NOVEL ANTIMICROBIAL PROTEINS

Abstract

The present invention relates to a polypeptide comprising a Gram negative endolysin and a peptide selected from the group consisting of an antimicrobial peptide, an amphipathic peptide, a cationic peptide, a sushi peptide or a defensin, wherein the endolysin in turn is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and sequences having at least 80% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NOG, SEQ ID NO:4, SEQ ID NOG, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and/or SEQ ID NO:9. 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 polypeptide comprising a Gram negative endolysin and a peptide selected from the group consisting of an antimicrobial peptide, an amphipathic peptide, a cationic peptide, a sushi peptide or a defensin, wherein the endolysin is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, with the provisos that: a) the polypeptide does not comprise the sequence according to SEQ ID NO:10, b) the peptide is selected from the group consisting of an antimicrobial peptide, an amphipathic peptide, a sushi peptide or a defensin, if the polypeptide comprises the sequence of SEQ ID NO:4, and c) the polypeptide does not comprise a cell wall binding domain of i) a modular Gram-negative endolysin or ii) a bacteriophage tail/baseplate protein, if the endolysin has a sequence according to: TABLE-US-00006 Host Phage name Protein ID Escherichia Enterobacteria phage CC31 YP_004009990.1 or a corresponding sequence merely lacking in addition the N-terminal methionine.

2. The polypeptide according to claim 1, wherein the endolysin is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9.

3. The polypeptide according to claim 2, wherein the endolysin is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9.

4. The polypeptide according to claim 1, wherein the peptide is an antimicrobial peptide or an amphipathic peptide.

5. The polypeptide according to claim 1, wherein the peptide 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 preferably 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; 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.

6. The polypeptide according to claim 5, wherein peptide comprises the sequence according to SEQ ID NO:37, SEQ ID NO:106 SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109 or SEQ ID NO:110.

7. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, or of SEQ ID NO:121.

8. The polypeptide according to claim 7, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:114 or of SEQ ID NO:115.

9. A polypeptide comprising the sequence of a peptide selected from the group consisting of SEQ ID NO: 81, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109 and SEQ ID NO: 110; and optionally the sequence of a muralytic enzyme.

10. The polypeptide according to claim 1, wherein the polypeptide degrades peptidoglycan of at least one Gram-negative bacterial species, in particular wherein the polypeptide degrades the peptidoglycan of E. coli bacteria and/or P. aeruginosa bacteria.

11. The polypeptide according to claim 10, wherein the polypeptide degrades the peptidoglycan of at least one Gram-negative bacterial species in absence of other outer membrane permeabilizing substances, in particular wherein the polypeptide degrades the peptidoglycan of E. coli bacteria and/or P. aeruginosa bacteria in absence of outer membrane permeabilizing substances.

12. The polypeptide according to claim 10, wherein the polypeptide exhibits in absence of outer membrane permeabilizing substances a minimal inhibitory concentration (MIC) of 20 μg/ml or less for E. coli strain RKI 06-08410.

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

14. A vector comprising a nucleic acid according to claim 13.

15. A host cell comprising a a nucleic acid according to claim 13.

16. A method for treatment of the human or animal by surgery or therapy or in diagnostic methods comprising administering to said human or animal the polypeptide of claim 1, wherein the polypeptide is administered without addition of further outer membrane permeabilizing substances.

17. A method of disinfecting a surface comprising contacting said surface with the polypeptide of claim 1, wherein the polypeptide is administered without addition of further outer membrane permeabilizing substances.

18. The polypeptide according to claim 9, wherein the polypeptide degrades peptidoglycan of at least one Gram-negative bacterial species, in particular wherein the polypeptide degrades the peptidoglycan of E. coli bacteria and/or P. aeruginosa bacteria.

19. A nucleic acid encoding a polypeptide according to claim 9.

20. A vector comprising a nucleic acid according to claim 19.

Description

FIGURE

[0118] In the following a brief description of the appended figure will be given. The figure is intended to illustrate an aspect of the present invention in more detail. However, it is not intended to limit the subject matter of the invention to such subject-matter only.

[0119] FIG. 1: illustrates positional requirements of preferred sequence motifs of selected peptide components of the inventive polypeptide. 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 the preferred sequence motif of the peptide component 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.

EXAMPLES

[0120] 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 example below. All such modifications fall within the scope of the appended claims.

Example 1

Minimal Inhibitory Concentration of Several Antibacterial Polypeptides Against E. coli in Presence and Absence of EDTA

[0121] The antibacterial activity of the following fusion proteins on E. coli in presence and absence of EDTA was assessed: [0122] SEQ ID NO:123, a fusion of Cecropin A. (A. aegyptii) peptide (SEQ ID NO: 43) with the endolysin of Vibrio phage VvAW1 (YP_007518361.1) [0123] SEQ ID NO:124, a fusion of Cecropin A. (A. aegyptii) peptide with a mutated cell wall binding domain of the modular KZ144 endolysin and Lys68 endolysin [0124] SEQ ID NO:125, a fusion of a modified peptide (SEQ ID NO:105) complying with the preferred sequence motif of the peptide component and an endolysin of Pseudomonas phage vB_PsyM_KIL1 (see YP_009276009.1) [0125] SEQ ID NO:114, a fusion of SMAP-29 peptide (SEQ ID NO:37) and the endolysin of Citrobacter koseri phage CkP1, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:7), [0126] SEQ ID NO:115, a fusion of a peptide comprising the preferred sequence motif for the peptide component (SEQ ID NO:106) and the endolysin of Enterobacteria phage CC31, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:8) [0127] SEQ ID NO:116, a fusion of the peptide according to SEQ ID NO: 107 and the endolysin of Citrobacter koseri phage CkP1, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:7), [0128] SEQ ID NO:117, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO:108 and the endolysin of Enterobacteria phage CC31, with again the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:8), [0129] SEQ ID NO:119, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO:81 and the endolysin of Serratia phage CHI14, with again the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:9), and [0130] SEQ ID NO:120, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO:109 and the endolysin of Citrobacter koseri phage CkP1, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:7).

[0131] The endolysin components of the polypeptide of SEQ ID NO:114 and the polypeptide of SEQ ID NO:115 (see SEQ ID NO:7 and SEQ ID NO:8) share a significant level of sequence identity (80%).

[0132] Likewise, the endolysin components of the polypeptide of SEQ ID NO:114 and the polypeptide of SEQ ID NO:119, (see SEQ ID NO:7 and SEQ ID NO:9) share a significant level of sequence identity (80%).

[0133] E. coli bacteria (E. coli strain RKI 06-08410; obtained from Robert Koch-Institut, Berlin, Germany) were 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, 150 mM NaCl, pH 7.4) and proteins were pipetted into a 96 well plate using different concentrations of proteins in an end volume of 20 μl with or without a final concentration of 500 μM EDTA. 180 μl of bacterial cell suspension or medium (Mueller-Hinton) as 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 minimal inhibitory concentration (MIC), which is the protein concentration in the well which showed the same OD600 value as the no-bacteria control, was determined.

[0134] The results in form of minimal inhibitory concentration (MIC in μg/ml) are shown in table 3 below.

TABLE-US-00004 TABLE 3 Antibacterial activity in presence and absence of EDTA MIC (μg/ml) MIC (μg/ml) SEQ ID NO with EDTA w/o EDTA SEQ ID NO: 123 ≤5 25 SEQ ID NO: 124 ≤5 >50 SEQ ID NO: 125 ≤5 >50 SEQ ID NO: 114 ≤5 10 SEQ ID NO: 115 ≤5 10 SEQ ID NO: 116 ≤5 15 SEQ ID NO: 117 ≤5 10 SEQ ID NO: 119 ≤5 7.5 SEQ ID NO: 120 ≤3.3 16.7

[0135] “≤” (e.g. ≤5, ≤3.3 or the like) means, that antibacterial activity was observed already at the first concentration tested (e.g., 5 μg/ml and 3.3 μg/ml, respectively). The MIC is thus at least the first tested concentration (e.g., 5 μg/ml and 3.3 μg/ml, respectively) and possibly lower. >50 means, that no antibacterial activity could be observed up to a concentration of 50 μg/ml.

[0136] All polypeptides tested showed good antibacterial activity against E. coli in presence of the outer membrane permeabilizer EDTA. However, in absence of EDTA, the antibacterial activity for three conventional fusion proteins dropped significantly. In contrast, the polypeptides according to the present invention retained a significant level of antimicrobial activity even in absence of EDTA.

Example 2

Minimal Inhibitory Concentration of Several Antibacterial Polypeptides Against P. aeruginosa in Presence and Absence of EDTA

[0137] The antibacterial activity on P. aeruginosa bacteria in presence and absence of EDTA was also assessed. The following polypeptides were used: [0138] SEQ ID NO:123 , a fusion of Cecropin A. (A. aegyptii) peptide (SEQ ID NO: 43) with the endolysin of Vibrio phage VvAW1 (YP_007518361.1) [0139] SEQ ID NO:124, a fusion of Cecropin A. (A. aegyptii) peptide with a mutated cell wall binding domain of the modular KZ144 endolysin and Lys68 endolysin [0140] SEQ ID NO:125, a fusion of a modified peptide (SEQ ID NO:105) complying with the preferred sequence motif of the peptide component and an endolysin of Pseudomonas phage vB_PsyM_KIL1 (see YP_009276009.1) [0141] SEQ ID NO:114, a fusion of SMAP-29 peptide (SEQ ID NO:37) and the endolysin of Citrobacter koseri phage CkP1, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:7), and [0142] SEQ ID NO:115, a fusion of a peptide comprising the preferred sequence motif for the peptide component (SEQ ID NO:106) and the endolysin of Enterobacteria phage CC31, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:8) [0143] SEQ ID NO:118, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO: SEQ ID NO:106 and the endolysin of Serratia phage CHI14, with again the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:9), [0144] SEQ ID NO:119, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO:81 and the endolysin of Serratia phage CHI14, with again the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:9), [0145] SEQ ID NO:120, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO:109 and the endolysin of Citrobacter koseri phage CkP1, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:7), [0146] SEQ ID NO:121, a fusion of a peptide comprising a preferred sequence according to SEQ ID NO:110 and the endolysin of Citrobacter koseri phage CkP1, with the additional technical modification of a C54S mutation to reduce aggregation (SEQ ID NO:7), and

[0147] Bacteria (P. aeruginosa PAO1) were 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, 150 mM NaCl, pH 7.4) and proteins were pipetted into a 96 well plate using different concentrations of proteins in an end volume of 20 μl with or without a final concentration of 500 μM EDTA. 180 μl of bacterial cell suspension or medium (Mueller-Hinton) as 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 in the well which showed the same OD600 value as the no-bacteria control was determined.

[0148] The results in form of minimal inhibitory concentration (MIC in μg/ml) are shown in table 4 below.

TABLE-US-00005 TABLE 4 Antibacterial activity in presence and absence of EDTA MIC (μg/ml) MIC (μg/ml) SEQ ID NO with EDTA w/o EDTA SEQ ID NO: 123 ≤5 >50 SEQ ID NO: 124 ≤5 >50 SEQ ID NO: 125 10 >50 SEQ ID NO: 114 ≤5 5 SEQ ID NO: 115 ≤5 10 SEQ ID NO: 118 ≤4.5 6.8 SEQ ID NO: 119 ≤5 7.5 SEQ ID NO: 120 ≤3.3 8.3 SEQ ID NO: 121 ≤5 15

[0149] “≤” (e.g. ≤5, ≤3.3 or the like) means, that antibacterial activity was observed already at the first concentration tested (e.g., 5 μg/ml and 3.3 μg/ml, respectively). The MIC is thus at least the first tested concentration (e.g. 5 μg/ml and 3.3 μg/ml, respectively) and possibly lower. >50 means, that no antibacterial activity could be observed up to a concentration of 50 μg/ml.

[0150] All polypeptides tested showed good antibacterial activity against P. aeruginosa in presence of the outer membrane permeabilizer EDTA. However, in absence of EDTA, the antibacterial activity for three conventional fusion proteins dropped significantly. In contrast, the polypeptides according to the present invention retained again a significant level of antimicrobial activity even in absence of EDTA.