells to Klebsiella quasipneumoniae DSM28212 cells with the help of the helper strain E. coli HB101 (pRK600). The resulting kanamycin-resistant clones were confirmed to have lost ampicillin resistance, and their genomic DNA was used for PCR amplification of the transposon-adjacent region and subsequently sequenced as described in Martinez-Garcia et al., (2011). Twenty-nine independent mutant clones were isolated, and transposon insertions were successfully mapped to 18 crebisin-resistant mutants. To confirm that the loss of crebisin sensitivity was indeed due to the mapped mutations, complementation assays were performed using ectopic expression of each wild-type gene.

[0109] For complementary assays, Klebsiella genomic regions containing the ExbB, ExbBD, OmpC, FhuA, TonB, and FimB gene ORFs, along with their 5' non-coding promoter regions, were PCR-amplified from Klebsiella quasipneumoniae DSM28212 genomic DNA with the help of Phusion DNA polymerase (Thermofisher Scientific Baltics) and ligated to pJET1.2 (Thermofisher Scientific Baltics). After sequencing, the cloned fragments were excised with fragment-specific restriction endonuclease pairs, ligated to pACYC184 (NEB), and transformed into their respective Klebsiella quasipneumoniae mutants. The primer sequences and cloning strategies used are shown in Table 4.

[0110] [Table 4]

[0111] Table 5 summarizes the results from mutant crebicin sensitivity studies and complementary assays. [Table 5]

[0112] Based on the results obtained, all crebicins except KvarIa are similar to group B colicins and utilize the TonB-dependent translocation pathway. All three M-type crebicins require FhuA, TonB, and ExbB for receptor-translocation, which is consistent with the fact that their E. coli homologs, colicins M.KpneA and KaerA, also depend on the TonB translocation pathway and require the functional OmpC. To date, we have been unable to identify any other putative receptors for these two crebicins (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 found that the only protein involved in KvarIa receptor-transposition was the outer membrane protein C (OmpC). OmpC mutants were selected for their resistance to KpneA and KaerA and appeared to be equally resistant to KvarIa. In summary, the inventors have demonstrated that the three M-type crebicins, KpneA, KpneM2, and KvarM, all require a translocation pathway involving the FhuA receptor and TonB to translocate via a mechanism similar to that of colicin M, enter the periplasm, and exert their activity.

[0114] Based on the similarity of their killing domains to colicin A, two klebicins named by the inventors, KpneA and KaerA, also appeared to depend on the TonB translocation pathway. This is in contrast to colicin A, which is translocated by the TolA-dependent pathway. Also, colicin A binds to BtuB, but no BtuB mutants resistant to KpneA or KaerA were isolated. However, both KpneA and KaerA require functional OmpC, an analog of OmpF that is also involved in colA translocation (Kleanthous 2010). So far, no other putative receptors for these two klebicins have been identified.

[0115] KvarIa appears to function in all TonB and ExbB mutants, differing from all other klebicins. Thus, based on the inventors' results, KvarIa does not use the TonB-dependent translocation pathway. Considering that all described colicins use either TonB or TolA as translocation factors, this protein is expected to be translocated by the Tol-dependent pathway. However, no single translocation mutant of Tol-dependent pathway-related genes resistant to KvarIa was isolated. Obtaining KvarIa-resistant transposon mutants was very difficult, and only some false-positive clones were isolated, so this may be related to the limitations of the method used. Under the conditions used for selection, mutations with a high fitness penalty may not have been obtained. Thus, so far, the outer membrane protein C (OmpC), the only protein involved in KvarIa's receptor-translocation, could be identified. OmpC mutants were selected by resistance to KpneA and KaerA and appeared to be equally resistant to KvarIa.

[0116] Further elucidation of the klebicin receptors and translocation factors is also important for the practical application of these klebicins. Klebicins are most promising for use in fighting antibiotic-resistant bacteria. However, 97.1% of carbapenem-resistant Klebsiella strains have been shown to not express or have low expression of OmpC or OmpF (Ye et al., 2018). In the study by the present inventors, carbapenem-resistant bacteria were not tested, but carbapenem-resistant Klebsiella has been shown to be expected to be resistant to KpneA, KaerA, and KvarIa, because all of these klebicins require a functional OmpC for their activity. The next step will be attempts to change the specificity of klebicins by manipulating the proteins, for example, by exchanging their receptor-translocations and killing the domains. On the other hand, in the current research situation, it can be concluded that there is a panel of six highly efficient plant-expressed klebicins 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 as pharmaceuticals as antibacterial agents against antibiotic-resistant Klebsiella.

[0117] (Example 10) MIC measurements of klebicins 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 inhibited the visible growth of the corresponding bacterial strain. To measure the MIC of individual crebicins, 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] Bacterial cultures grown overnight in MHB medium were dissolved in 1 ml of MHB. 595 The bacterial suspension was diluted to 0.5, and then diluted 1000-fold with 10 ml of the same medium. 90 μl aliquots of the diluted bacterial suspension were loaded into each well of a 96-well microplate already containing protein diluent using a multichannel pipette. Additional aliquots of the diluted bacterial suspension were plated on MHA medium (Müller-Hinton agar; 1.7% agar-containing MHB) for CFU enumeration in the initial bacterial inoculum. For bacterial growth, the microplates and agar plates were incubated at 30°C or 37°C for 20 hours, depending on the optimal growth conditions for the Klebsiella strain. Klebsiella pneumoniae subspecies ozaene DSM 16358, Klebsiella barriicola DSM 15968, and Klebsiella aerogenes DSM 30053 were incubated at 30°C, while Klebsiella quasinneumoniae subspecies similineumoniae DSM 28212 and Klebsiella oxytoka DSM 5175 were incubated at 37°C.

[0119] The MIC was determined by visual inspection of bacterial growth in the microplate wells. If the difference between two sets of trials was due to 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 nM (μM). [Table 7]

[0120] In most cases, the measured MIC values ​​of crebicin were less than 1–2 μg / ml. Nguen et al. (Scientific Reports (2018) 8:241) determined the MICs of 20 conventional antibiotics against 1497 strains of Klebsiella. Typically, the MIC values ​​determined in this study ranged from 0.5–32 μg / ml. Therefore, crebicin is equivalent to or superior to conventional antibiotics in terms of antibacterial activity calculated on a weight basis. Considering the difference in molecular weight (the MW of most antibiotics is less than 1 kDa), crebicin has significantly higher antibacterial activity when calculated on a molar basis.

[0121] (Example 11) Stability of crebicin KpneA, KaerA, KpneM, KpneM2, KvarM, and KvarIa during storage. To assess stability, purified, lyophilized crebicin protein samples were stored at -20°C, 5°C, and room temperature (approximately 23°C). Protein stability was evaluated based on antimicrobial 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. Crebicin was tested with the following strains: KpneM and KpneM2 with Klebsiella pneumoniae DSM16358, KvarIa with Klebsiella oxytoka DSM5175, and KpneA, KaerA, and KvarM with Klebsiella quasi pneumoniae DSM28212.

[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 the Bradford assay. For stability assessment in liquid culture, 5 μg of bacteriocin solution was added to CAA medium. 600 The bacteriocin was added to 1 ml of a suspension of a susceptible bacterial strain with a sensitivity of 0.3 (at point "0"). The bacteria mixed with the bacteriocin were incubated in a shaker for 4.5 hours. Klebsiella oxytoka DSM5175 and Klebsiella quasinneumoniae DSM28212 were incubated at 37°C, and Klebsiella pneumoniae DSM16358 was incubated at 30°C. Serial dilutions were performed in LB medium. The bacteria were plated on LB agar plates and incubated overnight at 30°C or 37°C, after which the CFU was calculated. Antimicrobial activity was expressed as CFU / mLΔlog relative to the untreated sample. 10 It was evaluated as such. In the radial diffusion assay, sequential 1:2 dilutions of the protein solubilized in PBS buffer were prepared. 5 μL of the protein dilution (1–1.8 μg of protein before dilution) was spotted onto a soft agar plate containing a highly susceptible bacterial strain. The residual activity of crebicin was evaluated after o / n incubation of the plate. Antimicrobial activity was evaluated as the highest dilution, taking into account specific energy units (AU), i.e., the difference in bacterial growth inhibition compared to the unaffected bacterial growth area determined by visual inspection of the plate by holding the plate in front of the light source. The highest dilution with growth inhibition was recorded as AU / μg bacteriocin activity. All experiments were performed three times.

[0123] At -20°C, despite a slight decrease in soluble protein concentrations (Figure 11G), the activity of all crebicin-containing proteins, KpneM, KpneM2, KvarM, KpneA, KaerA, and KvarIa, remained stable throughout the year (Figures 11A, D). When stored at 5°C, five of the six crebicins maintained their activity for one year; the activity of KpneA decreased (Figure 11E). The concentrations of KvarM and KpneM2 in solution decreased significantly, suggesting some degree of decrease in solubility during storage (Figure 11H). In general, crebicin exhibited poor stability during storage at room temperature. Nevertheless, the activity of crebicin KvarIa and KpneM remained stable for one year. The activity of KpneA, KvarM, and KpneM2 decreased dramatically (Figure 11C). This decrease in activity correlated with the protein concentration in solution, suggesting a decrease in solubility during storage (Figure 11I).

[0124] (Example 12) Evaluation of bacteriocin KvarIa activity against Klebsiella quasinensis in a mouse gastrointestinal model. Klebsiella pneumoniae is a normal component of the human gut microbiota. Gastrointestinal colonization has been considered a major reservoir for Klebsiella pneumoniae infection, particularly in patients in intensive care units (Gorrie et al., 2017). A prospective study in 1971 showed that 18.5% of patients who became colonized with multidrug-resistant Klebsiella pneumoniae after hospitalization were at a higher risk of developing subsequent infections caused by the same bacterium within 21 days compared to patients who did not become gut carriers (45% vs. 11%) (Martin and Bachman, 2018). Orally administered crebicin may be an efficient tool for eradicating asymptomatic, multidrug-resistant Klebsiella pneumoniae from the intestines of hospitalized patients. Since crebicin is rapidly inactivated by gastrointestinal enzymes when administered orally to hospitalized patients, it needs to be formulated to protect the stomach and ensure release in the small and large intestines. In this example, crebicin KvarIa was formulated with Eudragit S100 for delivery to the ileum and colon (release of crebicin at pH above 7) and force-administered orally to mice colonized with Klebsiella quasi pneumoniae in their intestines.

[0125] Kvaria cover A 5% Eudragit S100 solution was prepared by dissolving 0.5 g of Eudragit S100 (Evonik) in 10 ml of miliQ H2O and sonicating it in an ultrasonic bath at 25°C for 30 minutes. 250 μg of Kvar Ia was dissolved in 200 μg of 5% Eudragit S100. The resulting solution was freeze-dried at -51°C for 24 hours. Simulated gastric digestion, activity evaluation by radial diffusion assay. To investigate whether KvarIa coated with Eudragit-S100 is resistant to pepsin digestion, simulated gastric digestion experiments were performed. Protein exposure to simulated gastric juice (SGF, commercially available acidic pepsin extract) was performed using a low enzyme-to-substrate ratio. The method was derived from Moreno et al., (2005), Mandalari et al., (2009), and Eiwegger et al., (2006). Briefly, KvarIa produced in plants and KvarIa coated with Eudragit-S100 were mixed with SGF at the recommended concentration and incubated at 37°C for up to 60 minutes. Samples of the digested mixture were taken every few minutes for analysis. Digestion of protein fragments was assessed using SDS-PAGE. In parallel, residual antimicrobial activity was assessed using a radial diffusion assay. We used Coomassi staining on the gel to visualize proteolysis and estimate the median mass of peptide products; however, since Eudragit S100 distorted protein migration on the SDS-PAGE gel, this method was only used for uncoated KvarIa.

[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 1 mg of protein (pepsin:protein ratio 1:40) in the final digestion mixture containing 1 mg of protein and 0.025 mg of pepsin. The samples were placed in a shaker (200 rpm, 37°C). Aliquots (50 μl) of the reaction were taken at various time points (0.5, 5, 10, 20, 30, 60 minutes). Digestion was stopped by adding 0.5 M ammonium bicarbonate (10 μl NH4HCO3) to raise the pH to 6.5 in order to inactivate the pepsin. The Eudragit-coated KvarIa samples were adjusted to pH 8 to dissolve the Eudragit coating. Then, all samples were diluted with distilled water in a 1:2 ratio, and 5 μL aliquots of the diluted samples were dropped onto MHA plates containing Klebsiella quasinneumoniae DSM28212 for a 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. Coated KvarIa still showed detectable activity by agar diffusion assay 20 minutes after gastric digestion in vitro, while uncoated KvarIa was very rapidly inactivated in simulated gastric juice, having already lost its activity completely after 0.5 minutes of digestion (Figure 12A). SDS-PAGE profiles of uncoated KvarIa digest products clearly show that uncoated KvarIa is very rapidly digested by pepsin, and full-length protein is undetectable on the gel after 5 minutes of digestion (Figure 12B).

[0128] Colony formation in mouse intestines induced by Klebsiella quasinneumoniae DSM28212 and KvarIa treatment. Before the experiment, BALB / c mice (n=12) were acclimatized in individual cages for three days. To eradicate the gut microbiota, the mice were given drinking water containing ampicillin (2000 U / ml) and streptomycin (2 mg / ml) for three consecutive days. The ampicillin in the drinking water continued until the end of the experiment. On days 4-6 and 11-13 of the experiment, Klebsiella quasinneumoniae DSM28212 was administered orally. 9 CFU was administered to mice once daily. Five days after the last forced oral administration of Klebsiella quasinneumoniae (day 18), the mice were divided into four groups (n=3): Group 1 received forced oral administration of PBS, 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. Forced oral administration continued once daily from day 18 to day 21 (4 days). Fecal samples were collected before inoculation with Klebsiella quasinneumoniae and then daily from day 18 (immediately before the start of KvarIa treatment) to day 22 of the experiment. During treatment, the cages were changed daily. Throughout all experiments, the mice had free access to their normal diet, food, and water, but they were fasted for 6 hours prior to forced oral administration. The amount of Klebsiella quasipneumoniae DNA was quantified by real-time PCR using fecal samples collected on day 18 of the experiment (immediately before the start of KvarIa treatment) and day 22 of the experiment (one day after the final KvarIa treatment).

[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), 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 for real-time PCR: Klebsiella quasipneumoniae DSM28212 DNA (amplification of the Klebsiella hemolysin gene khe at cycle 13), Escherichia coli (E. coli) DNA - no khe amplification, and blanc - no khe amplification.

[0130]

Table 8

[0131] Colonization of the mouse intestine by Klebsiella quasipneumoniae DSM28212 before and after forced oral administration of klebicin According to the results of real-time PCR, it was confirmed that on the 18th day of the experiment (before the start of klebicin treatment), all mice showed the presence of Klebsiella quasipneumoniae DNA in their feces. On the 18th day of the experiment, the median CT values of each group of mice were in the range of 18.3 - 20.5 (Figure 14 and Table 9). Real-time PCR results showed that the number of Klebsiella quasinensis continued to increase in the feces of mice treated with PBS and uncoated KvarIa. On day 22, the day after the last Klebsiella treatment, the median CT value in the PBS group decreased from 19.97 to 17.23, and in the KvarIa-treated group it decreased from 20.05 to 18.36.

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

[0133] [Table 9]

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

[0135] Nucleic acid and amino acid sequences Sequence ID 1 (called KpneM) (Klebsiella pneumoniae EWD35590.1) MSETMVVATPTGFEPAGYGGGLFSPSTPNHSPQGQIFLQVTLPYYQSTKFCQDSMAWLAQYVKTHGAQDPLTIQVVANNIRYFLNADTNLCHNPKQNVWEAFHSEMTHSGPPPAKYDYHSMSLKQMSGN VVTPAAA FGHYLWGNGEARYVNLPDVGLKITPQMIPELMNIVNSGVTGHIPVDIKFVHDTSVSGGIVPAAYLGHITLRTEGTLDIQSGGAWTYNGVARAF NDTYDFNLGDFRGPIAESMTFLGSQFTGKQYEISMPGQINISGSGRR Sequence ID 2 (called KvarM) (Klebsiella variicola CTQ17225.1) MSDTMIVVATPTPGFSYASGLTYGGGAFAGAPANGPSEGQIFFQTVLPAYQSPNLCIGQLAWMTDYINKNGVGNPKTWEVISQNVLIFCSADTALVLNPRIAVYDGFHKTKWAPAKFNFKTQSQEKFSGNVTTPIAAFG HYLWGEGKPRTVDLSSVGLKIQANQIDPVMIAVKNNAAGTYQISGNFNRNTFIDGDIPGLYLGNITMKTEGTLKIDAKGNWNYNGVVRAFNDTYDANPSTHRSKSAEDLTTLLRLTQGTPYEIRIPGELKVSGSGKK

[0136] Sequence ID 3 (called KpneM2) (Klebsiera sp.WP_047066220) MSETLVVVAPAPSAPSMTYGGGLIYSSIPSGPNEGQIFFQTVLPAYSSPNFCTDRLRWMVKFINENGVGNPDTWKTLADVIRYYASADTAISKNPKTNPYDAWHKCPWPPASFDVKTMSVEKFSGSVNTPIVAFGHYL WGEGKPRSVDLSTVGLKVQANQIDPVMIAVKSYGAGTYQINGNFNRNTFDDGVIPGLYLGNITLKTEGTLKIEKNGSWNYNGVIRAFNDTYDANPSN HRSQAAEDLTTLLRITQGTPYEIRIPGEIKVSGSGKK Sequence ID 4 (called KaerM) (Klebsiella erogenes WP_015367360.1) MTDTLTVTATIPNGSSFNFQFEGMGNYYAAGSSTWDDPAMADAAHLYNAIQSMEDGSFTKALFADWLQFNAKGRENIPMINARFATMETMRFNDPGKAYFQFAQYNEYEGHTPGNNFTSGAFAPFLGLWHYIS GNGVETSLDITTIGLTFNQSNLTPVNDALKSQPPGNYPISSNFGKSVAEDNLYVAALLGRISMKTEGTLSIGESGEWSYNGVVRAYNDTYDANFDPSRGVIAQASTTVLSWFNGKPYPIALPGEIPVQLSGHR

[0137] Sequence ID 5 (called KpneA) (Klebsiella pneumoniae SAV78255.1) MPEETLTVVGGGNNSCNVSWGGGNGNNGGAGYSGKYGGTSYEGATSMLKLNDRVLIQLYLCNPLNPDYIGAPWGSDKDAESIIRANRDKPGKFKANIQNWKTSGTGSLGSPVVGKSYSSGDVDTYSVSFGKEKYNVLYNRKKDSFTTAYVDGGANKPEHSMKDQAIAVVKLYLLNE SQASVIDTTSGIITDSGKTLSGKLGDKYNTLAREAADNIKNFQGKKLRSFNDAMASINELANNPKMKLSQADKTVVSNALKQMDLSALADRFKGLEKAFTWGDRLLKAEKIRDGVVTGVTTGDWQKLAFEVEAMYLSGVAGAVALGITTAMISTVAVALSLPSVAVSALTVVAVIGI SILTSYIDADKAKALNNAVLGLFK

[0138] Sequence ID 6 (called KaerA) (Klebsiella erogenes WP_063414841.1) MANEDSMTVNGNAGSGVHWGGGSGNGNNGGAGSNGGANVALGGTMEVELGNGFTMIVDGTHPINPGIGGAPWSDDKSNKSAVDALNANKSKPAKFKANIQNYKSGTQGSLNSPAVNKSSSSGDVDTYAVSFGKEKYNV MYNRKKDSFTSGYVDGGATKPEHSMKDQAIAVVQLYLLNEKEKDVITTAAEIISSSGETISGKLGEKYKGLAQGVANDIRNFQGKKIRSFKDAMSSLEQFTKNPNMKLNQADKAALVNALNQVNLSTLADRFKGLERA FTWADRLLKAQKIKDGVVTGVTTGNWQPLALEVEAMYLSGVAGSVALGIVTGMISGLAALISIPALAVTAL TVTAVIGIAIATSYINADTAKALNNAVADLFK

[0139] Sequence ID 7 (called Koxy) (Klebsiella oxytoka WP_024273778) MAGFSYGGFGDGTTWSKERGTGPLPGGGSSGNSGNHSNTTPAEQKQINAIRADKNVRARLSNLIKAARKLNPSVKITVHAISPEGTMAISMEGLTATQARQAGLTGLVMGITVPGYIGSVGDFETGHKYNLKNPEKLNS IGVGTPLDGFNGGENIDTTPKKYRNWRATDEKSFYYVGTTVPMRLLHHLTVSRNKETDTYTMYFKAKDIKALYKIEVKNGDLDNMKLTTLAQGHPLFTAEFAKDIVRNFASVKNESDKEVLDKTSGVIISVGDKAGALLGEKY KALSREVASNIQNFQGKQIRTYDQAMASMNKLMTNPNMKIKAADKTAVINAWKAFNVEDMGNKFTALGRAFKVADYVTKGNNVREKSITGYETGNWGPLMREVESWTVSGLTSSVALAVFSATLGAMLVAAGVST AVVGIIGIIIAGLIGALIDDKFIDKLNNEIIRPAY

[0140] Sequence ID 8 (called KpneIa) (Klebsiella neumonier BAS34675) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPDKTTTTENVGGGKN GHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAKAEAEAAKAKAEAEAKAKAKAEAAAKAKAEAEAKAKAEAEAKAKAEAAAKAKAEAEAKAKAEAEAKAKAEAAAKAKAEAEAKAKAE AEAKAKAEAEAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKINAQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWRSFFV KIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI

[0141] Sequence ID 9 (called KvarIa) (Klebsiella variicola KDL88409) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPDRTTTTENVGGGKNGH NVTRSTTN GYVGPTMDVVDLVLELRKAIKEDNWRTFFVKIEAIAISFGATQLAALAFASLLGAPVGLLG YALIMAGIGALVSDDVVDAANKIIGI Sequence ID 10: ExbB Eco88I fwd AAACTCGGGTTGATGAACCTGTTTTTATACGTCT Sequence ID 11 ExbB Eco81I rev AAACCTGAGGTCAACCTACCCGTAATTTCTGCG

[0142] Sequence ID 12 ExbB Eco88I fwd AAACTCGGGTTGATGAACCTGTTTTTATACGTCT Sequence ID 13 ExbD Eco81I rev AAACCTGAGGTTATTTGGCTTTGACGGTCTC Sequence ID 14 FhuA Eco81I fwd AAACCTCAGGTTTAAGCCCTAAGACCAGACCC Sequence ID 15 FhuA Eco 81I rev AAACCTGAGGTTAGAAAACGGAAGGTGGGCGGTG Sequence ID 16 FimB Eco88I fwd AAACTCGGGGCTCCCGTAGCAAATAAAAACG Sequence ID 17 FimB Eco81I rev AAACCTGAGGGTTACTGAAGCAGCGACAGGCG Sequence ID 18 OmpCEco88I fwd AAACTCGGGCTTGTGGCTGAACGACTCATCA Sequence ID 19 OmpC Eco81I rev AAACCTGAGGTTAGAACTGGTAAACCAGGCCC

[0143] Sequence ID 20 TonB PsyI fwd AAAGACCGGGTCGGCAAAGCTCCTTATCAATAAACA Sequence ID 21 TonB BseSI rev AAAGTGCCCTCAGTTAATCTCGACGCCGTTG Sequence ID 22: Consensus sequence M(KpneM2, KvarM, KpneM, KaerM) MSXTXVVVATPXXXXXXXXTYGGGLFYXXXPXGPSEGQIFFQTVLPAYQSPNFCXDXLAWMADYINXNGVGNPXTWEVIAXNIRYFASADTALXXNPKXXVYDAFHKXXWPPAKXDXXTMSXEKFSGNVXTPIAAFG HYLWGXGKPRSVDLSTVGLKIQANQIDPVMIAVKSXXAGTYXISGNFNRNTFXDGXIPXXYLGNITXKTEGTLKIXXXGXWNYNGVVRAFNDTYDANPSXHRXXIAEDLTTLLXXXQGXPYEIRIPGEIKVSGSGKX

[0144] Sequence ID 23: Consensus sequence A(KaerA,KpneA) MXXEXXXXVXGXNXXXXVXWGGXXGNGNNGGAGXXGXXGXXXXXGXTXXXXLXBXXXXXXXXXXPJNPXXXGAPWXXXXSBKXAXXXJXANXXKP XKFKANIQNXKXXXXGSLXSPXVXKSXSSGDVDTYXVSFGKEKYNVXYNRKKDSFTXXYVDGGAXKPEHSMKDQAIAVVXLYLLNEXZXXVIXTXX XIIXXSGXTJSGKLGXKYXXLAXXXABBIXNFQGKKJRSFXDAMXSJXZXXXNPXMKLXQADKXXXXNALXQXBLSXLADRFKGLEXAFTWXDRL LKAZKIXDGVVTGVTTGBWQXLAXEVEAMYLSGVAGXVALGIXTXMISXXAXXJSJPXXAVXALTVXAVIGIXIXTSYIBADXAKALNNAVXXLFK

[0145] Sequence ID 24 Consensus Sequence Ia(KpneIa,KvarIa) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHD AMPDXTTTTENVGGGKNGHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAKXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXAXAKAKAEEAAKAKAEAXAKAKAEAXAKAKAEEAAKAKAEEAAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKIN AQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWRXFFVKIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI

[0146] Sequence ID 25 (array stretching saved in Sequence ID 22) TEGTL Sequence ID 26 (array stretching saved in Sequence ID 22) YNGV Sequence ID 27 (Array stretching saved in Sequence ID 22) RAFNDTYD Sequence ID 28 (array stretching saved in Sequence ID 23) GNGNNGGAG Sequence ID 29 (array stretched from sequence ID 23) GAPW Sequence ID 30 (array stretched from sequence ID 23) KFKANIQN

[0147] Sequence ID 31 (array stretched from sequence ID 23) SSGDVDTY Sequence ID 32 (array stretched from sequence ID 23) VSFGKEKYNV Sequence ID 33 (array stretched from sequence ID 23) YNRKKDSFT Sequence ID 34 (array stretched from sequence ID 23) YVDGGA Sequence ID 35 (array stretched from sequence ID 23) KPEHSMKDQAIAVV Sequence ID 36 (array stretched from sequence ID 23) LYLLNE

[0148] Sequence ID 37 (array stretched from sequence ID 23) SGKLG Sequence ID 38 (array stretched from sequence ID 23) NFQGKK Sequence ID 39 (array stretched from sequence ID 23) QADK Sequence ID 40 (array stretched from sequence ID 23) LADRFKGL Sequence ID 41 (array stretched from sequence ID 23) AFTW Sequence ID 42 (array stretched from sequence ID 23) DRLLKA Sequence ID 43 (array stretched from sequence ID 23) DGVVTGVTTG Sequence ID 44 (array stretched from sequence ID 23) EVEAMYLSGVAG

[0149] Sequence ID 45 (array stretched from sequence ID 23) VALGI Sequence ID 46 (array stretched from sequence ID 23) ALTV Sequence ID 47 (array stretched from sequence ID 23) AVIGI Sequence ID 48 (array stretched from sequence ID 23) TSYI Sequence ID 49 (array stretched from sequence ID 23) AKALNNAV Sequence ID 50 (array stretched from sequence ID 24) MPGFNYGGKGDGTNWSSERGTGPEPGGGSRGNGGDRDNSRGGAGNRGNWAGSGPLSAALINDSIAEALEKQLPRNTVEATSTPAYKKMRAAFDALPLDKQPEARAQITKAWQSAHDAMPD

[0150] Sequence ID 51 (array stretched from sequence ID 24) TTTTENVGGGKNGHNVTRSTPNWLKEKMKGLNQQVNNDLSGALAQHQKAEADARAKAEAAAKAK Sequence ID 52 (array stretched from sequence ID 24) AKAKAEAEAKAKAEA Sequence ID 53 (array stretched from sequence ID 24) AKAKAEA Sequence ID 54 (array stretched from sequence ID 24) AKAKAEAEAKAKAEAEAKAKAEADAVKDAVKFTADFYKEVFSVYGEKAEQLANLLATQAKGKNIRNIDDALKAYEKHKTNINKKINAQDRAAIAKALESVDVKEAAKNFAKFSKGLGYVGPTMDVVDLVLELRKAIKEDNWR

[0151] Sequence ID 55 (array stretched from sequence ID 24) FFVKIEAIAISFGATQLAALAFASLLGAPVGLLGYALIMAGIGALVSDDVVDAANKIIGI Sequence ID 56 khe gene amplification forward primer GATGAAACGACCTGATTGCATTC Sequence ID No. 57 khe gene amplification reverse primer CCGGGCTGTCGGGATAAG Sequence ID 58 khe gene amplification probe sequence (labeled with 6FAM at the 5' end and TAMRA at the 3' end) CGCGAACTGGAAGGGCCCG

[0152] References TIFF0007856871000013.tif220170 The contents of European Patent Application No. 19178 676.3, filed on June 6, 2019, including the detailed description, claims, drawings, and sequence listing, are incorporated herein by reference. Preferred embodiments of the present invention are as follows: [1] A protein having cytotoxic activity against Klebsiella, or a composition containing the protein, for use in therapeutic purposes. [2] The protein or composition for use according to [1], wherein the protein has lipid II cleavage activity or pore-forming ability in the cell membrane of Klebsiella cells. [3] A protein or composition for use according to [1] or [2] above, for use in a method of treating a target infection caused by Klebsiella, e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella variicola, wherein the Klebsiella may be antibiotic resistant, e.g., carbapenem resistant. [4] A protein or composition comprising the protein for use in a method of treating an infection of a target caused by Klebsiella, for example Klebsiella pneumoniae, Klebsiella oxytoka, Klebsiella quasinneumoniae, Klebsiella erogenes and / or Klebsiella barriicola, wherein the Klebsiella may be antibiotic resistant, for example carbapenem resistant. [5] A method for treating an infection caused by Klebsiella in a subject that requires treatment for such an infection, comprising administering to the subject a protein having cytotoxic activity against Klebsiella or a composition containing such protein. [6] 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 having cytotoxic activity against Klebsiella or a composition containing such protein. [7] The protein, composition, or method according to any one of the above [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 being preferably the N-terminal segment of the protein and the second segment being the C-terminal segment of the protein. [8] (A) The first segment is (A-ii) Amino acid residues 1-127 of sequence number 2 (KvarM), (Ai) Amino acid residues 1-128 of sequence number 1 (KpneM), (A-iii) Amino acid residues 1-123 of sequence number 3 (KpneM2), (A-iv) Amino acid residues 1-118 of sequence number 4 (KaerM), (Av) Amino acid residues 1-170 of sequence number 5 (KpneA), (A-vi) Amino acid residues 1-172 of sequence number 6 (KaerA), (A-vii) Amino acid residues 1-255 of sequence number 7 (Koxy), (A-viii) Amino acid residues 1-288 of sequence number 8 (KpneIa), or (A-ix) Amino acid residues 1-236 of sequence number 9 (KvarIa) Containing or consisting of the amino acid sequence; or (B) The first segment is (B-ii) Having at least 70% sequence identity with amino acid residues 1-127 of SEQ ID NO: 2 (Bi) The amino acid sequence of amino acid residues 1-128 of sequence number 1 has at least 70% sequence identity. (B-iii) Having at least 70% sequence identity with amino acid residues 1-123 of SEQ ID NO: 3, (B-iv) Having at least 70% sequence identity with amino acid residues 1-118 of sequence number 4, (Bv) Has at least 70% sequence identity with amino acid residues 1-170 of SEQ ID NO: 5 (B-vi) Having at least 70% sequence identity with amino acid residues 1-172 of sequence number 6, (B-vii) Having at least 70% sequence identity with amino acid residues 1-255 of sequence number 7, (B-viii) Having at least 70% sequence identity with amino acid residues 1-288 of sequence number 8, or (B-ix) Has at least 70% sequence identity with amino acid residues 1-236 of sequence number 9. Does it contain an amino acid sequence? or (C) The first segment is (C-ii) Compared to the amino acid sequence of amino acid residues 1-127 of SEQ ID NO: 2, there are 1-40 amino acid substitutions, additions, insertions and / or deletions. (Ci) Compared to the amino acid sequence of amino acid residues 1-128 of SEQ ID NO: 1, it has 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-iii) Compared to the amino acid sequence of amino acid residues 1-123 of SEQ ID NO: 3, there are 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-iv) Compared to the amino acid sequence of amino acid residues 1-118 of SEQ ID NO: 4, there are 1-40 amino acid substitutions, additions, insertions and / or deletions. (Cv) Compared to the amino acid sequence of amino acid residues 1-170 of SEQ ID NO: 5, it has 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-vi) Compared to the amino acid sequence of amino acid residues 1-172 of SEQ ID NO: 6, there are 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-vii) Compared to the amino acid sequence of amino acid residues 1-255 of SEQ ID NO: 7, it has 1-40 amino acid substitutions, additions, insertions and / or deletions. (C-viii) Compared to the amino acid sequence of amino acid residues 1-288 of SEQ ID NO: 8, it has 1-40 amino acid substitutions, additions, insertions and / or deletions, (C-ix) Compared to the amino acid sequence of amino acid residues 1-236 of SEQ ID NO: 9, it has 1-40 amino acid substitutions, additions, insertions, and / or deletions. The protein, composition, or method described in [7] above, 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 The protein, composition, or method according to [8], wherein in item (C), the number of substitutions, additions, insertions, and / or deletions of the amino acids 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 128-276 of sequence number 2 (KvarM), (Di) Amino acid residues 129-278 of sequence number 1 (KpneM), (D-iii) Amino acid residues 124-272 of sequence number 3 (KpneM2), (D-iv) Amino acid residues 119-266 of sequence number 4 (KaerM), (Dv) Amino acid residues 171-377 of sequence number 5 (KpneA), (D-vi) Amino acid residues 173-379 of sequence number 6 (KaerA), (D-vii) Amino acid residues 256-452 of sequence number 7 (Koxy), (D-viii) Amino acid residues 289-466 of sequence number 8 (KpneIa), or (D-ix) Amino acid residues 237-414 of sequence number 9 (KvarIa) Containing or consisting of the amino acid sequence; or (E) The second segment is (E-ii) Having at least 70% sequence identity with amino acid residues 128-276 of Sequence ID No. 2, (Ei) Having at least 70% sequence identity with amino acid residues 129-278 of sequence number 1, (E-iii) Having at least 70% sequence identity with amino acid residues 124-272 of sequence number 3, (E-iv) Having at least 70% sequence identity with amino acid residues 119-266 of sequence number 4, (Ev) The amino acid sequence of amino acid residues 171-377 of sequence number 5 has at least 70% sequence identity. (E-vi) Having at least 70% sequence identity with amino acid residues 173-379 of sequence number 6, (E-vii) Having at least 70% sequence identity with amino acid residues 256-452 of sequence number 7, (E-viii) Having at least 70% sequence identity with amino acid residues 289-466 of sequence number 8, or (E-ix) Has at least 70% sequence identity with amino acid residues 237-414 of sequence number 9. Does it contain an amino acid sequence? or (F) The second segment is (F-ii) Compared to the amino acid sequence of amino acid residues 128-276 of SEQ ID NO: 2, there are 1 to 30 amino acid substitutions, additions, insertions, or deletions. (Fi) Compared to the amino acid sequence of amino acid residues 129-278 of SEQ ID NO: 1, there are 1-30 amino acid substitutions, additions, insertions and / or deletions. (F-iii) Compared to the amino acid sequence of amino acid residues 124-272 of SEQ ID NO: 3, there are 1-30 amino acid substitutions, additions, insertions and / or deletions. (F-iv) Compared to the amino acid sequence of amino acid residues 119-266 of SEQ ID NO: 4, there are 1-30 amino acid substitutions, additions, insertions and / or deletions. (Fv) Compared to the amino acid sequence of amino acid residues 171-377 of SEQ ID NO: 5, there are 1-35 amino acid substitutions, additions, insertions and / or deletions. (F-vi) Compared to the amino acid sequence of amino acid residues 173-379 of SEQ ID NO: 6, there are 1-35 amino acid substitutions, additions, insertions and / or deletions. (F-vii) Compared to the amino acid sequence of amino acid residues 256-452 of SEQ ID NO: 7, there are 1-35 amino acid substitutions, additions, insertions and / or deletions. (F-viii) Compared to the amino acid sequence of amino acid residues 289-466 of sequence number 8, it has 1-35 amino acid substitutions, additions, insertions and / or deletions. (F-ix) Compared to the amino acid sequence of amino acid residues 237-414 of sequence number 9, it has 1-35 amino acid substitutions, additions, insertions and / or deletions. A protein, composition, or method according to any one of the above items [7], [8], and [9], comprising an amino acid sequence.

[11] The protein, composition, or method according to [9] or

[10] , wherein the first segment is one of items Ai~A-iv, Bi~B-iv, or Ci~C-iv, and the second segment is one of items Di~D-iv, Ei~E-iv, or Fi~F-iv.

[12] The protein, composition, or method according to

[11] , wherein each of the first segments is one or more of the items Ai to A-iv and the second segment is one of the items Di to D-iv; or each of the first segments is one of the items Bi to B-iv and the second segment is one of the items Ei to E-iv; or each of the first segments is one of the items Ci to C-iv and the second segment is one of the items Fi to F-iv.

[13] The protein is (a)(a-ii) Sequence ID 2 (KvarM), (ai) Sequence ID 1 (KpneM), (a-iii) Sequence ID 3 (KpneM2), (a-iv) Sequence ID 4 (KaerM), (av) Sequence ID 5 (KpneA), (a-vi) Sequence ID 6 (KaerA), (a-vii) Sequence ID 7 (Koxy), (a-viii) Sequence ID 8 (KpneIa), or (a-ix) Sequence ID 9 (KvarIa) The amino acid sequence; or (b)(b-ii) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 2, (bi) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, (b-iii) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 3, (b-iv) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 4, (bv) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 5, (b-vi) Having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 6, (b-vii) Having at least 70% sequence identity with the amino acid sequence of sequence number 7, (b-viii) Having at least 70% sequence identity with the amino acid sequence of sequence number 8, or (b-ix) Has at least 70% sequence identity with the amino acid sequence of Sequence 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 Sequence ID No. 2, (ci) Compared to the amino acid sequence of SEQ ID NO: 1, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (c-iii) Compared to the amino acid sequence of SEQ ID NO: 3, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (c-iv) Compared to the amino acid sequence of SEQ ID NO: 4, there are 1 to 80 amino acid substitutions, additions, insertions and / or deletions. (cv) Compared to the amino acid sequence of SEQ ID NO: 5, it has 1 to 110 amino acid substitutions, additions, insertions and / or deletions. (c-vi) Compared to the amino acid sequence of SEQ ID NO: 6, there are 1 to 110 amino acid substitutions, additions, insertions and / or deletions. (c-vii) Compared to the amino acid sequence of SEQ ID NO: 7, it has 1 to 130 amino acid substitutions, additions, insertions and / or deletions. (c-viii) Compared to the amino acid sequence of SEQ ID NO: 8, it has 1 to 130 amino acid substitutions, additions, insertions and / or deletions, or (c-ix) Compared to the amino acid sequence of SEQ ID NO: 9, it has 1 to 120 amino acid substitutions, additions, insertions, and / or deletions. amino acid sequence A protein, composition, or method according to any one of the above items [1] to [6], comprising or comprising an amino acid sequence consisting thereof.

[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 The protein, composition, or method according to

[13] , wherein in item (c), the number of substitutions, additions, insertions and / or deletions of the amino acids 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] A protein, composition, or method according to any one of the above [1] to

[14] , having cytotoxic activity against Klebsiella pneumoniae, Klebsiella oxytoka, 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, where each X represents 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, composition, or method according to any one of the above items [1] to

[15] , wherein the protein is one of those defined with respect to SEQ ID NOs: 1 to 4, 7, 8, or 22, preferably one of those defined with respect to SEQ ID NOs: 1 to 4 or 22.

[17] The cytotoxic activity of the protein is - The aforementioned protein, and - Comparison of amino acid sequences of SEQ ID NO: 1 protein However, when 20 microliters each of the protein and comparative protein solutions were spotted onto the bacterial colony of a susceptible Klebsiella strain on an agar plate, and the agar plate was incubated at 37°C for 16 hours, the activity was such that it produced spots free of surviving bacteria of the susceptible Klebsiella subspecies similipneumoniae SB30 (DSM 28212) of at least the same diameter. The protein, composition, or method according to any one of the above items [1] to

[16] , wherein the concentration of the protein in the solution is up to five times the concentration of the comparative protein solution.

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

[19] The protein described in

[18] , as further defined in any one of the preceding paragraphs [7] to

[17] .

[20] A composition comprising one or more proteins as defined in any one of the above items [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 the following items [7] to

[12] ,

[13] to

[17] and 18 to 19.

[22] A composition comprising one or more proteins, preferably a pharmaceutical composition, wherein the one or more proteins comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 24, where each X represents 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 one of those defined in SEQ ID NOs: 1-4, 7, 8, or 22, preferably one of those defined in SEQ ID NOs: 1-4 or 22; Preferably, the composition comprises a carrier.

[23] The composition according to any one of the above

[20] to

[22] , wherein the plant material is a plant or an extract thereof, and the plant material may be a plant expressing the one or more proteins, preferably a material derived from an edible plant expressing the one or more proteins.

[24] The composition according to any one of the above

[20] to

[23] , wherein the one or more proteins are formulated for oral delivery to the small or large intestine.

[25] An oral formulation comprising a protein as defined in any one of [1] to

[17] ,

[18] and

[19] above, or a composition as described in any one of

[20] to

[24] above, which can protect the protein from gastric conditions and release the protein in the intestine.

[26] A nucleic acid molecule or nucleic acid construct encoding a protein as defined in any one of [1] to

[17] ,

[18] or

[19] , preferably a protein as defined in any one of [7] to

[17] , preferably comprising a transcription promoter active in plant cells, and the nucleotide sequence encoding the protein for expression in cells, preferably in plant cells, under the control of the promoter.

[27] A nucleic acid molecule or nucleic acid construct encoding a protein as defined in any one of [1] to

[17] ,

[18] or

[19] , preferably a protein as defined in any one of [7] to

[17] , which is a viral (DNA or RNA) replicon containing the nucleotide sequence encoding the protein for expressing the nucleotide sequence in a cell, preferably a plant cell, or a nucleic acid molecule or nucleic acid construct encoding such a protein.

[28] A plant, plant tissue, or plant cell comprising a protein as defined in any one of the preceding paragraphs [1] to

[17] ,

[18] , and

[19] , or a nucleic acid molecule or nucleic acid construct as defined in paragraph

[26] or

[27] .

Claims

1. A pharmaceutical composition for use in a method of treating a target infection caused by Klebsiella, comprising a protein having cytotoxic activity against Klebsiella, wherein the protein comprises or consists of a first amino acid sequence segment and a second amino acid sequence segment directly bound in this order. (A) The first amino acid sequence segment consists of amino acid residues 1-127 of (A-ii) Sequence ID No. 2 (KvarM); or (B) The first amino acid sequence segment consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of amino acid residues 1 to 127 of (B-ii) Sequence ID No. 2; or (C) The first amino acid sequence segment consists of an amino acid sequence having 1 to 10 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of amino acid residues 1 to 127 of (C-ii) Sequence ID No. 2, and (D) Whether the second amino acid sequence segment consists of amino acid residues 128-276 of (D-ii) SEQ ID NO: 2 (KvarM); or (E) The second amino acid sequence segment consists of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of amino acid residues 128-276 of (E-ii) Sequence ID No. 2; or (F) The second amino acid sequence segment consists of an amino acid sequence having 1 to 15 amino acid substitutions, additions, insertions, or deletions compared to the amino acid sequence of amino acid residues 128 to 276 of (F-ii) Sequence ID No.

2. Pharmaceutical composition.

2. In item (B), sequence identity is at least 95%; and / or In item (C), the number of amino acid substitutions, additions, insertions, and / or deletions is 1 to 5 compared to the amino acid sequence of amino acid residues 1 to 127 of SEQ ID NO:

2. The pharmaceutical composition according to claim 1.

3. The pharmaceutical composition according to claim 1, wherein the first amino acid sequence segment is item A-ii and the second amino acid sequence segment is item D-ii; or, respectively, the first amino acid sequence segment is item B-ii and the second amino acid sequence segment is item E-ii; or, respectively, the first amino acid sequence segment is item C-ii and the second amino acid sequence segment is item F-ii.

4. A pharmaceutical composition for use in a method of treating a target infection caused by Klebsiella, comprising a protein having cytotoxic activity against Klebsiella, The aforementioned protein, (a) (a-ii) Amino acid sequence of Sequence ID No. 2 (KvarM); or (b) (b-ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of Sequence ID No. 2; or (c) (c-ii) Amino acid sequences having 1 to 30 amino acid substitutions, additions, insertions and / or deletions compared to the amino acid sequence of Sequence ID No. 2 containing, or comprising an amino acid sequence consisting thereof, Pharmaceutical composition.

5. In item (b), sequence identity is at least 95%; and / or In item (c), the number of amino acid substitutions, additions, insertions, and / or deletions is 1 to 20 compared to the amino acid sequence of SEQ ID NO:

2. The pharmaceutical composition according to claim 4.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the protein has cytotoxic activity against Klebsiella pneumoniae, Klebsiella oxytoka, Klebsiella granulomatis, Klebsiella quasipneumoniae, Klebsiella aerogenes and / or Klebsiella vallicola.

7. The protein contains the amino acid sequence of Sequence ID No. 22, where each X represents one of the 20 standard amino acid residues, or the absence of an amino acid residue; or The protein contains the amino acid sequence of SEQ ID NO: 2, A pharmaceutical composition according to any one of claims 1 to 6.

8. A pharmaceutical composition according to any one of claims 1 to 7, comprising one or more proteins as defined in any one of claims 1 to 7, and a carrier.

9. The pharmaceutical composition according to claim 8, wherein the plant material is a plant material or an extract thereof, and the plant material may be a plant-derived material expressing one or more of the aforementioned proteins.

10. The pharmaceutical composition according to claim 8 or 9, wherein the one or more proteins are formulated for oral delivery to the small or large intestine.

11. An oral formulation for use in a method of treating a target infection caused by Klebsiella, comprising a protein as defined in any one of claims 1 to 7, or a pharmaceutical composition as described in any one of claims 8 to 10, wherein the protein can be protected from gastric conditions and released in the intestine.

12. A pharmaceutical composition or formulation according to any one of claims 1 to 11, for use in a method for treating a target infection caused by Klebsiella, wherein the Klebsiella is antibiotic resistant.

13. A method for preventing or reducing contamination of an inanimate object by one or more species of Klebsiella, comprising the step of contacting the inanimate object with a protein having cytotoxic activity against Klebsiella or a composition comprising the protein, wherein the protein is defined in any one of claims 1 to 7.

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