Antimicrobial agents against <i>Staphylococcus aureus</i>

Abstract

The present invention relates to the field of antimicrobial agents active against Staphylococcus aureus bacteria. In particular, the present invention relates to polypeptides comprising the CHAP domain of LysK endolysin, the M23 endopeptidase domain of lysostaphin, the cell wall binding domain (CBD) of ALE-1, and a further peptide selected from the group consisting of an antimicrobial peptide, an amphipathic peptide, a cationic peptide, a hydrophobic peptide, a sushi peptide and a defensin. In addition, the present invention relates to nucleic acids encoding such polypeptides, vectors comprising such nucleic acids, and corresponding host cells, compositions and devices. Finally, the present invention relates to applications of the inventive polypeptides, in particular in the pharmaceutical field.

Claims

1. A polypeptide comprising: i) the CHAP domain of LysK endolysin or a variant thereof; ii) the M23 endopeptidase domain of lysostaphin; iii) the cell wall binding domain (CBD) of ALE-1, and iv) a further peptide selected from the group consisting of an antimicrobial peptide, an amphipathic peptide, a cationic peptide, a hydrophobic peptide, a sushi peptide and a defensin wherein said variant sequence exhibits mutation H8N and/or T43A compared to the amino acid sequence of SEQ ID NO:1, and wherein the CHAP domain of LysK endolysin is SEQ ID NO: 1; the M23-endopeptidase domain is SEQ ID NO: 3, and the CBD is SEQ ID NO: 5.

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

3. The polypeptide according to claim 1, wherein the polypeptide comprises: i) the amino acid sequence of SEQ ID NO:2, or of a variant sequence thereof exhibiting one or more mutations selected from the group consisting of K16E, K27N, H50Q, T85A, G153S, G153C compared to the amino acid sequence of SEQ ID NO:2, ii) the amino acid sequence of SEQ ID NO:4, and/or iii) the amino acid sequence of SEQ ID NO:6.

4. The polypeptide according to claim 3, wherein said variant sequence exhibits at least mutations K16E and H50Q compared to the amino acid sequence of SEQ ID NO:2.

5. The polypeptide according to claim 1, wherein the polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

6. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

7. The polypeptide according to claim 1, wherein the order of elements or of their respective variants is from the N- to the C-terminus: a) CHAP domain—M23-endopeptidase domain—CBD of ALE-1—peptide, or b) Peptide—CHAP domain—M23-endopeptidase domain—CBD of ALE-1.

8. The polypeptide according to claim 1, wherein the further peptide is: i) an antimicrobial peptide selected from the group consisting of the SEQ ID Nos. from SEQ ID NO: 40 to 94, ii) an amphipathic peptide selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, iii) a cationic peptide selected from the group consisting of the SEQ ID Nos. from SEQ ID NO: 11 to 39, iv) a sushi peptide according to SEQ ID NO: 95, or v) a hydrophobic peptide selected from the group consisting of SEQ ID NO: 96 and SEQ ID NO: 97.

9. The polypeptide according to claim 8, wherein the further peptide is selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 94.

10. The polypeptide according to claim 1, wherein the polypeptide comprises a sequence selected from the group consisting of SEQ ID NO:105, SEQ ID NO: 107, SEQ ID NO:109 and SEQ ID NO:111.

11. The polypeptide according to claim 1, wherein said polypeptide is capable of degrading the cell wall of Staphylococcus aureus bacteria.

12. A polypeptide comprising a variant sequence of SEQ ID NO:1, wherein said variant sequence exhibits mutation H8N and/or T43A compared to the amino acid sequence of SEQ ID NO:1.

13. A polypeptide comprising a variant sequence of SEQ ID NO:2, wherein said variant sequence exhibits one or more mutations selected from the group consisting of K16E, K27N, H50Q, T85A, G153S, G153C compared to the amino acid sequence of SEQ ID NO:2.

14. The polypeptide according to claim 13, wherein said variant sequence exhibits at least mutations K16E and H50Q compared to the amino acid sequence of SEQ ID NO:2.

15. The polypeptide according to claim 13, wherein said variant sequence exhibits at least mutations K16E, K27N, H50Q, T85A, and G153S compared to the amino acid sequence of SEQ ID NO:2.

16. The polypeptide according to claim 13, wherein said polypeptide further comprises the sequence of at least one catalytic domain of a peptidoglycan hydrolase and/or exhibits antibacterial activity.

17. The polypeptide according to claim 16, wherein said polypeptide comprises the sequence of SEQ ID NO: 108 or SEQ ID NO: 110.

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

19. A vector comprising a nucleic acid according claim 18.

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

21. A composition comprising a polypeptide according to claim 1, wherein the composition is an aqueous solution, a powder, a suppository, an emulsion, a suspension, a gel, a lotion, a cream, salve, ointment, injectable solution, syrup, spray, inhalant, a coating composition, a stent coating composition, or a catheter coating composition, or a biomaterial.

22. A device comprising a polypeptide according to claim 1, wherein the device is a medical device.

23. A method for the treatment of the human or animal body by surgery or therapy or in diagnostic methods practiced on the human or animal body comprising administering to a subject in need thereof a polypeptide according to claim 1.

24. The method according to claim 23, wherein the polypeptide treats or prevents bacterial infections in a subject.

25. The method according to claim 23, wherein the polypeptide treats a wound of a subject, or for the treatment of dermatitis or otitis.

26. A method for disinfecting an inanimate surface, composition and/or object, comprising contacting said surface, composition or object with a polypeptide according to claim 1.

27. A method for preventing contamination of an inanimate surface, composition and/or object with bacteria, comprising contacting said surface, composition or object with a polypeptide according to claim 1.

28. The polypeptide according to claim 13, wherein said polypeptide further comprises the sequence of at least one catalytic domain of a peptidoglycan hydrolase and/or exhibits antibacterial activity against S. aureus.

29. The composition comprising a polypeptide according to claim 21, wherein the biomaterial is a bone cement.

30. The composition comprising a polypeptide according to claim 21, wherein the aqueous solution is a buffer or a physiological solution.

31. The composition comprising a polypeptide according to claim 21, wherein the coating composition is an implant coating composition.

32. The method according to claim 24, wherein the polypeptide treats or prevents a bacterial infection in a subject.

33. The method according to claim 25, wherein the wound is an acute wound or chronic wound.

34. The method according to claim 25, wherein the acute wound is an iatrogenic wound.

35. A method for disinfecting a nosocomial environment or a doctor's office comprising contacting a surface, composition or object with a polypeptide according to claim 1.

36. The method according to claim 27, wherein the bacteria is Staphylococcus aureus.

37. The method according to claim 32, wherein the bacterial infection is by Staphylococcus aureus bacteria.

Description

EXAMPLES

(1) In the following a specific example illustrating embodiments and aspects of the invention is 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: Antibacterial Activity on S. aureus of Two Fusion Proteins of the Invention

(2) Becker et al. (Sci Rep. 2016 Apr. 28; 6:25063) reported construction of fusion protein L-K with reduced incidence of resistant S. aureus strain development. In an attempt to provide other improved fusion proteins against S. aureus, the inventors generated two fusion proteins, each comprising the CHAP domain of LysK endolysin, the M23 endopeptidase domain of lysostaphin; and the cell wall binding domain (CBD) of ALE-1. In addition, one of the fusion proteins did comprise the cathepsin G (77-83) peptide (SEQ ID NO:94), the other the cationic peptide KNK (SEQ ID NO:38). The resulting fusion proteins (SEQ ID NO:105 and SEQ ID NO: 107) were assayed for antibacterial activity and resistance development. For this purpose MIC (minimal inhibitory concentration) assays (see below) were carried out for several culture cycles.

(3) MIC assay S. aureus Sp10 was grown in (Luria-Bertani) medium and diluted 1:10 in Mueller-Hinton medium. At an optical density OD.sub.600 of about 0.6, bacteria were diluted in the same medium 1:10 followed by a 1:500 dilution. Protein buffer (20 mM HEPES, 500 mM NaCl, pH 7.4) and proteins or gentamycin were pipetted into a 96 well plate, using different concentrations of proteins/gentamycin and an end volume of 20 μl. The proteins used were the fusion proteins according to SEQ ID NO:105 and SEQ ID NO: 107. 180 μl of bacterial cells or a medium (Mueller-Hinton) control were given to the 96 well plate and mixed. The plate was incubated for 18-22 hours at 37° C. and the bacterial growth was determined measuring the OD.sub.600 values of the wells. The well with the lowest concentration of protein/gentamycin showing the same OD.sub.600 value as the medium control was taken as MIC. For the next cycle, the bacterial solution from the sub-MIC well was used. The sub-MIC well is the well in which the next lower concentration to the MIC concentration was tested, i.e. the well with the highest concentration of protein/gentamycin but still OD.sub.600 above the medium control. For further cycles of the resistance assay, the bacteria from this sub-MIC well of the previous cycle were taken for the next over-night culture.

(4) The results are given in tables 3a and b below.

(5) TABLE-US-00003 TABLE 3a Minimal inhibitory concentration (MIC; μg/ml) SEQ ID SEQ ID NO: Cycle NO: 105 107 Gentamycin 1 4 4 0.8 6 4 4 4 12 6 6 6

(6) Table 3b below illustrates said results of table 3a as fold change over the initial MIC (cycle 1).

(7) TABLE-US-00004 TABLE 3b Fold change of MIC SEQ ID SEQ ID NO: Cycle NO: 105 107 Gentamycin 1 1 1 1 6 1 1 5 12 1.5 1.5 7.5

(8) As evident from table 3b above, both fusion proteins of the invention are less prone to resistant strain development over time than gentamycin.

(9) Moreover, both fusion proteins of the invention showed in comparison to the results reported for the fusion protein L-K of the prior art (see Becker et al. (Sci Rep. 2016 Apr. 28; 6:25063) surprisingly reduced incidence of resistant strain development after more cycles (1.5 fold change MIC for both fusions proteins of the present invention after 12 cycles vs. 2 fold change MIC for L-K fusion protein of Becker et al. after 10 cycles (see Becker et al. FIG. 1B), while exhibiting in parallel a higher antibacterial activity (initial MIC of 4 μg/ml for the fusion proteins of the present invention vs. 7.8 μg/ml for the L-K fusion protein of Becker et al.).

Example 2: Variants of Polypeptides According to the Present Invention

(10) The inventors of the present invention also created variants of the above mentioned polypeptides. For this purpose, mutations where introduced in the sequence of SEQ ID NO: SEQ ID NO:105.

(11) TABLE-US-00005 TABLE 4 Clone Mut1 Mut2 Mut3 Mut4 1 K17E H51Q 2 T127I 3 K28N T86A G154C 4 N126S A16I 5 G85D G119S 6 A16I F36L A46V I80T 7 G166S E172R T173N A174R 8 E172G T173N A174R 9 V26A Q114P 10 N138D G154S K171I

(12) Table 5 below illustrates the results of these mutations on the activity for the fusion protein of SEQ ID NO:105. As used herein, strain DSM 346 refers to Staphylococcus aureus strain DSM 346 (Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany). S64 and S69 are Staphylococcus aureus strains S64 and S69 obtained from Prof. Rob Lavigne (Katholieke Universiteit Leuven, Belgium).

(13) TABLE-US-00006 TABLE 5 MIC Clone Strain [μg/mL] 1 DSM 346 4 S64 6 S69 6 2 DSM 346 2 S64 4 S69 4 3 DSM 346 4 S64 8 S69 8 4 DSM 346 2 S64 4 S69 4 5 DSM 346 4 S64 4 S69 4 6 DSM 346 6 S64 20 S69 12 7 DSM 346 6 S64 10 S69 8 8 DSM 346 4 S64 6 S69 6 9 DSM 346 4 S64 4 S69 4 10 DSM 346 4 S64 6 S69 8

(14) All mutants retained antibacterial activity on S. aureus.

(15) Some of the above mentioned mutations were also verified for the sequence of SEQ ID NO: 107. In addition, some of the above mutations were combined. In addition, mutation G154S was used instead of G154C. Table 6 below illustrates the mutations tested for the fusion protein of SEQ ID NO: 107.

(16) TABLE-US-00007 TABLE 6 Clone Mut1 Mut2 Mut3 Mut4 Mut 5 Mut 6 11 K17E H51Q 12 K17E H51Q E172G T173N A174R 13 K28N T86A G154S 14 K28N T86A G154S E172G T173N A174R 15 K17E H51Q K28N T86A G154S

(17) Table 7 below illustrates the results of these mutations on the activity for the fusion protein of SEQ ID NO: 107.

(18) TABLE-US-00008 TABLE 7 MIC Clone Strain [μg/mL] 11 DSM 346 4 S64 4 S69 12 12 DSM 346 8 S64 10 S69 10 13 DSM 346 4 S64 4 S69 4 14 DSM 346 10 S64 8 S69 8 15 DSM 346 2 S64 4 S69 4

(19) All mutants retained antibacterial activity on S. aureus.

(20) In addition, thermal stability was assessed for two of said mutants in comparison to the unmodified fusion protein. The thermal stability assay was carried out with the strain Staphylococcus aureus DSM 346. The proteins were diluted to a concentration of 0.3 mg/ml followed by an incubation for 20 minutes at different temperatures (see table 8 below). A standard MIC assay was carried out after this incubation time. The higher the temperature after which the protein still shows (a high) activity, the better is the thermal stability of a protein.

(21) TABLE-US-00009 TABLE 8 MIC [μg/mL] 44.6° 47.1° 49.7° 52.3° 54.9° 57.4° 60° RT C. C. C. C. C. C. C. SEQ ID 6 20 25 30 30 >30 >30 >30 NO: 107 Clone 11 4 6 10 10 16 >30 >30 >30 Clone 15 4 4 4 4 4 4 6 8

(22) Clones 11 and 15 thus showed increased thermal stability in comparison to the unmodified fusion protein.