Modified peptides

Abstract

The present invention relates to the field of antimicrobial agents. In particular, the present invention relates to polypeptides comprising the sequence of a peptidoglycan hydrolase and a peptide sequence heterologous to the peptidoglycan hydrolase wherein said heterologous peptide sequence comprises a specific sequence motif which is 16, 17, 18, 19 or 20 amino acids in length. The present invention relates also to corresponding nucleic acids, vectors, bacteriophages, host cells, compositions and kits. The present inventions also relates to the use of said polypeptides, nucleic acids, vectors, bacteriophages, host cells, compositions and kits in methods for treatment of the human or animal body by surgery or therapy or in diagnostic methods practiced on the human or animal body. The polypeptides, nucleic acids, vectors, bacteriophages, host cells, compositions and kits according to the invention may also be used as an antimicrobial in, e.g., food or feed, in cosmetics, or as disinfecting agent.

1 . A fusion protein comprising the sequence of:
 a) a peptidoglycan hydrolase, and   b) a peptide sequence heterologous to the peptidoglycan hydrolase, wherein said heterologous peptide sequence comprises a sequence motif which:
 i) is 16, 17, 18, 19 or 20 amino acids in length; 
 ii) comprises at least 40% and at most 60% amino acids selected from a first group of amino acids consisting of lysine, arginine and histidine,
 wherein each amino acid is selected independently from said first group, 
 wherein each amino acid selected from this first group is arranged in said sequence motif either alone, pairwise together with a further amino acid selected from the first group, or in a block with 2 further amino acids selected from the first group, but does not occur in a block with 3 or more amino acids selected from the first group, wherein at least 2 pairs of amino acids selected from the first group are present in said sequence motif, and wherein at most one block with 3 of the amino acids selected from the first group in a row is present in said sequence motif, with the additional proviso, that if such block with 3 amino acids of the first group is present in said sequence motif, then the amino acids at positions −12, −11, −8, −5, −4, +6, +7, +10, +13, and +14 relative to the first amino acid of the 3 amino acid block are, provided the respective position may be found in said sequence motif, not selected from said first group, 
 
 iii) comprises at least 40% and at most 60% amino acids selected from a second group of amino acids consisting of alanine, glycine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, tyrosine and valine,
 wherein each amino acid is selected independently from said second group, 
 wherein at least three different amino acids are selected from this second group, if the sum of amino acids of selected from the first group and selected from the second group yield 100% of the sequence motif; 
 wherein the sequence motif does not comprise the sequence AFV, if the sequence motif contains at least two single, non-adjacent phenylalanine residues and at least one of these phenylalanine residues is directly preceded by a lysine residue, and wherein the sequence motif does not comprise the sequence AALTH (SEQ ID NO:2), if the sequence motif contains at least three single, non-adjacent histidine residues, 
 
 iv) wherein the remaining amino acids of said sequence motif, if any are present in the motif, are selected from a third group consisting of asparagine, aspartic acid, glutamine, glutamic acid, methionine, or cysteine, wherein each of said amino acids is selected independently from said third group, and wherein glutamine may be selected only once and wherein the selection may furthermore not comprise a combination of glutamine and glutamic acid, 
 v) wherein the sequence motif complies with one of the sequence motifs depicted in  FIG. 1 , and wherein “X” denotes that the sequence motif does not exhibit at the respective position an amino acid selected from the first group, and 
   c) wherein said fusion protein does not comprise the sequence of SEQ ID NO:1.   
     
     
         2 . (canceled) 
     
     
         3 . The fusion protein according to  claim 1 , wherein “X” denotes that the sequence motif exhibits at said position an amino acid selected from the second group. 
     
     
         4 . The fusion protein according to  claim 1 , wherein the sequence motif is 17, 18 or 19 amino acids in length. 
     
     
         5 . The fusion protein according to  claim 1 , wherein the amino acids selected from the third group are selected from asparagine, aspartic acid, glutamine, and glutamic acid. 
     
     
         6 . The fusion protein according to  claim 1 , wherein the sequence motif does not contain more than one amino acid selected from the third group, preferably wherein the sequence motif does not contain any amino acid selected from the third group. 
     
     
         7 . The fusion protein according to  claim 1 , wherein the sequence motif does not comprise a block consisting of 3 amino acids of the first group. 
     
     
         8 . The fusion protein according to  claim 1 , wherein the peptide sequence is an artificial peptide sequence not occurring in nature. 
     
     
         9 . The fusion protein according to  claim 1 , wherein the peptidoglycan hydrolase is Lys394, KZ144, OBPgpLys endolysin or a tail baseplate protein of Vibrio phage ICP1, in particular wherein the fusion protein comprises the sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27. 
     
     
         10 . The fusion protein according to  claim 1 , wherein the sequence motif is helical. 
     
     
         11 . The fusion protein according to  claim 1 , wherein a proline residue is located within 1 to 5 amino acid residues N-terminal or C-terminal of the sequence motif. 
     
     
         12 . The fusion protein according to  claim 11 , wherein said proline residue is located between the sequence of the peptidoglycan hydrolase and the sequence motif. 
     
     
         13 . The fusion protein according to  claim 1 , wherein the sequence motif is situated N-terminal of the sequence of the peptidoglycan hydrolase. 
     
     
         14 . The fusion protein according to  claim 1 , wherein the fusion protein comprises a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41 SEQ ID NO:42 and SEQ ID NO:43. 
     
     
         15 . A polypeptide comprising the sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23. 
     
     
         16 . A nucleic acid encoding the fusion protein according to  claim 1 . 
     
     
         17 . A vector comprising a nucleic acid according to  claim 16 . 
     
     
         18 . A bacteriophage comprising a nucleic acid according to  claim 16 . 
     
     
         19 . A host cell comprising a fusion protein according to  claim 1 . 
     
     
         20 . A composition comprising a fusion protein according to  claim 1 . 
     
     
         21 . The composition according to  claim 20 , wherein the composition is a pharmaceutical composition comprising a pharmaceutical acceptable diluent, excipient or carrier or wherein the composition is a cosmetic composition comprising an acceptable diluent, excipient or carrier. 
     
     
         22 . A kit comprising a fusion protein according to  claim 1 . 
     
     
         23 . A method of treatment of the human or animal body by surgery or therapy or in diagnostic methods practiced on the human or animal body, in particular for use in a method of treatment or prevention of bacterial infections, comprising administering to a subject in need thereof a polypeptide according to  claim 1 . 
     
     
         24 . A method of providing an antimicrobial in food, feed, or cosmetics, or as disinfecting agent comprising introducing into food, feed or cosmetics, or applying to a surface in need of disinfecting, a polypeptide according to  claim 1 . 
     
     
         25 . A method for the treatment or prevention of Gram-negative bacterial contamination of foodstuff, of food processing equipment, of food processing plants, of surfaces coming into contact with foodstuff, of feedstuff, of feed processing equipment, of feed processing plants, of surfaces coming into contact with feedstuff, of medical devices, or of inanimate surfaces in hospitals, doctor's offices and other medical facilities, comprising treating foodstuff, food processing equipment, food processing plants, surfaces coming into contact with foodstuff, feedstuff, feed processing equipment, feed processing plants, surfaces coming into contact with feedstuff, medical devices, or inanimate surfaces in hospitals, doctor's offices and other medical facilities with a polypeptide according to  claim 1 .