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.

Claims

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.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0112] In the following a brief description of the appended figure will be given. The figure is intended to illustrate the present invention in more detail. However, it is not intended to limit the scope of the invention to these specific examples.

[0113] FIG. 1 illustrates positional requirements of preferred sequence motifs of the present invention. The table indicates for sequence motifs of 16 (white) to 20 (dark grey) amino acids in length positions at which no amino acid selected from the first group may be present (respective positions are labelled with “X”). At said positions (i.e. those labelled with “X”), only amino acids selected from the second, or as the case may be, from the third group may be present. More preferably, only amino acids selected from the second group are present at said positions. Amino acids selected from the first group of the sequence motif may only be present at positions which are not labelled with an “X”. However, at said non-labelled positions, amino acids of the second, or as the case may be, third group may also be present. Altogether 18 alternatives, each for a length of 16, 17, 18, 19 or 20 amino acids are provided. The table also clearly specifies the position where potentially a triplet amino acid of the first group may be present (three positions in a row without “X”). For alternative 1 this would be positions 8 to 10. As required for a sequence motif of the polypeptide of the present invention, the amino acids at positions −5 (i.e. position #3), −4 (i.e. position #4), +6 (i.e. position #14), +7 (i.e. position #15), and +10 (i.e. position #18) relative to the first amino acid of the 3 amino acid block (i.e. position #8) are not to be selected from the first group. The relative positions −12, −11, −8, +13, and +14 cannot be found in the first alternative and are thus not taken into account.

[0114] FIGS. 2a-e illustrate in more detail the positional requirements of preferred sequence motifs. “X” denotes that the sequence motif does not exhibit at the respective position an amino acid selected from the first group. FIG. 2a: positional requirements for sequence motifs of 16 amino acids in length. FIG. 2b: positional requirements for sequence motifs of 17 amino acids in length. FIG. 2c: positional requirements for sequence motifs of 18 amino acids in length. FIG. 2d: positional requirements for sequence motifs of 19 amino acids in length. FIG. 2e: positional requirements for sequence motifs of 20 amino acids in length.

EXAMPLES

[0115] 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, accompanying figure and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Adaption of the Antimicrobial Peptide Cecropin A (A. aegypti) to the Sequence Motif of the Present Invention Increases Antibacterial Activity

[0116] The antimicrobial peptide Cecropin A (A. aegypti) (GGLKKLGKKLEGAGKRVFNAAEKALPVVAGAKALRK; SEQ ID NO:44) has been proposed in the art as candidate for fusions with, e.g., endolysins (see WO 2010/149792). However, a fusion of Cecropin A (A. aegypti) with KZ144 endolysin is not as effective against P. aeruginosa and E. coli bacteria as is a fusion of SMAP-29 peptide with KZ144. Furthermore, Cecropin A (A. aegypti) does not comply with the sequence motif of the present invention, as Cecropin A (A. aegypti) exhibits no sequence motif fulfilling the requirement, that at least 40% amino acids from the first group must be present. The inventor thus reasoned, that introduction of further amino acids of said group might increase antibacterial activity.

[0117] To test this hypothesis, the inventor fused Cecropin A (A. aegypti) to Lys394 endolysin, yielding a fusion protein comprising the sequence of SEQ ID NO:45. In parallel, a similar fusion protein was created, in which the Cecropin A (A. aegypti) peptide sequence was C-terminally truncated and mutated at various positions (peptide: GGLKKLGKKLKKAGKRVFKAAKKAL; SEQ ID NO: 11) and fused to Lys394 endolysin. The resulting fusion protein comprises the sequence of SEQ ID NO:32. Due to introduction of additional lysine residues, the modified Cecropin A (A. aegypti) sequence now complied with the sequence motif of the present invention. Both fusion proteins were tested for their antibacterial activity towards K. pneumoniae bacteria.

[0118] Bacteria were grown in (Luria-Bertani) medium and diluted 1:10 in Mueller-Hinton medium. At 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 were pipetted into a 96 well plate, using different concentrations of proteins and an end volume of 20 μl including 500 μM EDTA final concentration. 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 OD600 values of the wells. The MIC which is the protein concentration of the well which showed the same OD600 value as the no-bacteria control was determined.

TABLE-US-00001 TABLE 1 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 45 SEQ ID NO: 32 K. pneumoniae 25 ≤5 ATCC 13883

[0119] The fusion of Cecropin A (A. aegypti) to Lys394 endolysin (SEQ ID NO:45) showed antibacterial activity, with a MIC of 25 μg/ml. For the fusion with the mutated Cecropin A (A. aegypti) sequence (SEQ ID NO:32) the MIC was much lower. ≤5 μg/ml means, that already at the (lowest) starting concentration no bacterial growth could be observed. Lower concentrations have not been tested, i.e. the actual MIC could be even lower than 5 μg/ml. Designing a Cecropin A (A. aegypti) variant complying with the sequence motif of the present invention thus improved the antibacterial activity of the original antimicrobial peptide.

Example 2: Improve in Antibacterial Activity is Independent of Endolysin Moiety

[0120] To test whether the increase in antibacterial activity is unique to the combination of peptide and endolysin utilized in example 1, the inventor tested the same peptides (i.e. SEQ ID NO: 11 and SEQ ID NO:44) in a fusion with another endolysin, OBPgpLys. The resulting polypeptides (SEQ ID NO:46 and SEQ ID NO:33) were tested essentially as described in example 1 but on P. aeruginosa PAO1 bacteria.

TABLE-US-00002 TABLE 2 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 46 SEQ ID NO: 33 P. aeruginosa 17.5 12.5 PAO1

[0121] The fusion of cecropin A (A. aegypti) to OBPgpLys endolysin (SEQ ID NO:46) showed antibacterial activity, with a MIC of 17.5 μg/ml. For the fusion with the mutated cecropin A (A. aegypti) sequence (SEQ ID NO:33) the MIC was significantly lower (12.5 μg/ml). Hence, the improve in antibacterial activity is not dependent on the sequence of endolysin used.

Example 3: Adaption of the Peptide BMAP-28 to the Sequence Motif of the Present Invention Increases Antibacterial Activity

[0122] BMAP-28, a bovine peptide of the cathelicidin family (GGLRSLGRKILRAWKKYGPIIVPIIRIG; SEQ ID NO:47), was fused to a derivative of KZ144 endolysin (SEQ ID NO:25), yielding a fusion protein comprising SEQ ID NO:48. In parallel, a similar fusion protein was created, in which the peptide sequence of BMAP-28 was mutated at two positions (peptide: RGLRRLGRKILRAWKKYGPIIVPIIRIG; SEQ ID NO: 12) and fused to the same derivative of KZ144 endolysin (fusion protein: SEQ ID NO:34). Due to introduction of two arginine amino acids in the N-terminal region of BMAP-28 peptide, said sequence now complied with the sequence motif of the present invention. Both fusion proteins were tested for their antibacterial activity on E. coli bacteria.

[0123] Bacteria were grown in (Luria-Bertani) medium and diluted 1:10 in Mueller-Hinton medium. At 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 were pipetted into a 96 well plate, using different concentrations of proteins and an end volume of 20 μl including 500 μM EDTA final concentration. 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 OD600 values of the wells. The MIC which is the protein concentration of the well which showed the same OD600 value as the no-bacteria control was determined.

TABLE-US-00003 TABLE 3 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 48 SEQ ID NO: 34 E. coli >30 10 03-07953

[0124] “>30” means, that for the non-mutated fusion protein with the original BMAP-28 peptide (SEQ ID NO:48) no antibacterial activity could be observed up to a concentration of 30 μg/ml. Antibacterial activity at higher concentrations is possible, but was not experimentally verified. In contrast, significant antibacterial activity was observed for the fusion protein with the mutated BMAP-28 peptide fragment, with a MIC of 10 μg/ml. This result emphasizes the importance of the sequence motif identified by the inventor and shows that designing respective polypeptides will facilitate generation of new antibacterial agents.

Example 4: The Type of Positively Charged Amino Acid in the Sequence Motif is Only of Little Significance

[0125] In a further experiment the, inventor compared a fusion protein composed of the MSI 78 (4-20) fragment (KFLKKAKKFGKAFVKIL; SEQ ID NO:49) and Lys394 endolysin (fusion protein with SEQ ID NO:50) with a similar fusion protein, in which a modified MSI 78 (4-20) peptide (RFLRRARRFGRAFVRIL; SEQ ID NO: 13) was fused to Lys394 endolysin (fusion protein: SEQ ID NO:35). In the modified MSI 78 (4-20) peptide (SEQ ID NO: 13) the lysine residues of the MSI 78 (4-20) peptide have been substituted with arginine residues. Both fusion proteins were tested for their antibacterial activity on E. coli bacteria.

[0126] Bacteria were grown in (Luria-Bertani) medium and diluted 1:10 in Mueller-Hinton medium. At 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 were pipetted into a 96 well plate, using different concentrations of proteins and an end volume of 20 μl including 500 μM EDTA final concentration. 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 OD600 values of the wells. The MIC which is the protein concentration of the well which showed the same OD600 value as the no-bacteria control was determined.

TABLE-US-00004 TABLE 4 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 50 SEQ ID NO: 35 E. coli 10.2 6 03-07953

[0127] Both fusion proteins showed antibacterial activity in essentially the same range. Thus, the type of positively charged amino acid selected from the first group in the sequence motif of the invention (e.g. K or R) is of minor importance.

Example 5: Adaption of the Peptide Magainin to the Sequence Motif of the Present Invention Improves Antibacterial Activity

[0128] Magainin, an antimicrobial peptide from Xenopus laevis (GIGKFLHSAKKFGKAFVGEIMNS; SEQ ID NO:51), was fused to Lys394 endolysin (SEQ ID NO:24), yielding a fusion protein comprising SEQ ID NO:52. In parallel, a similar fusion protein was created. The peptide sequence of magainin was truncated and coupled with a linker (peptide: GIKKFLKSAKKFGKAFKKVIRGGGGS; SEQ ID NO: 14). Said peptide sequence was fused to Lys394 endolysin (fusion protein: SEQ ID NO:36). Both fusion proteins were tested for their antibacterial activity on P. aeruginosa PAO1 bacteria as described in example 2.

TABLE-US-00005 TABLE 5 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 52 SEQ ID NO: 36 P. aeruginosa >30 ≤5 PAO1

[0129] “>30” means again, that for the non-mutated fusion protein with the original magainin peptide (SEQ ID NO:52) no antibacterial activity could be observed up to a concentration of 30 μg/ml. Antibacterial activity at higher concentrations is possible, but was not experimentally verified. In contrast, significant antibacterial activity was observed for the fusion protein with the mutated magainin peptide fragment, with a MIC of ≤5 μg/ml.

Example 6: Adaption of the Peptide HPA-NT3 to the Sequence Motif of the Present Invention Increases Antibacterial Activity

[0130] HPA-NT3, a Helicobacter pylori-derived peptide (FKRLKKLFKKIWNWK; SEQ ID NO:53), was fused to a derivative of KZ144 endolysin (SEQ ID NO:25), yielding a fusion protein comprising SEQ ID NO:54. In parallel, a similar fusion protein was created, in which the peptide sequence of HPA-NT3 was adapted to the sequence motif of the present invention (peptide: KRLKKLAKKIWKWGRRGPGS; SEQ ID NO: 15) and fused to the same derivative of KZ144 endolysin (fusion protein: SEQ ID NO:37). Both fusion proteins were tested for their antibacterial activity on P. aeruginosa PAO1 bacteria as described in example 2.

TABLE-US-00006 TABLE 6 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 54 SEQ ID NO: 37 P. aeruginosa >18 12.5 PAO1

[0131] “>18” means, that for the non-mutated fusion protein with the original HPA-NT3 peptide (SEQ ID NO:54) no antibacterial activity could be observed up to a concentration of 18 μg/ml. Antibacterial activity at higher concentrations is possible, but was not experimentally verified. In contrast, antibacterial activity was observed for the fusion protein with the mutated HPA-NT3 peptide (SEQ ID NO: 15), with a MIC of 12.5 μg/ml. Adapting the antimicrobial peptide to the motif of the present invention thus increased antibacterial activity of the fusion protein.

Example 7: De Novo Generation of an Artificial Antimicrobial Peptide Starting from a Sequence Motif of Stonustoxin

[0132] In an attempt to further verify suitability of the identified sequence motif, the inventor tried to render a peptide sequence previously not known for any antimicrobial activity into a useful peptide sequence for fusion with an endolysin. For this purpose, the inventor relied on amino acids 298-326 of the alpha subunit of stonustoxin (IPLIHDKISNFQQIFQDYMLTVQKKIAEK; SEQ ID NO:55). Stonustoxin is a component of the reef stonefish venom. Effects of the venom include severe pain, shock, paralysis, and tissue death. Antimicrobial activities are however not known.

[0133] SEQ ID NO:55 was fused to a derivative of KZ144 endolysin, yielding a fusion protein comprising SEQ ID NO:56. In parallel, a similar fusion protein was created, in which the stone fish sequence was mutated at various positions (peptide: IKLIKRVIKKFKKIFRKYPLTVKKGIAVG; SEQ ID NO: 16) and fused to the same derivative of KZ144 endolysin (fusion protein: SEQ ID NO:38). Due to exchange of several amino acids in the stone fish sequence, the first 18 amino acids of said sequence now complied with the sequence motif of the present invention. In particular, the percentage of positively charged amino acids in said sequence motif has been increased (with lysine and arginine residues) and the proline residue removed. Both fusion proteins were tested for their antibacterial activity on P. aeruginosa bacteria.

[0134] Bacteria were grown in (Luria-Bertani) medium and diluted 1:10 in Mueller-Hinton medium. At 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 were pipetted into a 96 well plate, using different concentrations of proteins and an end volume of 20 μl including 500 μM EDTA final concentration. 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 OD600 values of the wells. The MIC which is the protein concentration of the well which showed the same OD600 value as the no-bacteria control was determined.

TABLE-US-00007 TABLE 7 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 56 SEQ ID NO: 38 P. aeruginosa >91 17 PAO1

[0135] >91 means, that for the non-mutated fusion protein (SEQ ID NO:56) with the original stonustoxin peptide (SEQ ID NO:55) no antibacterial activity could be observed up to a concentration of 91 μg/ml. Antibacterial activity at higher concentrations cannot be ruled out, but was not tested. This is as expected, because the stonustoxin fragment used in said fusion is not known for any antimicrobial activity and KZ144 endolysin alone is in principle inactive on P. aeruginosa. In contrast, unexpected de novo antibacterial activity was observed for the fusion protein with the mutated stonustoxin peptide fragment, with a MIC as low as 17. This result emphasizes the importance of the sequence motif identified by the inventor and shows that designing respective polypeptides will facilitate generation of new antibacterial agents.

Example 8: De Novo Generation of an Artificial Antimicrobial Peptide Starting from a Sequence Motif of CagL Protein

[0136] The inventor created two further de novo antimicrobial peptides on basis of amino acids 26-48 of the CagL protein of Helicobacter pylori (GLKQLDSTYQETNQQVLKNLDE; SEQ ID NO:57). CagL protein is specialized adhesin of Helicobacter pylori that is targeted to the pilus surface, where it binds to integrin α5β1 and mediates receptor-dependent delivery of CagA protein into gastric epithelial cells. An antimicrobial activity has not been reported.

[0137] SEQ ID NO:57 was fused to a derivative of KZ144 endolysin, yielding a fusion protein comprising SEQ ID NO:58. In parallel, two similar fusion proteins were created, in which the CagL sequence was mutated at various positions (peptide1: GLKKLKRVYRKWVKAVKKVLKLGGGGS; SEQ ID NO: 17, including a C-terminal linker; peptide2: GLKVLKKAYRRIRKAVRKILKA; SEQ ID NO: 18) to conform with the motif of the present invention. The peptides were fused to the same derivative of KZ144 endolysin (fusion proteins: SEQ ID NO:39 and SEQ ID NO:40). Both fusion proteins were tested for their antibacterial activity on P. aeruginosa bacteria as described in example 2.

TABLE-US-00008 TABLE 8 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 58 SEQ ID NO: 39 SEQ ID NO: 40 P. aeruginosa >90 12.5 15 PAO1

[0138] >90 means, that for the non-mutated fusion protein (SEQ ID NO:58) with the original CagL peptide (SEQ ID NO:57) no antibacterial activity could be observed up to a concentration of 90 μg/ml. This is as expected, because the CagL fragment used in said fusion is not known for any antimicrobial activity and KZ144 endolysin alone is inactive on P. aeruginosa. In contrast, unexpected de novo antibacterial activity was observed for both fusion proteins with the mutated CagL peptide fragment, with a MIC as low as 12.5 and 15 μg/ml.

Example 9: De Novo Generation of an Artificial Antimicrobial Peptide Starting from a Sequence Motif of IE1 Protein

[0139] The next de novo antimicrobial peptide was created on basis of amino acids 178-198 of IE1 protein (YKEKFMVCLKQIVQYAVNS; SEQ ID NO:59). IE1 derives from human cytomegalovirus and antimicrobial activities are not known.

[0140] SEQ ID NO:59 was fused again to the derivative of KZ144 endolysin, yielding a fusion protein comprising SEQ ID NO:60. In parallel, a fusion protein was created, in which the IE1 sequence was mutated at various positions (peptide: YKRAFKKVLKRIRRYAKRS; SEQ ID NO: 19) and fused to the same derivative of KZ144 endolysin (fusion protein: SEQ ID NO:41). Both fusion proteins were tested for their antibacterial activity on P. aeruginosa bacteria as described in example 2.

TABLE-US-00009 TABLE 9 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 60 SEQ ID NO: 41 P. aeruginosa >30 15-20 PAO1

[0141] >30 means, that for the non-mutated fusion protein (SEQ ID NO:60) with the original IE1 peptide (SEQ ID NO:59) no antibacterial activity could be observed up to a concentration of 30 μg/ml. Antibacterial activity at higher concentrations cannot be ruled out, but was not tested and would not be expected, because the IE1 fragment used in said fusion is not known for any antimicrobial activity. In contrast, unexpected de novo antibacterial activity was observed for the fusion protein with the mutated IE1 peptide fragment, with a MIC between 15 and 20 μg/ml.

Example 10: Generation of a Further Fusion Protein Comprising a Peptide with a Sequence Motif of the Present Invention

[0142] The inventor created also a further fusion protein comprising the sequence of SEQ ID NO:42. Said fusion protein comprises a peptide conforming with the present invention (SEQ ID NO: 20). The fusion protein was tested for antibacterial activity on P. aeruginosa bacteria as reported in example 2.

TABLE-US-00010 TABLE 10 Minimal inhibitory concentration of the tested fusion protein Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 42 P. aeruginosa ≤5 PAO1

[0143] Significant antibacterial activity was observed for the fusion protein with the novel peptide, with a MIC of ≤5 μg/ml.

Example 11: Generation of a Further Variations of a Fusion Protein Comprising a Peptide with a Sequence Motif of the Present Invention

[0144] The inventor created further fusion proteins comprising a peptide conforming with the motif of the present invention (SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68). The fusion proteins were tested for antibacterial activity on E. coli bacteria.

TABLE-US-00011 TABLE 11 Minimal inhibitory concentration of the tested fusion protein Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO: 64 E. coli ≤5 ≤5 ≤5 ≤5 DSMZ 11753 Bacterial strain SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 68 E. coli ≤5 7.5 ≤5 ≤5 DSMZ 11753

[0145] Antibacterial activity was observed for all fusion proteins.

Example 12: Adaption of Peptide MW2 of Briers et al. To Sequence Motif of the Present Invention

[0146] Briers et al. (MBio. 2014; 5(4):e01379-14) reported creation of various fusion proteins, including inter alia peptide MW2 (SEQ ID NO:69). Said peptide does not comply with the sequence motif of the present invention. Starting from this peptide the inventor created a fusion protein comprising said peptide and the derivative of KZ144 endolysin (SEQ ID NO:25), resulting in a fusion protein according to SEQ ID NO:70. In addition, the inventor created a number of derivatives of the peptide of SEQ ID NO:69 (SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74). These derivatives match the sequence motif of the present invention, while MW2 does not. The resulting fusion proteins are provided in SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78. The fusion proteins were tested for antibacterial activity on P. aeruginosa PAO1 bacteria as described in example 2.

TABLE-US-00012 TABLE 12 Minimal inhibitory concentration of the tested fusion protein Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 70 SEQ ID NO: 75 SEQ ID NO: 76 SEQ ID NO: 77 SEQ ID NO: 78 P. aeruginosa >30 10 10 25 20 PAO1

[0147] Antibacterial activity was observed for all fusion proteins. Noteworthy, the fusion proteins on basis of the four derivatives of MW2 peptide (i.e. adapted to the sequence motif of the present invention) yielded improved antibacterial activity as compared to the fusion protein with the “wildtype” MW2 peptide.

Example 13: Use of the Peptide Magainin in Combination with a Further Peptidoglycan Hydrolase

[0148] The inventor also combined the two peptides of example 5 with a further peptidoglycan hydrolase, namely a tail baseplate protein of Vibrio phage ICP1 (SEQ ID NO:27). The resultant fusion proteins comprise the sequences of SEQ ID NO:79 and SEQ ID NO:43. The fusion proteins were tested for antibacterial activity on E. coli bacteria.

TABLE-US-00013 TABLE 13 Minimal inhibitory concentration of the tested fusion proteins Minimal inhibitory concentration (MIC; μg/ml) Bacterial strain SEQ ID NO: 79 SEQ ID NO: 43 E. coli 1 ≤0.5 DSMZ 11753

[0149] The resulting fusion proteins exhibited both antibacterial activity. The peptide complying with the sequence motif of the present invention (SEQ ID NO: 14) provided again better activity than the wild-type peptide (SEQ ID NO:51).

Example 14: Further Peptides

[0150] In a final set of experiments the inventor created three further fusion proteins, each comprising a endolysin sequence and a peptide complying with the sequence motif according to the present invention. The three peptides were SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23. All three resulting fusion proteins showed excellent antibacterial activity.