NEW ANTIMICROBIAL AGENTS AGAINST ENTEROCOCCUS BACTERIA

Abstract

The present invention relates to the field of antimicrobial agents active against Enterococcus bacteria. In particular, the present invention relates to a polypeptide comprising a first and a second amino acid sequence, wherein the first amino acid sequence is a sequence selected from SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO: 5; and derivatives thereof; and wherein the second amino acid sequence is an antimicrobial peptide, amphipathic peptide, cationic peptide, hydrophobic peptide, sushi peptide or defensin. In addition, the present invention relates to nucleic acids encoding such polypeptides, vectors comprising such nucleic acids, and corresponding host cells. Finally, the present invention relates to applications of the inventive polypeptides, nucleic acids, vectors, and/or host cells, in particular in the pharmaceutical field.

Claims

1. A polypeptide comprising a first and a second amino acid sequence, wherein the first amino acid sequence is a sequence selected from the following group of sequences: i) an amino acid sequence according to SEQ ID NO:2; ii) an amino acid sequence according to SEQ ID NO:3; and iii) an amino acid sequence according to SEQ ID NO: 5; and wherein the second amino acid sequence is an antimicrobial peptide, amphipathic peptide, cationic peptide, hydrophobic peptide, sushi peptide or defensin.

2. (canceled)

3. The polypeptide according to claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO:3 and a further amino acid sequence, wherein said further amino acid sequence is an enzyme capable of degrading the cell wall of bacteria, in particular of Gram-positive bacteria.

4. The polypeptide according to claim 3, wherein said enzyme is an amino acid sequence according to SEQ ID NO: 104.

5. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2 and of SEQ ID NO:3.

6. (canceled)

7. The polypeptide according to claim 1, wherein the second amino acid sequence is: i) an antimicrobial peptide selected from the group consisting of SEQ ID NO: 4, SEQ ID Nos. from SEQ ID NO: 31 to 83 and SEQ ID NO: 100, ii) an amphipathic peptide selected from the group consisting of SEQ ID NO: 87, SEQ ID NO: 88 and SEQ ID NO: 89, iii) a cationic peptide selected from the group consisting of the SEQ ID Nos. from SEQ ID NO: 6 to 30, iv) a sushi peptide according to SEQ ID NO: 84, or iii) a hydrophobic peptide selected from the group consisting of SEQ ID NO: 85 and SEQ ID NO: 86.

8. The polypeptide according to claim 1, wherein the second amino acid sequence is an amino acid sequence according to SEQ ID NO: 4 or SEQ ID NO: 100.

9. The polypeptide according to claim 1, wherein i) the first amino acid sequence is SEQ ID NO: 5, and wherein the second amino acid sequence is SEQ ID NO: 4, ii) wherein the first amino acid sequence is SEQ ID NO: 5, and wherein the second amino acid sequence is SEQ ID NO: 100, or iii) wherein the first amino acid sequence is SEQ ID NO: 3, and wherein the second amino acid sequence is SEQ ID NO: 100.

10. The polypeptide according to claim 1, wherein the polypeptide degrades the peptidoglycan of Enterococcus bacteria, in particular of Enterococcus faecalis and/or Enterococcus faecium bacteria.

11. The polypeptide according to claim 10, wherein the polypeptide is at an about physiological pH, such as pH 7.4, more active than the wildtype enzyme (SEQ ID NO:1) and/or exhibits essentially the same or increased activity at about physiological pH compared to more acidic pH, such as pH 5.25 or 6.

12. A composition comprising an endolysin according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

13. The composition according to claim 12, wherein the composition is bone cement or comprises biomaterial.

14. A method for treating the human or animal body by surgery or therapy comprising administering to said subject a polypeptide according to claim 1.

15. The method according to claim 14, wherein the polypeptide or composition is used for the treatment or prevention of bacterial infections, in particular for the treatment or prevention of bacterial infections with Enterococcus faecalis and/or Enterococcus faecium bacteria.

16. The method according to claim 14, wherein the polypeptide or composition is used at about physiological pH, e.g. at a pH of about 7.2 to 7.6, more preferably at a pH of about 7.4.

17. A method of disinfecting inanimate surfaces, compositions and/or objects, in particular in the nosocomial environment or in a doctor's office, comprising contacting said inanimate surfaces, compositions and/or objects with a polypeptide according to claim 1.

18. A method of preventing contamination of inanimate surfaces, compositions and/or objects with bacteria, in particular for preventing contamination with Enterococcus faecalis and/or Enterococcus faecium bacteria, comprising contacting said inanimate surfaces, compositions and/or objects with a polypeptide according to claim 1.

19. The method according to claim 17, wherein the polypeptide or composition is used at about physiological pH, e.g. at a pH of about 7.2 to 7.6, more preferably at a pH of about 7.4.

20. A nucleic acid encoding a polypeptide according to claim 1.

21. A vector comprising a nucleic acid according to claim 20.

22. A host cell comprising a polypeptide according to claim 1.

23. A host cell comprising a nucleic acid according to claim 20.

24. A host cell comprising a vector according to claim 21.

25. The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 92, 93, 94, 101, 102, 103, and 105.

Description

EXAMPLES

[0093] In the following, specific examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Increased Antibacterial Activity on E. faecalis Bacteria Compared to the Wildtype Endolysin

[0094] E. faecalis bacteria were grown over night in LB (Luria-Bertani) medium and diluted 1:10 in same medium. At optical density OD.sub.600 of about 0.6 bacteria were pelleted, washed in buffer (10 mM HEPES, pH 7.4) and diluted 1:10 in same buffer. 50 l bacteria solution was mixed with 50 l of protein solution (10 g in 20 mM HEPES, 300 mM NaCl, pH 7.4) or control (20 mM HEPES, 300 mM NaCl, pH 7.4). The proteins used were the wildtype endolysin (wt) according to SEQ ID NO:1 and a polypeptide according to the present invention comprising SEQ ID NO: 93. Samples were incubated for 60 min at 37 C. with gently agitation. 25 l of an undiluted sample and a 1:10 dilution series in 1PBS buffer was plated on LB agar plates which were incubated over night at 37 C. Colonies were counted and the growth reduction was determined.

[0095] Table 3 below shows the growth reduction of E. faecalis HC-1909-5 after incubation with the wildtype endolysin and the polypeptide according to the present invention. Incubation with endolysin led to a bacterial elimination of 99.99% (4 log). More than 99.999% (>5 log) was achieved after incubation with the fusion protein. Four 1:10 dilutions were done leading to a test counting limit of 5 log reduction.

TABLE-US-00003 TABLE 3 Protein Wt endolysin 4 log SEQ ID NO: 93 5 log

Example 2: Increased Antibacterial Activity on E. faecalis Bacteria Compared to the Wildtype Endolysin Over Broad pH Range

[0096] E. faecalis bacteria were grown over night in LB (Luria-Bertani) medium and diluted 1:10 in same medium. At optical density OD.sub.600 of about 0.6 bacteria were pelleted, washed in different buffers. The buffers used were the following: 100 mM Malic acid disodium salt, pH 5 and pH 6 and mixtures of 1 M Na.sub.2HPO.sub.4 and 1 M NaH.sub.2PO.sub.4 for pH 7.4 and pH 8. Bacteria were then diluted 1:10 in different buffers (look above). 50 l bacteria solution was mixed with 50 l of protein solution (10 g in 20 mM HEPES, 300 mM NaCl, pH 7.4) or control (20 mM HEPES, 300 mM NaCl, pH 7.4). The proteins used were the wildtype endolysin (wt) according to SEQ ID NO:1 and a polypeptide according to the present invention comprising SEQ ID NO: 93. The end pH values after mixing bacteria and proteins were the following: pH 5.25, pH 6.5, pH 7.4 and pH 7.75. Samples were incubated for 60 min at 37 C. with gently agitation. 25 l of an undiluted sample and a 1:10 dilution series in 1PBS buffer was plated on LB agar plates which were incubated over night at 37 C. Colonies were counted and the growth reduction was determined. Four 1:10 dilutions were done leading to a test counting limit of 5 log reduction.

[0097] Table 4 below shows the growth reduction of E. faecalis strain HC-1909-5 under different pH conditions after incubation with the wildtype endolysin and the polypeptide according to the present invention. 99-99.9% (2-3 log) bacterial elimination were visible for the wildtype endolysin from pH 5.25-pH 7.75 with decreasing activity at basic pH. 99.99-99.997% (4-4.5 log) bacterial elimination was reached with the fusion protein with only slightly decreased activity at basic pH.

TABLE-US-00004 TABLE 4 wildtype SEQ ID NO: SEQ ID NO: [pH] endolysin 93 102 5.25 3 log 4.5 log 3.5 log 6 3 log 4.5 log 4 log 7.4 2 log 4.5 log 3.5 log 7.75 2.5 log 4 log 2.5 log

[0098] Similar results (e.g. essentially constant anti-bacterial activity over a broad pH range, in particular without significant activity loss at a physiological pH of 7.4) can also be achieved with other polypeptides of the invention, such as polypeptides comprising additional a His-tag (e.g. SEQ ID NO: 94 or SEQ ID NO: 103), or with polypeptides comprising the cell wall binding domain (CBD) of SEQ ID NO:1 and an additional catalytic domain, e.g. a sequence according to SEQ ID NO:104. An example for such polypeptide is a polypeptide comprising SEQ ID NO: 105.

Example 3: Broad Antibacterial Activity Against Diverse Set of Enterococci Strains

[0099] Bacteria were grown over night in LB (Luria-Bertani) medium and diluted 1:10 in same medium. At optical density OD.sub.600 of about 0.6 bacteria were pelleted, washed in buffer (10 mM HEPES, pH 7.4) and diluted 1:10 in same buffer. 50 l bacteria solution was mixed with 50 l of protein solution (10 g in 20 mM HEPES, 300 mM NaCl, pH 7.4) or control (20 mM HEPES, 300 mM NaCl, pH 7.4). The proteins used were the wildtype endolysin (wt) according to SEQ ID NO:1 and a polypeptide according to the present invention comprising SEQ ID NO: 93. Samples were incubated for 60 min at 37 C. with gently agitation. 25 l of an undiluted sample and a 1:10 dilution series in 1PBS buffer was plated on LB agar plates which were incubated over night at 37 C. Colonies were counted and the growth reduction was determined. Four 1:10 dilutions were done leading to a test counting limit of 5 log reduction.

[0100] Table 5 below shows the growth reduction of different Enterococcus faecalis, Enterococcus faecium and other Enterococcus strains. On average, 99.9987% (4.9 log) bacterial elimination was determined.

TABLE-US-00005 TABLE 5 Strain Growth reduction E. faecalis HC-1909-5 5 log E. faecium NCIMB 11181 5 log E. faecalis DSM 20478 5 log E. faecalis va96529 5 log E. faecium va80443 5 log E. faecalis 12470 5 log E. faecium 13368 5 log E. faecalis NWV 16205 5 log E. faecalis NMV 10462 5 log E. faecium 90-2 5 log E. faecalis NWV 21315 5 log E. faecalis NWV 16205 5 log E. faecium NWV 6818 5 log E. faecium NWV 8780 5 log E. faecalis DSM 20478 5 log E. faecium 13372 5 log E. faecium 13371 5 log E. faecium 13370 5 log E. faecium 13369 5 log E. faecalis 13350 5 log E. faecalis 13293 5 log E. faecalis 12643 5 log E. faecalis 12583 5 log E. faecalis url0856 5 log E. faecalis va32880_3 5 log E. spec. Colja 9 4 log E. faecalis DSM 12956 5 log E. faecalis DSM 2981 5 log E. faecalis DSM 2570 5 log E. faecalis DSM 6134 4 log E. faecalis Bobby 2 5 log E. spec. 02.15-1.54 4 log E. faecalis 5 log