Antimicrobial agents

Abstract

The present invention relates to antimicrobial agents against Gram-positive bacteria, in particular to fusion proteins composed of an enzyme having the activity of degrading the cell wall of Gram-positive bacteria and an additional peptide stretch fused to the enzyme at the N- or C-terminus. Moreover, the present invention relates to nucleic acid molecules encoding said fusion protein, vectors comprising said nucleic acid molecules and host cells comprising either said nucleic acid molecules or said vectors. In addition, the present invention relates to said fusion protein for use as a medicament, in particular for the treatment or prevention of Gram-positive bacterial infections, as diagnostic means or as cosmetic substance. The present invention also relates to the treatment or prevention of Gram-positive bacterial contamination of foodstuff, of food processing equipment, of food processing plants, of surfaces coming into contact with foodstuff, of medical devices, of surfaces in hospitals and surgeries. Further, the present invention relates to a pharmaceutical composition comprising said fusion protein.

1 . A fusion protein comprising an enzyme having Gram-positive cell wall degrading activity and a peptide segment fused to the enzyme at the N- or C-terminus or at both termini. 
     
     
         2 . The fusion protein according to  claim 1 , wherein said fusion protein comprises an additional amino acid residue on the N-terminus. 
     
     
         3 . The fusion protein according to  claim 1 , wherein said fusion protein exhibits an amino acid sequence according to SEQ ID NO: 63 to 90. 
     
     
         4 . The fusion protein according to  claim 1 , wherein said fusion protein comprises a tag or additional protein on the C- and/or N-terminus. 
     
     
         5 . The fusion protein according to  claim 4 , wherein said tag or additional protein is linked to the fusion protein by one or more additional amino acid residues. 
     
     
         6 . The fusion protein according to any of the preceding claims, wherein the peptide segment is linked to the fusion protein by one or more additional amino acid residues. 
     
     
         7 . The fusion protein according to  claim 1 , wherein the enzyme is an endolysin, autolysin or a bacteriocin. 
     
     
         8 . The fusion protein according to  claim 7 , wherein the enzyme exhibits an amino acid sequence according to SEQ ID NO: 57 to 61. 
     
     
         9 . The fusion protein according to  claim 1 , wherein the Gram-positive cell wall degrading activity is against a bacterium selected from the group consisting of the bacteria listed in Table 1. 
     
     
         10 . The fusion protein according to  claim 1 , wherein the peptide segment is a cationic. 
     
     
         11 . The fusion protein according to  claim 32 , wherein the sushi peptide exhibits an amino acid sequence according to SEQ ID NO: 54. 
     
     
         12 . The fusion protein according to  claim 10 , wherein the cationic peptide comprises at least one amino acid residue selected out of the group consisting of arginine, histidine and lysine residues. 
     
     
         13 . The fusion protein according to  claim 32 , wherein the antimicrobial peptide exhibits an amino acid sequence according to SEQ ID NO:1 to 11 or 48 to 53. 
     
     
         14 . The fusion protein according to  claim 32 , wherein the hydrophobic peptide exhibits an amino acid sequence according to SEQ ID NO: 12, 50, 55 or 56. 
     
     
         15 . The fusion protein according to  claim 10 , wherein at least about 70% of the amino acid residues of said cationic peptide are either arginine, histidine or lysine residues, or wherein at least about 70% of the amino acid residues of said peptide are either arginine or lysine, or wherein the amino acid residues of said peptide are either arginine or lysine residues. 
     
     
         16 . The fusion protein according to  claim 15 , wherein the cationic peptide exhibits an amino acid sequence according to SEQ ID NO: 13 or 24 to 44. 
     
     
         17 . The fusion protein according to  claim 32 , wherein the amphipatic peptide comprises at least one positively charged amino acid residue selected from the group consisting of lysine, arginine and histidine residues, and is combined with at least one hydrophobic amino acid residue selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline and glycine residues. 
     
     
         18 . The fusion protein according to  claim 17 , wherein at least about 70% of the amino acid residues in said amphipatic peptide are either arginine or lysine residues and at least about 30% of the amino acid residues in said amphipatic peptide are valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, histidine, threonin, serine, proline or glycine residues. 
     
     
         19 . The fusion protein according to  claim 18 , wherein the amphipatic peptide exhibits an amino acid sequence according to SEQ ID NO: 1 to 5, 23, 48 or 49. 
     
     
         20 . The fusion protein according to  claim 1 , wherein the peptide segment comprises about 5 to about 100 amino acid residues. 
     
     
         21 . An isolated nucleic acid molecule encoding a fusion protein according to  claim 1 . 
     
     
         22 . A vector comprising a nucleic acid molecule encoding a fusion protein according to  claim 1 . 
     
     
         23 . A host cell comprising a nucleic acid molecule encoding a nucleic acid according to  claim 1 . 
     
     
         24 . The host cell according to  claim 23 , wherein the cell is a bacterial cell or a yeast cell. 
     
     
         25 . The host cell according to  claim 24 , wherein the yeast cell is  Pichia pastoris  cell. 
     
     
         26 . A method of treating or diagnosing a disease or condition comprising contacting a subject or sample with a fusion protein according  claim 1 . 
     
     
         27 . The method of  claim 26 , wherein said disease is a Gram-positive bacterial infection. 
     
     
         28 . A method for disinfecting a surface or article comprising contacting said surface or article with a fusion protein according to  claim 1 . 
     
     
         29 . A method of treating or preventing Gram-positive bacterial contamination of a foodstuff, a food processing equipment, a food processing plant, a surface coming into contact with foodstuff, a medical device, of or a surface or surgical field in a hospital comprising contacting a said foodstuff, equipment, plant, surface, device or surgical field with the fusion protein according to  claim 1 . 
     
     
         30 . method for diagnosing and infection or condition in medicine, food, feed or an environment comprising contacting a sample of said medicine, food, feed or environment with the fusion protein according to  claim 1 . 
     
     
         31 . A pharmaceutical composition comprising a fusion protein according to  claim 1 . 
     
     
         32 . The fusion protein according to  claim 10 , wherein the cationic peptide segment is a polycationic peptide, amphipatic peptide, sushi peptide, defensin, hydrophobic peptide or an antimicrobial peptide. 
     
     
         33 . The fusion protein of  claim 20 , wherein the peptide segment comprises about 5 to 50 amino acid residues. 
     
     
         34 . The fusion protein of  claim 20 , wherein the peptide segment comprises about 5 to 30 amino acid residues.