Antimicrobial fusion proteins comprising an endolysin and an amphipathic peptide segment

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.

Claims

1. A fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

2. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein according to claim 1.

3. A vector comprising the nucleic acid molecule according to claim 2.

4. An isolated host cell comprising the nucleic acid molecule according to claim 2.

5. An isolated host cell comprising the vector according to claim 3.

6. A method of treating a Gram-positive bacterial infection or contamination comprising contacting a subject, surface or sample having said infection or contamination with the fusion protein according to claim 1.

7. The method of claim 6, wherein the subject, surface or sample is foodstuff, food processing equipment, food processing plants, surfaces coming into contact with foodstuff, medical devices, or surfaces in hospitals and surgeries.

8. A pharmaceutical composition comprising the fusion protein according to claim 1.

Description

EXAMPLE 1: CLONING, EXPRESSION AND PURIFICATION OF CPL-1, PLY511, LYSK, LYSOSTAPHIN (LSS) AND PA6-GP20 ENZYMES MODIFIED WITH VARIOUS PEPTIDE STRETCHES ON THE N-TERMINUS OR THE C-TERMINUS

(1) Enzymes

(2) Cpl-1 according to SEQ ID NO: 57 is an endolysin originating from Streptococcus pneumoniae phage Cpl-1. The endolysin Cpl-1 is encoded by the nucleic acid molecule according to SEQ ID NO: 91. The nucleic acid molecule according to SEQ ID NO: 91 was synthetically produced with a BamH I (5′-GGA TCC-3′) restriction site at the 5′-end of the nucleic acid molecule and an Xho I (5″-CTC GAG-3′) restriction site at the 3′-end of the nucleic acid molecule.

(3) Ply511 according to SEQ ID NO: 58 is an endolysin originating from Listeria monocytogenes phage A511. The endolysin Ply511 is encoded by the nucleic acid molecule according to SEQ ID NO: 92. The nucleic acid molecule according to SEQ ID NO: 92 was synthetically produced with a BamH I (5′-GGA TCC-3′) restriction site at the 5′-end of the nucleic acid molecule and an Xho I (5″-CTC GAG-3′) restriction site at the 3′-end of the nucleic acid molecule.

(4) LysK according to SEQ ID NO: 59 is an endolysin originating from Staphylococcus aureus phage K. The endolysin LysK is encoded by the nucleic acid molecule according to SEQ ID NO: 93. The nucleic acid molecule according to SEQ ID NO: 93 was synthetically produced with a BamH I (5′-GGA TCC-3′) restriction site at the 5′-end of the nucleic acid molecule and an Xho I (5′-CTC GAG-3′) restriction site at the 3′-end of the nucleic acid molecule.

(5) Lysostaphin (Lss) according to SEQ ID NO: 60 is a bacteriocin originating from Staphylococcus simulans. The bacteriocin Lss is encoded by the nucleic acid molecule according to SEQ ID NO: 94. The nucleic acid molecule according to SEQ ID NO: 94 was synthetically produced with a BamH I (5′-GGA TCC-3′) restriction site at the 5′-end of the nucleic acid molecule and an Xho I (5′-CTC GAG-3′) restriction site at the 3′-end of the nucleic acid molecule.

(6) PA6-gp20 according to SEQ ID NO: 61 is an endolysin originating from Propionibacterium acnes phage. The endolysin PA6-gp20 is encoded by the nucleic acid molecule according to SEQ ID NO: 123. The nucleic acid molecule according to SEQ ID NO: 123 was synthetically produced with a BamH I (5′-GGA TCC-3′) restriction site at the 5′-end of the nucleic acid molecule and an Xho I (5′-CTC GAG-3′) restriction site at the 3′-end of the nucleic acid molecule.

(7) The following peptide stretches in table 5 were used for production of fusion proteins with the enzymes Cpl-1, Ply511, LysK, Lysostaphin (Lss) and PA6-gp20:

(8) TABLE-US-00005 TABLE 5 Nucleic acid molecule encoding Peptide stretch the peptide stretch Pseudin 1 SEQ ID NO: 95 (SEQ ID NO: 51) WLBU2-Variant SEQ ID NO: 96 (SEQ ID NO: 55) LL-37 SEQ ID NO: 97 (SEQ ID NO: 5) Indolicidin SEQ ID NO: 98 (SEQ ID NO: 7) Magainin SEQ ID NO: 99 (SEQ ID NO: 4) Pleurocidin SEQ ID NO: 100 (SEQ ID NO: 1) Cecropin A SEQ ID NO: 101 (A. aegypti) (SEQ ID NO: 9) Buforin II SEQ ID NO: 102 (SEQ ID NO: 3) Sarcotoxin IA SEQ ID NO: 103 (SEQ ID NO: 11) PK SEQ ID NO: 104 (SEQ ID NO: 13) Pentapeptide SEQ ID NO: 105 (SEQ ID NO: 12) PK2 SEQ ID NO: 106 (SEQ ID NO: 31)

(9) The nucleic acid molecules encoding the respective peptide stretches were synthetically produced with a Nde I (5′-CAT ATG-3′) restriction site at the 5′-end of the nucleic acid molecule and a BamH I (5′-GGA TCC-3′) restriction site at the 3′-end of the nucleic acid molecule, except the nucleic acid molecule encoding the PK and PK2 for ligation with the bacteriocin Lss, which was produced with a Nco I restriction site plus two additional nucleotides (5′-CCA TGG GC-3′) at the 5′-end of the nucleic acid molecule.

(10) Fusion proteins are constructed by linking at least two nucleic acid sequences using standard cloning techniques as described e.g. by Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual. Therefore the nucleic acid molecules encoding the peptide stretches were cleaved in a digest with the respective restriction enzymes Nde I and BamH I and in case of the nucleic acid molecule encoding the peptide stretch PK and PK2 for ligation with the Lss the digest was performed with the restriction enzymes Nco I and BamH I. Subsequently the cleaved nucleic acids encoding the peptide stretches were ligated into the pET21 b expression vector (Novagen, Darmstadt, Germany), which was also cleaved in a digest with the respective restriction enzymes Nde I and BamH I before. The cleaved nucleic acid molecule encoding the peptide stretch PK and PK2 for ligation with Lss was ligated into a modified pET32 b expression vector (unmodified vector obtainable from Novagen, Darmstadt, Germany), which was also cleaved in a digest with the respective restriction enzymes Nco I and BamH I before. The modification of the pET32b expression vector refers to the deletion of the sequence encoding a S-tag and the central His.sub.6-tag.

(11) Afterwards, the nucleic acid molecules encoding the enzymes Cpl-1, Ply511, PA6-gp20, LysK and Lss were cleaved in a digest with the restriction enzyme BamH I and Xho I, so that the endolysin could be ligated into the pET21b expression vector (Novagen, Darmstadt, Germany) and the modified pET32 b expression vector, respectively, which were also cleaved in a digest with the respective restriction enzymes BamH I and Xho I before.

(12) In the case of the peptide stretch PK, which was ligated to the C-terminus of the Lysostaphin and the LysK, the resulting fusion protein has a His.sub.6-tag on the N-terminus, wherein the His.sub.6-tag is linked to the N-terminus by a linker. For the cloning of the respective nucleic acid molecules the pET32 b expression vector (Novagen, Darmstadt, Germany) was used.

(13) Thus, the nucleic acid molecule encoding the peptide stretch is ligated into the respective vector at the 5′-end of the nucleic acid molecule encoding the respective enzyme. Moreover, the nucleic acid molecule encoding the respective enzyme is ligated into the respective plasmid, so that a nucleic acid molecule encoding a His.sub.6-tag consisting of six histidine residues is associated at the 3″-end of the nucleic acid molecule encoding the endolysin.

(14) As some fusion proteins may either be toxic upon expression in bacteria, or not homogenous due to protein degradation, the strategy might be to express these fusion proteins fused or linked to other additional proteins. Example for these other additional protein is thioredoxin, which was shown to mediate expression of toxic antimicrobial peptides in E. coli (TrxA mediating fusion expression of antimicrobial peptide CM4 from multiple joined genes in Escherichia coli. Zhou L, Zhao Z, Li B, Cai Y, Zhang S. Protein Expr Purif. 2009 April; 64(2):225-230). In the case of the fusion protein consisting of the N-terminal PK or PK2 peptide and the bacteriocin Lss, the peptide was ligated into the modified pET32 b expression vector, so that an additional thioredoxin is associated at the 5′-end of the peptide. The thioredoxin could be removed from the expressed fusion protein by the use of enterokinase, therefore between the nucleic acid molecule encoding the peptide and the one encoding the thioredoxin is an enterokinase restriction site introduced.

(15) The sequence of the endolysin-peptide-fusions was controlled via DNA-sequencing and correct clones were transformed into E. coli BL21(DE3) and in E. coli BL21 (DE3) pLysS cells (Novagen, Darmstadt, Germany) for protein expression.

(16) Recombinant expression of the fusion proteins according to SEQ ID NO: 107 to 122 and 124 is performed in E. coli BL21 (DE3) and E. coli BL21 (DE3) pLysS cells (Novagen, Darmstadt, Germany). The cells were growing until an optical density of OD600 nm of 0.5-0.8 was reached. Then the expression of the fusion protein was induced with 1 mM IPTG (isopropylthiogalactoside) and the expression was performed at 37° C. for a period of 4 hours.

(17) E. coli BL21 cells were harvested by centrifugation for 20 min at 6000 g and disrupted via sonication on ice. Soluble and insoluble fraction of the E. coli crude extract were separated by centrifugation (Sorvall, SS34, 30 min, 15 000 rpm). All proteins were purified by Ni.sup.2+ affinity chromatography (Akta FPLC, GE Healthcare) using the C-terminal His.sub.6-tag, encoded by the pET21b or pET32b vectors.

(18) Some proteins were expressed using a modified pET32b vector (S-tag and central His.sub.6-tag deleted) as described above, which fuses thioredoxin on the N-terminus of the proteins of interest. The vector also contains an enterokinase cleavage site right before the protein of interest. This site allows the proteolytic cleavage between thioredoxin and the protein of interest, which can purified via the remaining C-terminal His.sub.6-tag. For antimicrobial function of the fusion protein it may be necessary to remove the thioredoxin by proteolytic cleavage. Therefore the fusion protein was cleaved with 2-4 units/mg recombinant enterokinase (Novagen, Darmstadt, Germany) to remove the thioredoxin following the protocol provided by the manufacturer. After enterokinase cleavage the fusion protein was purified via His.sub.6-tag purification as described below.

(19) The Ni′ affinity chromatography is performed in 4 subsequent steps, all at room temperature: 1. Equilibration of the Histrap FF 5 ml column (GE Healthcare) with up to 10 column volumes of Washing Buffer (20 mM imidazole, 1 M NaCl and 20 mM Hepes on pH 7.4) at a flow rate of 3-5 ml/min. 2. Loading of the total lysate (with wanted fusion protein) on the Histrap FF 5 ml column at a flow rate of 3-5 ml/min. 3. Washing of the column with up to 10 column volumes of Washing Buffer to remove unbound sample followed by a second washing step with 10% Elution buffer (500 mM imidazole, 0.5 M NaCl and 20 mM Hepes on pH 7.4) at a flow rate of 3-5 ml/min. 4. Elution of bounded fusion proteins from the column with a linear gradient of 4 column volumes of Elution Buffer (500 mM imidazole, 0.5 M NaCl and 20 mM Hepes on pH 7.4) to 100% at a flow rate of 3-5 ml/min.

(20) Purified stock solutions of fusion proteins in Elution Buffer (20 mM Hepes pH 7.4; 0.5 M NaCl; 500 mM imidazole) were at least 90% pure as determined visually on SDS-PAGE gels (data not shown).

EXAMPLE 2: ANTIMICROBIAL ACTIVITY OF CPL-1 ENZYMES MODIFIED WITH VARIOUS PEPTIDE STRETCHES ON THE N-TERMINUS

(21) The fusion proteins Cpl 1 with the N-terminal peptide stretches Pseudin 1, WLBU2-Variant, LL-37, Indolicidin, Magainin, Pleurocidin, Cecropin A (A. aegypti), Buforin II, Sarcotoxin IA and PK were produced as described in example 1. The antimicrobial activity of said fusion protein against Streptococcus pneumoniae DSMZ 11967 and Streptococcus pneumoniae DSMZ 14378 were tested by using the plating test described below. The measured activity of the fusion protein is shown in Table 6.

(22) The results presented in Table 6 show high antimicrobial activity of all fusion proteins against Streptococcus pneumoniae DSMZ 11967 and Streptococcus pneumoniae DSMZ 14378.

(23) Plating Assay:

(24) Exponentially growing cells of e.g. Streptococci, Listeria, Propionibacteria or Staphylococci were taken (1 ml) cooled on ice and washed with distilled water. The bacteria were resuspended in 20 mM Tris pH 7.0, 1 mM MgCl.sub.2, 0.5 M Saccharose. Fusion proteins were diluted in resuspension buffer, adding sucrose to a final concentration of 0.5 M and incubated (final concentration of the fusion protein about 10 μg/ml) with the respective bacteria for 60 minutes at room temperature. After that bacteria were plated on appropriated agar plates (e.g. Streptococci: Columbia blood agar) containing 0.5 M sucrose and the resulting colonies were counted after incubation.

(25) The residual colonies were counted after an overnight incubation at 37° C. Based on the counted cell numbers the antibacterial activity as logarithmic units (=log.sub.10N.sub.0/N.sub.i with N.sub.0=number of untreated cells and N.sub.i=number of treated cells) was calculated. All samples were replicated at least in four fold.

(26) TABLE-US-00006 TABLE 6 Peptide stretch Activity against Activity against (N-terminal unless Streptococcus pneumoniae Streptococcus pneumoniae Fusion protein Enzyme part otherwise indicated) DSMZ 11967 DSMZ 14378 SEQ ID NO: 107 Cpl-1 Pseudin 1 +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 51) SEQ ID NO: 108 Cpl-1 WLBU2-Variant ++ ++ (SEQ ID NO: 57) (SEQ ID NO: 55) SEQ ID NO: 109 Cpl-1 LL-37 +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 5) SEQ ID NO: 110 Cpl-1 Indolicidin +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 7) SEQ ID NO: 111 Cpl-1 Magainin +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 4) SEQ ID NO: 112 Cpl-1 Pleurocidin +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 1) SEQ ID NO: 113 Cpl-1 Cecropin A +++ +++ (SEQ ID NO: 57) (A. aegypti) (SEQ ID NO: 9) SEQ ID NO: 114 Cpl-1 Buforin II +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 3) SEQ ID NO: 115 Cpl-1 Sarcotoxin IA +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 11) SEQ ID NO: 116 Cpl-1 PK +++ +++ (SEQ ID NO: 57) (SEQ ID NO: 13) Abbreviations: +: 1 log; ++: 2-3 log; +++: 4 or more logs.

EXAMPLE 3: ANTIMICROBIAL ACTIVITY OF PLY511 ENZYME MODIFIED WITH THE PENTAPEPTIDE ON THE N-TERMINUS

(27) The fusion protein Ply511 with the N-terminal peptide stretch pentapeptide according to SEQ ID NO: 12 was produced as described in example 1. The antimicrobial activity of said fusion protein against Listeria monocytogenes DSMZ 15675 and Listeria monocytogenes DSMZ 20600 was tested by using the plating test described in example 2. The measured activity of the fusion protein is shown in Table 7.

(28) The results presented in Table 7 show high antimicrobial activity of the fusion protein pentapeptide: Ply511 against Listeria monocytogenes DSMZ 15675 and Listeria monocytogenes DSMZ 20600.

(29) TABLE-US-00007 TABLE 7 Peptide stretch Activity against Activity against (N-terminal unless Listeria monocytogenes Listeria monocytogenes Fusion protein Enzyme part otherwise indicated) DSMZ 15675 DSMZ 20600 SEQ ID NO: 117 Ply511 Pentapeptid +++ +++ (SEQ ID NO: 58) (SEQ ID NO: 12) Abbreviations: +: 1 log; ++: 2-3 log; +++: 4 or more logs.

EXAMPLE 4: ANTIMICROBIAL ACTIVITY OF LSS AND LYSK ENZYME MODIFIED WITH POLYCATIONIC PEPTIDES ON THE N-TERMINUS OR C-TERMINUS

(30) The fusion proteins Lss and LysK, respectively, with the N-terminal peptide stretch PK according to SEQ ID NO: 13, the fusion protein Lss with the N-terminal peptide stretch PK2 according to SEQ ID NO:31, as well as the fusion proteins Lss and LysK, respectively, with the C-terminal peptide stretch PK were produced as described in example 1. The antimicrobial activity of said fusion proteins against Staphylococcus aureus DSMZ 346 and Staphylococcus epidermidis DSMZ 20041 was tested by using the plating test described in example 2, as well as by using the lysis test as described in the following.

(31) Lysis Test

(32) The Lysis test was used for the modified LysK and Lysostaphins to examine the antimicrobial effect of these fusion proteins.

(33) Staphylococcal cells of were grown in BHI medium until and optical density at 600 nm of 0.7-1 was reached indicating exponential growth. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl (pH 7.4), 60 mM NaCl, 2 mM CaCl2. Cells were resuspended at an optical density at 600 nm of 1.0 and incubated with fusion proteins. Activity was measured spectrophotometrically at 600 nm.

(34) The measured activity of the fusion protein is shown in Table 8.

(35) The results presented in Table 8 show high antimicrobial activity of the fusion proteins Lss with the N-terminal peptide PK or PK2 against Staphylococcus aureus DSMZ 346 andStaphylococcus epidermidis DSMZ 20041. But also the other fusion proteins show antimicrobial activity against the two tested bacterial strains.

(36) TABLE-US-00008 TABLE 8 Peptide stretch Activity against Activity against (N-terminal unless Staphylococcus aureus Staphylococcus epidermidis Fusion protein Enzyme part otherwise indicated) DSMZ 346 DSMZ 20041 SEQ ID NO: 118 LysK PK + + (SEQ ID NO: 59) (SEQ ID NO: 13) SEQ ID NO: 119 Lysostaphin PK +++ +++ (SEQ ID NO: 60) (SEQ ID NO: 13) SEQ ID NO: 120 Lysostaphin PK2 +++ +++ (SEQ ID NO: 60) (SEQ ID NO: 31) SEQ ID NO: 121 LysK PK (C-terminal) + + (SEQ ID NO: 59) (SEQ ID NO: 13) SEQ ID NO: 122 Lysostaphin PK (C-terminal) + + (SEQ ID NO: 60) (SEQ ID NO: 13) Abbreviations: +: 1 log; ++: 2-3 log; +++: 4 or more logs.

EXAMPLE 5: ANTIMICROBIAL ACTIVITY OF PA6-GP20 ENZYME MODIFIED WITH THE HYDROPHOBIC PEPTIDE STRETCH WALMAGH 1

(37) The fusion protein PA6-gp20 with the N-terminal peptide stretch Walmagh 1 according to SEQ ID NO: 56 was produced as described in example 1. The antimicrobial activity of said fusion protein against Propionibacterium acnes DSMZ 1897 and Propionibacterium acnes DSMZ 16379 was tested by using the plating test described in example 2. The measured activity of the fusion protein is shown in Table 9.

(38) The results presented in Table 9 show antimicrobial activity of the fusion protein against both bacterial strains of Propionibacterium acnes.

(39) TABLE-US-00009 TABLE 9 Peptide stretch Activity against Activity against (N-terminal unless Propionibacterium acnes Propionibacterium acnes Fusion protein Enzyme part otherwise indicated) DSMZ 1897 DSMZ 16379 SEQ ID NO: 124 PA6-gp20 Walmagh 1 ++ ++ (SEQ ID NO: 61) (SEQ ID NO: 56) Abbreviations: ++: 2-3 log;

(40) The fusion proteins in Table 6 to 9 without any tag and linker were also tested with the activity assays described above. They all showed antimicrobial activity against the used bacterial strains in Table 6 to 9.