Antibacterial method

Abstract

The present invention provides a method of killing, damaging or preventing the replication of bacteria comprising administering or applying a bacteriocin to said bacteria, wherein said bacteriocin is a peptide comprising the amino acid sequence MKFKFNPTGTIVKKLTQYEIAWFKNKHGYYPWEIPRC and related sequences, wherein the bacteria is selected from E. faecium, E. faecalis, E. hirae, S. pseudointermedius and/or S. hemolyticus; and/or in said method said bacteria are subjected to a stress condition. Also provided are related methods and uses such as methods of treatment. Also provided are novel truncation and fusion proteins variants such as MIKKFPNPYTLAAKLTTYEINWYKQQYGRYPWERPVA and MKFKFNPTGTIVKKLTQYEINWYKQQYGRYPWERPVA and their use as bacteriocins in various methods and uses.

Claims

1. A bacteriocin peptide of less than 50 amino acids comprising an amino acid sequence selected from: a) MKFKFNPTGTIVKKLTQYEINWYKQQYGRYPWERPVA (SEQ ID NO: 4); and b) a sequence with at least 85% sequence identity to sequence a), wherein sequence b) comprises at least the consensus sequence KXXXGXXPWE (SEQ ID NO: 40).

2. The bacteriocin peptide of claim 1, consisting of an amino acid sequence selected from: a) MKFKFNPTGTIVKKLTQYEINWYKQQYGRYPWERPVA (SEQ ID NO: 4); and b) a sequence with at least 85% sequence identity to sequence a), wherein sequence b) comprises at least the consensus sequence KXXXGXXPWE (SEQ ID NO: 40).

3. A method of killing, damaging or preventing the replication of bacteria comprising administering or applying the bacteriocin of claim 1 to said bacteria.

4. The method of claim 3 wherein said bacteria is E. faecium, E. faecalis, E. hirae, S. pseudointermedius and/or S. hemolyticus.

5. The method of claim 3 wherein said bacteria is E. faecium, E. faecalis and/or S. hemolyticus.

6. The method of claim 4, wherein said method is performed in vitro.

7. The method of claim 4, wherein the method is for disinfecting or decontaminating an item, of bacteria present on said item, wherein the method comprises covering, impregnating, or coating said item with said bacteriocin or applying said bacteriocin to said item, wherein optionally in said method said bacteria are subjected to heating at between 40 and 50° C. before, during and/or after administering or applying said bacteriocin.

8. The method of claim 7, wherein said item is i) a medical device, instrument, implement or equipment, a prosthetic, an implant, a scaffold, or a tissue or wound dressing, or ii) a personal health care product.

9. The method of claim 8, wherein said personal health care product is toothpaste, mouthwash, skin cream, lotion or spray.

10. A method of treating or preventing a bacterial infection in a subject or patient comprising administering the bacteriocin of claim 1 to said subject or patient or to a part of said subject's or patient's body.

11. The method of claim 10, wherein said subject or patient is a mammal.

12. The method of claim 10, wherein said bacterial infection is an infection on the skin and said bacteriocin is administered topically.

13. The method of claim 12, wherein said bacterial infection is caused by E. faecium, E. faecalis and/or S. hemolyticus.

14. The method of claim 10, wherein said bacteriocin is provided in a host cell which produces said bacteriocin.

15. The method of claim 10, wherein said bacteriocin is co-administered or co-applied with one or more additional antibacterial agents, wherein optionally said bacteriocin is in the form of a composition comprising said bacteriocin and said one more additional antibacterial agents.

16. The method of claim 15, wherein said one or more additional antibacterial agents are selected from one or more bacteriocins or antibiotics.

Description

(1) FIG. 1 shows the NMR structure of EntK1 (A, B) compared with LsbB (C, D) (Ovchinnikov et al., 2014, J. Biol. Chem., 289:23838-23845) in 50% TFE. The structures ensembles of the 20 lowest energy structures superimposed are shown in A and C and cartoon representations of the lowest energy structures are shown in B and D.

(2) FIG. 2 shows the effect of heating on the development of EntK1 and EntEJ97 resistant mutants. Bacteriocin (20 μl of 1 mg/ml) was applied to soft agar containing indicator cells on plates, which was then incubated overnight at indicated temperatures for growth and developing the inhibition zones and resistant colonies. The mutants still appear after culture at 30° C. but not at the elevated temperature of 45° C.

EXAMPLES

Example 1: Structural Analysis of EntK1, Analysis of Inhibitory Spectra of Bacteriocins, Analysis of Resistant Mutants and Effect of Stress Conditions on Mutants

Materials and Methods

(3) Bacterial strains, growth conditions, bacteriocins and antimicrobial assays. All the strains (see below) used in minimal inhibitory concentration (MIC) assays (Varahan et al., 2014, M. Bio. 5:e01726-01714) were grown in BHI medium (Oxoid) at 30° C. without shaking. A collection of E. faecium strains isolated from blood in patients from different European hospitals was received from Department of Medical Microbiology, University Medical Center Utrecht, the Netherlands. LsbB, EntK1, EntEJ97 and BHT-B were synthesized by Pepmic Co., LTD, China with 98-99% purity. Synthesized peptides were solubilized to concentrations of 10.0-0.1 mg/ml in 0.1% (vol/vol) trifluoroacetic acid and stored at −20° C. until use. Garvicin ML was purified to 95% as described by (Borrero et al., 2011, Appl. Environ. Microbiol., 77:369-373). Bacteriocin activity was determined by microtiter plate assay as previously described (Holo et al., 1991, J. Bacteriol., 173:3879-3887). The MIC was defined as the minimal bacteriocin concentration that inhibited the growth of the indicator strain by at least 50% (50% of the turbidity of the control culture without bacteriocin) in 200 μl culture.
CD Spectroscopy
Circular dichroism (CD) spectra were recorded using a Jasco J-810 spectropolarimeter (Jasco International Co) calibrated with D-camphor-10-sulfonate (Icatayama Chemical). All measurements were made using a quartz cuvette (Sterna) with 0.1 cm path length. Samples were scanned five times with a scanning rate of 50 nm/min with a bandwidth of 0.5 nm and response time of 1 s over the wavelength range 190-250 nm. Spectra were recorded at different (30 and 50%) trifluoroethanol (TFE) (Aldridge) concentrations at 25° C. The approximate α-helical content of the protein was estimated from its molar ellipticity at 222 nm (Scholtz et al., 1991, Biopolymers 31:1463-1470).
NMR Spectroscopy
The experiments were run on a sample containing 1.0 mM EntK1, 50% D3-TFE (99.5% D) (Aldrich), Milli-Q water and 0.2 mM of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (Larodan).
2D NOESY (Jeener et al., 1979, J. Magn. Reson., 71:4546-4553), 2D TOCSY (Braunschweiler and Ernst, 1983, J. Magn. Reson., 53:521-528), 2D DQCOSY, .sup.15N-HSQC (Davis et al., 1992, J. Magn. Reson., 98:207-216) and .sup.13C-HSQC (Hurd, 1991, J. Magn. Reson., 91:648-653) were recorded. The data was acquired on a 600 MHz Bruker Avance II spectrometer with four channels and a 5 mm TCI cryoprobe (Bruker Biospin). NOESY spectra with mixing times between 120 ms and 300 ms were obtained for both samples. TOCSY mixing times of 15-80 ms were used. The experiments were run at 298.15 K. Spectra were processed using the Topspin program (Bruker Biospin). 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as a chemical shift standard, and .sup.15N and .sup.13C data were referenced using frequency ratios as described in (Wishart et al., 1995, J. Biomol. NMR., 6:135-140).
For visualization, assignment and integration of the spectra the computer program CARA was used (Keller, 2004, The Computer Aided Resonance Assignment Tutorial. CANTINA Verlag, Goldau, Switzerland). The spectra were assigned using standard methods.
Dihedral angle restraints were obtained from the chemical shift values using the program TALOS-N(Shen and Bax, 2013, J. Biomol. NMR, 56:227-241). Nuclear Overhauser effect (NOE) distance restraints were calculated from the peak volumes in the NOESY spectra with NOESY mixing time of 200 ms.
All structure calculations were made using the structure calculation program CYANA 2.1 (Guntert et al., 1997, J. Mol. Biol., 273:283-298; Herrmann et al., 2002, J. Mol. Biol., 319:209-227). The annealing macro in CYANA calculated 100 structures. The 20 structures with the lowest energy were kept and analyzed further. The root-mean-square deviation (RMSD) was calculated and the structures were visualized using MolMol (Koradi et al., 1996, J. Mol. Graph., 14:51-55, 29-32).
Generation of bacteriocin resistant mutants of E. faecalis and E. faecium. EntEJ97 and EntK1 resistant mutants were obtained by a spot-on-lawn assay using 20 μl of bacteriocin with concentrations of 1.0-0.1 mg/ml. After overnight incubation at 30° C., resistant colonies of E. faecalis LMGT3358 (resistant to EntEJ97) and E. faecium LMGT2787 (resistant to EntEJ97 and EntK1) were picked randomly from the BHI agar plates. The level of resistance against EntEJ97 and EntK1 was determined by a microtitre plate assay (Holo et al., 1991, supra). DNA from the bacteriocin resistant mutants was extracted from overnight cultures with a GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich) and PCR of the rseP gene was performed using primers Ent, EF (F, M, R) (Table 1). For sequencing of E. faecium LMGT2787 mutants with transposons inside rseP, additional primers (EF_R2, T1_F, T2_F and T3_F) were created (Table 1). PCR products were purified with NucleoSpin Extract II (Macherey-Nagel, Duren, Germany) and sent to GATC Biotech, Germany, for sequencing.
Construction of S. pneumoniae transformants and mutants of rseP.
To express the E. faecalis rseP gene heterologously in S. pneumoniae, the gene was placed by homologous recombination in the genome of strain SPH131, behind the ComS-inducible P.sub.comX promoter (ComRS system) (Berg et al., 2011, J. Bacteriol., 193:5207-5215). The P.sub.comX-rseP construct was created by overlap extension PCR (Higuchi et al., 1988, Nature. 332:543-546). First, the E. faecalis LMGT3358 rseP was amplified using the primer pair ds171/ds172 with genomic E. faecalis DNA as template. The P.sub.comX promoter and its ˜1000 bp upstream and downstream regions were amplified using the primer pairs khb31/khb36 and khb33/khb34, respectively. Genomic DNA derived from strain SPH131 served as template. The P.sub.comX with its upstream region was fused to the 5′ end of the E. faecalis rseP gene using the primers khb31 and ds172. The P.sub.comX downstream fragment was fused to the 3′ end of the rseP gene using primer pair ds171 and khb34. Finally, these two fragments were fused using primer khb31 and khb34 giving rise to P.sub.comX-rseP. The Janus cassette (Sung et al., 2001, Appl. Environ. Microbiol., 67:5190-5196) in strain SPH131 was replaced with the P.sub.comX-rseP fragment by natural transformation, giving rise to strain ds218.
S. pneumoniae has a gene homologous to the lactococcal rseP. To avoid the potential background noise of the S. pneumoniae rseP, this gene was removed from the genome in strain ds218 using the Janus cassette (Sung et al., 2011, supra). An rseP deletion cassette was constructed by amplifying Janus with the primers kan484F and RpsL41.R (Johnsborg et al., 2008, Mol. Microbiol. 69:245-253) where genomic DNA from strain RH426 (Johnsborg et al., 2008, supra) served as template. The Janus cassette was then fused to the ˜1000 bp upstream (primers th009 and th010 with genomic RH1 (Johnsborg et al., 2008, supra) DNA as template) and downstream region (primers th011 and th012 with genomic RH1 DNA as template) of rseP using primers th009 and th012. The resulting fragment was used to transform strain ds218 resulting in the replacement of S. pneumoniae rseP with Janus generating strain ds219. Janus was then removed from strain ds219 by transforming with a fragment consisting of the rseP flanking regions only. This fragment was constructed by amplifying the rseP ˜1000 bp upstream region using primers th009 and ds87, while the ˜1000 bp downstream region was amplified with the primers ds88 and th012. The upstream and downstream fragments were then fused using the primers pair th009/th012. The resulting fragment was used to replace the Janus in strain ds219 resulting in strain ds220.
To create a strain expressing point mutated rseP genes we replaced the E. faecalis 3358 rseP in strain ds220 with Janus cassette giving rise to strain ds221. This Janus cassette was amplified with the primer pair khb31/khb34 and genomic DNA from strain SPH131 served as template. Selected residues in RseP were substituted with alanines by a PCR approach, using primer pairs listed in Table 1. The resulting DNA pair fragments were subsequently fused using the primers khb31 and khb34. The resulting fragment was used to replace Janus cassette in strain ds221 giving rise to strains ms1-5 (Table 2). Ectopic expression of the rseP gene in strain ds220 and ms1-5 was induced with the synthetic ComS peptide (NH2-LPYFAGCL-COOH) (Genosphere Biotechnologies) as described by Berg et al. (2011, supra).

Results

(4) Structural analysis of EntK1 by CD and NMR-spectroscopy.

(5) CD spectra of EntK1 showed that it was unstructured in water but became structured in TFE solution (data not shown). Maximum structuring was obtained in 50% TFE (55% α-helical content). This concentration was consequently used in the NMR experiment.

(6) The NMR spectra were assigned using standard methods as described in Materials and Methods. Chemical shift indexing indicates that there is an α-helix in EntK1 from residue 8 to 27 and TALOS-N torsion angle predictions (Shen & Bax, 2013, supra) indicated backbone torsion angles (ϕ- and Ψ-angle) consistent with α-helical regions from residue 8 to 25 (data not shown).
A total of 561 (15.1 per residue) unique NOE connectivities were assigned (data not shown). In the presence of TFE, the observed connectivities indicated an α-helical region stretching from residue 8 to 26.
Structures of EntK1 based on the experimentally obtained constrains were calculated using CYANA. A superimposition of the structure ensemble of the 20 lowest energy structures of EntK1 and the cartoon depiction of the lowest energy structure of EntK1 are shown in FIGS. 1A and B respectively. The NMR structure of EntK1 contains an α-helix from residue 8 to residue 24. The structure ensemble has been deposited to the Protein Data Bank with access code: 5L82 and the NMR data have been deposited to the Biomagnetic Resonance Data Bank with the access code 34006.
The inhibitory spectra of EntK1 and two homologous bacteriocins.
A sizable collection of Gram-positive bacteria from different species and genera was used as indicators to assess the inhibition spectra of the bacteriocins EntK1, EntEJ97 and LsbB (Table 3, 4). EntQ, which is a member of the LsbB family bacteriocins, was not tested because synthetic EntQ peptides had poor activity, likely due to formation of disulphide bridges between and inside the molecules and was only active when mixed with reducing agents (DTT, 2-mercaptoethanol). As seen in Table 3, the antimicrobial spectra of EntK1, LsbB, and EntEJ97 differ greatly. LsbB was only active against L. lactis IL1403. EntK1 was active mostly against E. faecium and E. hirae and some lactococcal strains. EntEJ97 showed the broadest activity spectrum, inhibiting E. faecium, E. faecalis, L. lactis, S. aureus, L. garvieae, and L. monocytogenes. It is important to note that EntK1 appeared to have significantly lower MIC values (i.e., being more potent) than EntEJ97 against E. faecium (Table 3). Similarly, all three bacteriocins were active against L. lactis IL1403, but LsbB was about 340 and 70 times more potent than EntK1 and EntEJ97, respectively (Table 3). Since EntK1 and EntEJ97 were active against E. faecium, we wanted to test these two bacteriocins against E. faecium strains isolated from blood in patients from different European hospitals, including VRE strains. The results showed that both bacteriocins could inhibit the nosocomial strains with EntK1 being more active that EntEJ97 as in the case of non-nosocomial isolates (Table 4).
Susceptible E. faecalis and E. faecium can be adapted to provide resistant mutants in response to EntEJ97 and EntK1.
The strains E. faecalis LMGT3358 and E. faecium LMGT2787 were exposed to various concentrations of EntEJ97 and EntK1 at 30° C. and many resistant colonies appeared within the inhibition zones on agar plates (see below). For E. faecalis, twelve EntEJ97 resistant colonies were randomly selected and examined for resistance to EntEJ97 by microtiter assays. For E. faecium the same was done for six EntEJ97 and six EntK1 mutants.
Two levels of E. faecalis resistance to EntEJ97 were found: seven colonies were highly resistant (HR)—at least 500 times more compared to the WT. The second set of five colonies showed a lower resistance (LR) level, being 16-32 times more resistant than the WT E. faecalis. For E. faecium only HR (500 times) type mutants were found. When all these mutants were challenged with the non-related bacteriocins BHT-B (Hyink et al., 2005, FEMS Microbiol. Lett., 252:235-241) and garvicin ML (Borrero et al., 2011, supra), the mutants were found as sensitive as the WT strains (data not shown). These results clearly imply the involvement of specific resistance mechanism(s) toward EntEJ97/EntK1 amongst these mutants.
Highly resistant mutants of E. faecalis and E. faecium have mutations in the rseP gene.
We investigated whether the Enterococcus homologue of the lactococcal RseP (receptor for LsbB), might serve the same function for the EntEJ97/EntK1 bacteriocins. The DNA regions containing rseP in all the EntEJ97/EntK1-resistant mutants (high and low) were therefore obtained by PCR and sequenced. All HR mutants contained a mutation within the rseP gene. The E. faecalis, resistant mutants contained either one or three consecutive 8-bp CAAAAAAT sequence repeats in the rseP gene, while the WT rseP has two such repeats. All the E. faecium HR mutants carried a transposon within the rseP gene (data not shown). In all cases, mutations caused frameshift in the rseP gene and premature termination, indicating that a functional ResP is necessary for the sensitivity toward EntEJ97/EntK1. Surprisingly, no mutations were found in rseP from all EntEJ97 LR mutants, indicating that additional genes can affect the sensitivity of the bacterium to these bacteriocins.
RseP expression in S. pneumoniae confers sensitivity to EntEJ97/EntK1.
In order to confirm that the expression of rseP is sufficient to confer sensitivity to EntEJ97 and EntK1, the rseP gene from E. faecalis LMGT3358 was inserted into the genome of the distantly related and non-sensitive host S. pneumoniae strain SPH131 as described in (Berg et al., 2011, supra). The endogenous rseP of S. pneumoniae SPH131 was removed to avoid potential background noise. In the final construct of S. pneumoniae ds220 (lacking the endogenous rseP) enterococcal rseP expression was under the control of the inducible promoter P.sub.comX (Berg et al., 2011, supra). While non-induced cells were not sensitive to EntEJ97 and EntK1, they became sensitive to the bacteriocins upon induction of rseP (Table 5). The results indicate that the enterococcal rseP is indeed directly involved in the sensitivity to EntEJ97 and EntK1.
The active site of RseP is partly involved in the sensitivity to the EntEJ97/EntK1 bacteriocins.
Members of the RseP protein family have a conserved proteolytic motif (HExxH, where x is any amino acid) located within the first transmembrane helix (Koide et al., 2007, J. Biol. Chem., 282:4553-4560). To analyze whether this active site is important for bacteriocin sensitivity, we changed the invariant residues in this motif —H18-E19-F20-G21-H22—one by one and altogether in the enterococcal RseP (invariant residues underlined) to alanine and then assessed for bacteriocin sensitivity. In addition, Y24 was replaced with alanine to serve as a control. The result showed that all strains expressing an RseP protein in which conserved residues were changed to alanines became more resistant to EntEJ97 and EntK1, especially when all three conserved residues were replaced with alanines (30 times more resistant to EntEJ97 than the WT) (Table 5). When a residue outside the active center was replaced with alanine (Y24>A) no changes in the bacteriocin sensitivity were observed. This result demonstrates that the active site of RseP is essential for bacteriocin activity. However, since none of the mutants became completely resistant to EntEJ97, there must be one or more additional sites in RseP involved in bacteriocin interaction.
EntK1 and EntEJ97 resistant mutants become sensitive at elevated growth temperature.
We examined how elevated growth temperature, which is a stress factor, influences the development of EntEJ97 and EntK1 resistance in E. faecium and E. faecalis. Resistant mutants only appeared at 30° C. but not at 45° C. (FIG. 2). Cell counts of cultures grown for 8 hours at 30° C. and 45° C. in BHI broth showed that the cell number at 30° C. was only about 1.2 times higher than that at 45° C. (data not shown), implying that the lack of resistant mutants at the elevated temperature was not due to poor growth. The EntEJ97/EntK1 resistant mutants with a non-functional rseP gene (described above) obtained at 30° C. could not grow at 45° C., while WT cells grew well (data not shown).

(7) TABLE-US-00002 TABLE 1 Primers used in the experiments. Primer Oligonucleotide sequence (5′ .fwdarw. 3′) (SEQ ID NO) Reference Ent_F CGAAGTGGTCAAGTCCAATGGT (SEQ ID NO: 6) This study Ent_M GTGCGGATTGCGCCACTTGAC (SEQ ID NO: 7) This study Ent_R GATGACTTAAGACTTCTGCATCAT (SEQ ID NO: 8) This study EF_F GCTCTTAGCAAGATTTGATGGC (SEQ ID NO: 9) This study EF_M CGTCCACACTGACTACCTCATC (SEQ ID NO: 10) This study EF_R CTTAGACCGTTTCGACAGTTTGC (SEQ ID NO: 11) This study EF_R2 TGCAATCTGTCGACGTGACAC (SEQ ID NO: 12) This study T1_F AGCTAGCTCAAAGGAAGAGGC (SEQ ID NO: 13) This study T2_F TGCAATCTGTCGACGTGACAC (SEQ ID NO: 14) This study T3_F GCTCGAACAGCTAAGAATGCCT (SEQ ID NO: 15) This study th009 ACGTTTGAGCAATTTCCTTCC (SEQ ID NO: 16) This study th010 CACATTATCCATTAAAAATCAAACAGCGTTTCCTCCGTCTTTTG (SEQ ID NO: 17) This study th011 GTCCAAAAGCATAAGGAAAGTCGAGGAATATTATGAAACAAAG (SEQ ID NO: 18) This study th012 CATTTCCAACTAGAAGGGCTG (SEQ ID NO: 19) This study ds171 ATTTATATTTATTATTGGAGGTTCAATGAAAACAATTATCACATTCA (SEQ ID NO: This study 20) ds172 ATTGGGAAGAGTTACATATTAGAAATTAAAAGAAAAAGCGTTGAATATC (SEQ ID This study NO: 21) ds87 AGCGTTTCCTCCGTCTTTTG (SEQ ID NO: 22) This study ds88 CAAAAGACGGAGGAAACGCTTCGAGGAATATTATGAAACAAAG (SEQ ID NO: 23) This study Kan484.F GTTTGATTTTTAATGGATAATGTG (SEQ ID NO: 24) (Johnsborg et al 2008) RpsL41.R CTTTCCTTATGCTTTTGGAC (SEQ ID NO: 25) (Johnsborg et al 2008) khb31 ATAACAAATCCAGTAGCTTTGG (SEQ ID NO: 26) (Berg et al 2011) khb33 TTTCTAATATGTAACTCTTCCCAAT (SEQ ID NO: 27) (Berg et al 2011) khb34 CATCGGAACCTATACTCTTTTAG (SEQ ID NO: 28) (Berg et al 2011) khb36 TGAACCTCCAATAATAAATATAAAT (SEQ ID NO: 29) (Berg et al 2011) 466p1 GGTATTCTTGTCCTCGTAGCTGAATTTGGCCACTTTTATTTTGC (SEQ ID This study NO: 30) 467p2 GCAAAATAAAAGTGGCCAAATTCAGCTACGAGGACAAGAATACC (SEQ ID This study NO: 31) 468p1 ATTCTTGTCCTCGTACATGCATTTGGCCACTTTTATTTTGCAAAAC (SEQ This study ID NO: 32) 469p2 GTTTTGCAAAATAAAAGTGGCCAAATGCATGTACGAGGACAAGAAT (SEQ This study ID NO: 33) 470p1 GTACATGAATTTGGCGCTTTTTATTTTGCAAAACGAGC (SEQ ID NO: 34) This study 471p2 GCTCGTTTTGCAAAATAAAAAGCGCCAAATTCATGTAC (SEQ ID NO: 35) This study 472-p1 GAATTTGGCCACTTTGCTTTTGCAAAACGAGC (SEQ ID NO: 36) This study 473-p2 GCTCGTTTTGCAAAAGCAAAGTGGCCAAATTC (SEQ ID NO: 37) This study 474p1 ATTCTTGTCCTCGTAGCTGCATTTGGCGCCTTTTATTTTGCAAAACGAGC This study (SEQ ID NO: 38) 475p2 GCTCGTTTTGCAAAATAAAAGGCGCCAAATGCAGCTACGAGGACAAGAAT This study (SEQ ID NO: 39) (Johnsborg et al., 2008, Mol. Microbiol. 69: 245-253; Berg et al., 2011, J. Bacteriol., 193: 5207-5215)

(8) TABLE-US-00003 TABLE 2 S. pneumoniae strains used in the experiments. Strain Genotype/relevant features.sup.a Reference/source RH1 S. pneumoniae, R704, but ebg::spc; Johnsborg et al., 2008 Ery.sup.r Spc.sup.r RH426 S. pneumoniae, contains the Janus Johnsborg et al., 2008 cassette; Ery.sup.r Kan.sup.r SPH131 S. pneumoniae, contains the ComRS Berg et al., 2011 system, Janus cassette is placed behind P.sub.comX; Ery.sup.r Kan.sup.r ds218 S. pneumoniae SPH131 but Δjanus:: This study rseP from E. faecalis LMGT5833 ds219 S. pneumoniae ds218 but This study ΔrseP.sub.wt::janus ds220 S. pneumoniae ds219 but This study Δjanus ds221 S. pneumoniae ds220 but This study Δ rseP::janus ms1 S. pneumoniae ds221 but This study Δjanus::EF-rseP-H18>A ms2 S. pneumoniae ds221 but This study Δjanus::EF-rseP-E19>A ms3 S. pneumoniae ds221 but This study Δjanus::EF-rseP-H22>A ms4 S. pneumoniae ds221 but This study Δjanus::EF-rseP-Y24>A ms5 S. pneumoniae ds221 but This study Δjanus::EF-rseP-HExxH>AAxxA .sup.aCm, chloramphenicol; Ery, erythromycin; Spc, spectinomycin; Kan, kanamycin; Sm, streptomycin.

(9) TABLE-US-00004 TABLE 3 MIC 50 values (nM) of LsbB family bacteriocins against different bacterial species. Indicator strain LsbB EntK1 Ent- EJ97 Staphylococcus. aureus LMGT 3310 >7000 >5500 590 S. aureus LMGT 3260 >7000 >5500 2300 S. aureus LMGT 3266 >7000 >5500 1175 S. aureus LMGT 3258 >7000 >5500 >4700 S. aureus LMGT 3289 >7000 >5500 >4700 S. aureus LMGT 3272 >7000 >5500 1175 S. epidermidis LMGT 3026 >7000 >5500 >4700 Enterococcus. faecalis LMGT 3199 >7000 5500 145 E. faecalis LMGT 3571 >7000 >5500 295 E. faecalis LMGT 3572 >7000 >5500 145 E. faecalis LMGT 3330 >7000 >5500 145 E. faecalis LMGT 3143 >7000 >5500 145 E. faecalis LMGT 3359 >7000 >5500 145 E. faecalis LMGT 3200 >7000 >5500 295 E. faecalis LMGT 3386 >7000 >5500 295 E. faecalis LMGT 3370 >7000 5500 145 E. faecalis LMGT 3567 >7000 >5500 295 E. faecalis LMGT 3358 >7000 2700 75 E. faecium LMGT 3599 >7000 85 145 E. faecium LMGT 2787 >7000 20 145 E. faecium LMGT 3193 >7000 85 145 E. faecium LMGT 3110 >7000 45 295 E. faecium LMGT 3104 >7000 10 145 E. faecium LMGT 3313 >7000 85 145 E. faecium LMGT 3192 >7000 20 295 E. faecium LMGT 2769 >7000 45 145 E. hirae LMGT 3236 >7000 45 75 Lactococcus garvieae LMGT 1546 >7000 1370 295 L. garvieae LMGT 2217 >7000 340 295 L. curvatus LMGT 2355 >7000 5500 145 L. lactis IL1403 0.5 170 37 L. lactis LMGT 2233 >7000 >5500 145 L. lactis LMGT 2084 >7000 >5500 295 L. lactis LMGT 2095 >7000 >5500 145 L. cremoris LMGT 2057 >7000 >5500 145 Lactobacillus sakei LMGT 2334 >7000 >5500 >4700 Bacillus cereus LMGT 2805 >7000 >5500 >4700 B. cereus LMGT 3025 >7000 >5500 2300 B. cereus LMGT 2731 >7000 >5500 >4700 B. cercus LMGT 2711 >7000 2750 >4700 B. cercus LMGT 2735 >7000 >5500 >4700 Listeria monocytogenes LMGT 2651 >7000 >5500 2300 L. monocytogenes LMGT 2605 >7000 >5500 1175

(10) TABLE-US-00005 TABLE 4 MIC 50 values (nM) of EntK1 and EntEJ97 against E. faecium strains isolated from blood in patients from different hospitals in Europe. E. faecium indicator strain EntK1 EntEJ97 AH137 10 75 UW6920 45 145 P032 inv-4 20 37 602589 45 145 P040 INV-25 20 75 A11 EFH2/00 45 75 130409015079* 20 37 928379 A* 20 75 PO1402593* 20 75 14-597963* 45 145 *Vancomycin-resistant strains

(11) TABLE-US-00006 TABLE 5 Sensitivity of S. pneumonia clones to EntEJ97, EntK1 and LsbB. Strain MIC (nM)* S. pneumoniae Mutation EntEJ97 EntK1 LsbB ds220 non-induced WT rseP >4500 >5500 >7000 ds220 induced WT rseP 18 700 >7000 ms1 H18>A 300 >5500 >7000 ms2 E19>A 300 >5500 >7000 ms3 H22>A 300 >5500 >7000 ms4 Y24>A 18 700 >7000 ms5 HExxH>AAxxA 600 >5500 >7000 ds 221 no rseP >4500 >5500 >7000 *Bacteriocins were active only when rseP (WT or mutated) was induced with ComS (2 μM). Without ComS the cells were resistant to the bacteriocins. ComS was not toxic to the cells even at 20 μM.

Discussion

(12) Structural analyses by CD spectroscopy showed that EntK1 was unstructured in water but became structured when exposed to membrane-mimicking environments (DPC-micelles or TFE).

(13) The structure of EntK1 in 50% TFE is very similar to that of LsbB (FIG. 1). Both bacteriocins have an N-terminal part mostly composed of an amphiphilic α-helical motif, and an unstructured C-terminal half. EntK1's α-helix is longer than the one of LsbB, being 16-19 residues, versus 13-15 residues in LsbB. A closer look at the α-helices of EntK1 and LsbB showed that both are amphiphilic with the basic amino acids along one side of the helix and nonpolar residues along the other side (data not shown). The α-helical part of the bacteriocins is believed to be involved in a pore-formation mechanism, where the hydrophobic part is facing the hydrophobic core of the membrane or a protein (receptor), while the hydrophilic part is facing the pore to cause cellular leakage. The C-terminal unstructured parts of EntK1 and LsbB are about the same lengths, 10-13 residues and more similar in sequence. Structure prediction servers (PONDR® VL-XT, JPred 4) indicated that EntEJ97 also contains an α-helix from Lys10 to Gly 35 with an unstructured C-terminal tail of 10 residues, which corresponds with our data on LsbB and EntK1 structures.
Besides EntK1 and LsbB, structures of three other leaderless bacteriocins have been obtained so far—Enterocin 7, Lacticin Q and Aureocin A53. Unlike EntK1 and LsbB these bacteriocins are structured in water. Enterocin 7—a two-peptide bacteriocin, where both peptides have a similar fold, consists of N-terminal, middle and C-terminal helices (Lohans et al., 2013, Biochemistry, 52:3987-3994). Lacticin Q and aureocin A53 both consist of 4 helices surrounding a hydrophobic core (Acedo et al., 2016, Biochemistry, 55:733-742). The structures of enterocin 7, lacticin Q and aureocin A53 resemble the structure of circular bacteriocins, which are also highly structured in aqueous solutions. EntK1 and LsbB structures are very different from those bacteriocins by being unstructured in water and containing only one single helix (FIG. 1).
EntEJ97 and EntK1 employ the enterococcal RseP (in addition to the lactococcal one) as receptor. The lactococcal Zn-dependent metallopeptidase RseP has been shown to be responsible for the sensitivity to LsbB in L. lactis IL1403 (Uzelac et al., 2013, J. Bacteriol., 195:5614-5621). According to our study, this conclusion is based on the fact that mutants of E. faecalis LMGT3358/E. faecium LMGT2787 that are highly resistant to EntEJ97/EntK1, contain frameshift mutations in rseP, and, more conclusively, heterologous expression of the enterococcal rseP rendered resistant pneumococcal cells sensitive to these bacteriocins (Table 5). However, at least in the case of E. faecalis LMGT3358, EntEJ97 LR mutants had intact rseP. Both types of resistance were specific to EntEJ97 and EntK1 because all resistant cells were sensitive to the non-related bacteriocins BHT-B (leaderless type) and garvicin ML (circular type). Furthermore, both (high and low) types of resistance share one common feature—they emerge at the standard growth temperature of 30° C. but not at the elevated 45° C., indicating that both resistance types are linked to the stress response (FIG. 2).
RseP deletion mutants have shown increased susceptibility to lysozyme and other treatments compared to the susceptibility of the WT strain (Varahan et al., 2103, J Bacteriol., 195:3125-3134) and RseP plays a role in the bacterial stress response (Kim, 2015, J. Microbiol., 53:306-310, Alba et al., 2002, Genes Dev., 16:2156-2168). In our study, enterococcal EntK1 and EntEJ97 resistant mutants with frame shift rseP obtained at 30° C. could not grow at 45° C., while the WT cells grew well. However, in addition, resistance types in which no mutations in resP existed were also susceptible to stress.
The putative active site of enterococcal RseP proteases is located near the N—terminal part of the molecule and contains a consensus sequence motif HExxH, in which the two histidine residues are thought to coordinate a zinc atom together with a conserved glutamate residue. E. coli RseP active site is located ˜14 Å into the lipid membrane surface (Feng et al., 2007, Science, 318:1608-1612). According to our results, replacing the conserved residues with alanines in E. faecalis RseP increased cells resistance to EntEJ97 and EntK1 especially when all three conserved residues were replaced. However, even this version of RseP did not give total resistance to EntEJ97 unlike the HR mutants of E. faecalis, meaning that the active site of RseP is only partly and/or indirectly involved in receptor/target recognition by the EntEJ97/EntK1 bacteriocins. Our results strongly suggest that the proteolytic active site of RseP is somehow involved in the receptor function and thereby the bacteriocin activity.
The study of EntK1 and EntEJ97 inhibition spectrum showed an interesting detail—EntK1 had generally narrower antibacterial spectrum than EntEJ97. Against E. faecium strains EntK1 was much more potent (i.e., lower MIC values) than EntEJ97 (Table 3, 4). EntEJ97 was equally active against E. faecalis and E. faecium (Table 3, 4). However, multiple sequence alignment analysis (using Clustal Omega) of many RseP (30 from E. faecalis and 30 from E. faecium) did not show any pronounced differences between the two RseP groups (data not shown). Thus, the properties of RseP that define the different levels of sensitivity toward EntEJ97 and EntK1 await further investigation.

Example 2: Preparation of EntK1 and EntEJ97 Variants and their Effects on Various Bacteria

Materials and Methods

(14) Bacterial strains, growth conditions, bacteriocins and antimicrobial assays.

(15) All the strains (see below) used in minimal inhibitory concentration (MIC) assays (Ovchinnikov et al., 2017, Front Microbiol., 8, p774, doi:10.3389/fmicb.2017.00774) were grown in BHI medium (Oxoid) at 30° C. without shaking. The tested strains were obtained from the LMG collection. EntK1 (SEQ ID NO:1), EntEJ97 (SEQ ID NO:2), GarKS, EntEJ97short (SEQ ID NO:3), K1-EJ (also known as EntK1J97) hybrid (SEQ ID NO:4) and EJ-K1 (SEQ ID NO:5) hybrid and were synthesized by Pepmic Co., LTD, China with 98-99% purity. NisinZ and micrococcin P1 were purified from source. Synthesized peptides were solubilized to concentrations of 10.0-0.1 mg/ml in 0.1% (vol/vol) trifluoroacetic acid and stored at −20° C. until use.
Garvicin KS is made up of 3 peptides:

(16) TABLE-US-00007 (SEQ ID NO: 43) MGAIIKAGAKIVGKGVLGGGASWLGWNVGEKIWK (SEQ ID NO: 44) MGAIIKAGAKIIGKGLLGGAAGGATYGGLKKIFG; and (SEQ ID NO: 45) MGAIIKAGAKIVGKGALTGGGVWLAEKLFGGK
NisinZ has the sequence: ITSISLCTPGCKTGALMGCNMKTATCNCSIHVSK (SEQ ID NO:46)
Micrococcin P1 has the structure:

(17) ##STR00001##

(18) Bacteriocin activity was determined using a microtitre plate assay. The plates were incubated at 30° C. for 8 h and the growth was measured spectrophotometrically at 600 nm (A.sub.600) with 15 min intervals using SPECTROstarNano (BMG LABTECH, Germany). The MIC was defined as the bacteriocin concentration that inhibited the growth of the indicator strain by at least 50% in 200 μl culture (i.e., 50% of the turbidity of the control culture without bacteriocin).

Results

(19) The MIC values that were obtained for EntK1, EntEJ97 and the hybrid peptides against selected pathogenic bacteria isolates are shown in Tables 6A and B. The results show that EntEJ97, EntK1 and their variants EntEJ97short, K1-EJ hybrid and EJ-K1 hybrid are effective against S. pseudointermedius, S. haemolyticus, E. hirae, E. faecium, E. faecalis, as well as S. aureus and Listeria. Particular efficacy was observed against S. pseudointermedius (for EntEJ97 short) and S. haemolyticus (EntEJ97short and K1-EJ hybrid) compared to other bacteriocins (Table 6B). EntEJ97short was particularly effective against E. hirae, E. faecalis and S. pseudointermedius and K1-EJ proved particularly effective against E. hirae, E. faecium, E. faecalis and S. haemolyticus.

(20) TABLE-US-00008 TABLE 6 MIC values for EntK1, EntEJ97 and hybrid peptides against selected pathogenic bacteria isolates A μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml Sa ATCC μg/ml μg/ml μg/ml μg/ml Bacteriocin SP 4012 SP 4013 SP 4014 SH 4015 SH 4016 KAVA 33591 Sa 3328 Sa 3242 Sa3324 Sa3323 GarKS 6.25 6.25 6.25 6.25 6.25 6.25-12.5 12.5 3.1 3.1 6.25 6.25 NisinZ 0.4 0.4 0.4 1.6-3.1 1.6-3.1 1.6-3.1 0.8 0.8 3.1 1.6 6.25 EntEJ97 12.5 12.5 12.5 12.5 12.5 >50 >50 6.25 6.25 12.5 6.25 EntEJ97short 3.1 3.1 1.6-3.1 1.6-3.1 1.6-3.1 >50 >50 3.1 12.5 6.25 3.1-6.25 K1-EJ hybrid 25 12.5 12.5 0.8 0.8-1.6 >50 >50 3.1 3.1 6.25 3.2-6.25 EJ-K1 hybrid >50 >50 >50 50 50 >50 >50 6.25 12.5 12.5 3.2-6.25 micrococcinP1 0.4 0.4 0.8 1.6 1.6 >12.5 0.4 0.1 0.2 0.1 0.1  B Bacteriocin E. hirae (4) E. faecium (2) E. faecalis (2) S. pseudointermedius (3) S. aureus (5) Listeria spp. (3) EJ97 0.4-1.6  0.8-1.6 3.1 12.5 6.25->50 12.5-25 EJshort 0.1-0.4  12.5-25 1.6  1.6-31  3.1->50 >50 K1-EJ hybrid 0.1-0.4 <0.05-0.4 1.6-3.1 12.5-25  3.1->50 6.25-25 EJ-K1 hybrid 3.2-6.3 3.15 25-50 50   6.25->50  12.5->50 K1 0.2-0.8 <0.02-0.4 >25 ND >25 >50 SP = S. pseudointermedius; SH = S. haemolyticus; KAVA = micrococcin P1 (used as control); Sa = S. aureus ND: Not determined