Combination of bactericidal agent with a lysosomotropic alkalinising agent for the treatment of a bacterial infection

Abstract

The present invention relates to the field of medicine, specifically the field of bacterial infection and treatment thereof.

Claims

1. A method of treatment of an intracellular and/or persistent Staphylococcus infection in a subject in need thereof, comprising: a) administration of an effective amount of a first agent, wherein said first agent increases the intracellular pH of a host cell and/or of an intracellular compartment of a host cell; and b) administration of an effective amount of a second agent, wherein said second agent is a bactericidal agent, wherein the first agent is a lysosomotropic alkalinizing agent and enhances the activity of the second agent, and wherein the second agent is a bacterial lysin or autolysin, or a bacteriophage lysin.

2. The method of treatment according to claim 1, wherein the Staphylococcus infection is an S. aureus infection.

3. The method of treatment according to claim 1, wherein the host cell is a eukaryotic host cell within the subject in need of treatment and/or wherein the intracellular compartment is a phagolysosome.

4. The method of treatment according to claim 1, wherein the first agent is selected from the group consisting of chloroquine, bafilomycin A1 and ammonium chloride.

5. The method of treatment according claim 1, wherein the second agent is a bactericidal agent capable of entering the host cell and/or the intracellular compartment of the host cell.

6. The method of treatment according to claim 1, wherein the second agent further comprises a protein transduction domain.

7. The method of treatment according to claim 1, wherein the second agent further comprises an antimicrobial peptide.

8. The method of treatment according to claim 6, wherein the protein transduction domain is selected from the group consisting of SEQ ID NO: 12-25.

9. The method of treatment according to claim 7, wherein the antimicrobial peptide is selected from the group consisting of SEQ ID NO: 70-90.

10. The method of treatment according to claim 1, wherein the second agent comprises a chimeric bactericidal polypeptide having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 27-47.

Description

FIGURE LEGENDS

(1) FIG. 1.

(2) Induction of S. aureus SCVs and non-replicating persisters by low pH and bacterial regrowth through pH increase.

(3) MRSA S. aureus strains 6850 (a), JE2 (b) and Cowan (c) were inoculated in media buffered at different pH as indicated. Colony phenotypes of viable bacteria were determined and the percentage of SCVs plotted over time. Three independent experiments done in triplicates are presented as mean±SEM.

(4) Low pH-induced MRSA S. aureus strains 6850 (d), JE2 (e) and Cowan (f) persisters were re-inoculated in various buffered pH media as indicated and growth was followed over time. Three independent experiments done in triplicates presented as mean±SEM.

(5) FIG. 2

(6) Intracellular Persistence of S. aureus within Phagolysosomes.

(7) A549 cells were infected with S. aureus Cowan and extracellular bacteria were killed by addition of flucloxacillin. The number (a) and phenotype (b) of viable intracellular persisting bacteria were determined at indicated time points. Data are pooled from two experiments performed in triplicates, mean±SEM.

(8) FIG. 3

(9) Reduction of S. aureus persisters through phagolysosome alkalinization. S. aureus Cowan-infected A549 cells were treated with flucloxacillin alone (control) or supplemented with lysosomotropic alkalinizing agents (chloroquine (a), bafilomycin A1 (b) and ammonium chloride (c)). Colony phenotypes of viable intracellular persisting bacteria were determined and enumerated at indicated time points. Data were pooled from three independent experiments done in triplicates, mean±SEM. Two-way ANOVA found the factors time and treatment to be significant (p-value<0.01).

(10) FIG. 4

(11) Reduction of S. aureus Persisters by Chloroquine in an In Vivo Infection Model.

(12) Mice were infected with S. aureus Cowan intraperitoneally. Six hours and two days post-infection mice were treated with 1 mg flucloxacillin and 0.2 mg chloroquine (+CQ). Mice treated with flucloxacillin only served as control (−CQ). †, sacrifice (a). Colony phenotypes of bacteria recovered from target tissues (b), peripheral blood and peritoneal lavage (c) were determined and enumerated. Each point represents one mouse. Horizontal bars indicate mean±SEM, n=11 mice per group. PL, peritoneal lavage. Two-way ANOVA found the factor treatment to be significant (p-value<0.01).

(13) FIG. 5

(14) Intracellular targeting of S. aureus in osteosarcoma cells by mixtures of engineered endolysins. (A) Cells infected for 3 h with S. aureus Newman (MOI 0.1) treated by endolysin mixtures for 1 h and 4 h. (B) Cells infected for 3 h with S. aureus Newman (MOI 0.1) treated by endolysin mixtures for 1 h and 4 h in the presence of 20 μM chloroquine. (C) Cells infected for 3 h with S. aureus Cowan (MOI 0.01) treated by endolysin mixtures for 1 h and 4 h. (D) Cells infected for 3 h with S. aureus Cowan (MOI 0.01) treated by endolysin mixtures for 1 h and 4 h in the presence of 20 μM chloroquine.

(15) FIG. 6

(16) Intracellular targeting of S. aureus in osteosarcoma cells (MOI 1.0) by mixtures of engineered endolysins. (A) Cells infected for 24 h with S. aureus Newman treated by endolysin mixtures for 4 h. (B) Cells infected for 24 h with S. aureus Newman treated by endolysin mixtures for 4 h in the presence of 20 μM chloroquine. (C) Cells infected for 72 h with S. aureus Cowan treated by endolysin mixtures for 4 h. (D) Cells infected for 72 h with S. aureus Cowan treated by endolysin mixtures for 4 h in the presence of 20 μM chloroquine.

(17) FIG. 7

(18) Activity of bactericidal agents according to the invention comprising a functional enzymatic domain from a cell wall lytic enzyme and further comprising a protein transduction domain on the N-terminal side of the molecule: (A) R9-CHAP-CBD, (B) R9-M23-CBD, (C) TAT-Ami-CBD, (D) TAT-CHAP-CBD, and (E) TAT-M23-CBD.

EXAMPLES

(19) The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

(20) Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2′.sup.d edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

Example 1

(21) Induction of Staphylococcus aureus persisters by low pH, awakening by phagolysosomal alkalinization and effective treatment by phagolysosomal alkalinization combined with a bactericidal agent.

(22) 1.1 pH-Dependent Induction of S. aureus Small Colony Variants (SCVs)

(23) The well-defined MSSA strains 6850 and Cowan and MRSA strain JE2 were grown in 4.0, 5.5, 6.5 and 7.4 pH media, mimicking the pH found in physiologic sites such as lysosomes, abscesses and blood. Directly after inoculation S. aureus showed a large colony phenotype, independent of the pH. The frequency of SCVs significantly increased over time in pH 4.0 growth medium and reached 39% (JE2 and 6850) and 28% (Cowan) after five days. In contrast pH 7.4 growth medium sustained SCVs below 2% in all strains tested (FIG. 1a-c). An intermediate percentage of SCVs was found at pH 5.5 and 6.5. Thus, we showed a clear correlation between low pH and SCV formation. This method permitted easy and controlled formation of unstable, non-genetically modified SCVs in various S. aureus strains.

(24) 1.2 Induction of Non-Replicating S. aureus by Low pH

(25) The pH-dependent growth of S. aureus Cowan was followed over time by labeling the bacterial cell wall with fluorescent cell wall binding domains (CBDs). Immediately after staining, bacterial cell walls were fully labeled. After three days in pH 4.0 growth medium, the majority of bacteria still exhibited fluorescent cell walls, consistent with absent bacterial replication. In contrast, bacteria grown at pH 7.4 proliferated extensively, demonstrated by highly fragmented and 89 reduced fluorescent cell wall labeling. Scanning electron microscopy (SEM) of bacteria originating from a small colony, obtained under low-pH conditions, showed impaired cell division resulting in rod-shaped S. aureus, in contrast to large colony bacteria showing normal cell division.

(26) 1.3 Growth Resumption of Low pH-Adapted S. aureus Persisters

(27) Our findings indicated that both, SCVs and non-replicating persisters, are induced by low pH. In clinics, the presence of these persisting bacteria correlates with increased recurrence of infection which implies that bacteria revert to a highly virulent and fast-growing form 14. We therefore tested in vitro whether low pH-induced SCVs and/or non-replicating persisters can restore normal growth in neutral pH. Non-replicating S. aureus persisters were induced and kept at pH 4.0 for three days and then transferred to pH 4.0, 5.5, 6.5 or 7.4 growth media. Bacteria in pH 7.4 and 6.5 resumed growth after approximately 12 hours (FIG. 1d-f) whereas bacteria kept at low pHs (<6.5) remained in a nonproliferating state (FIG. 1d-f). These data suggest that both persisting phenotypes were reversible adaptions to low pH as supported by the capability of regrowth upon neutralization of pH.

(28) 1.4 Intracellular Induction of S. aureus SCVs

(29) We investigated whether internalized S. aureus exhibited a SCV phenotype. MSSA strain Cowan is highly invasive, but not cytotoxic which allowed maintaining this strain intracellularly over several days in the lung epithelial cell line A549. Extracellular bacteria were killed by adding a high dose of flucloxacillin to the infected host cells. Flucloxacillin is typically used to treat S. aureus endocarditis in patients. Absence of extracellular bacteria was confirmed by sterility of culture supernatants. Host cells were lysed to release intracellular bacteria and colony counts, as well as colony phenotypes, were determined at various time points. Five hours after infection, 0.2% of all viable intracellular bacteria had a SCV phenotype (FIG. 2a-b). The number of viable intracellular persisting bacteria decreased during the course of infection while the frequency of SCVs increased and reached 5.6% after seven days.

(30) 1.5 Phagolysosomal Localization of Persisting S. aureus

(31) Our data indicated that acidity favored SCV formation, suggesting that the acidic phagolysosomal milieu may have the same effect. A549 cells were infected with S. aureus Cowan. Intracellular bacteria were localized within LAMP-2 antibody positive vesicles, visualized by fluorescence microscopy. LAMP-2 (CD 107b) is highly expressed in phagolysosomes, suggesting that intracellular persisting S. aureus predominantly resided within phagolysosomes.

(32) 1.6 Reduction of S. aureus SCVs Through Phagolysosomal Alkalinization

(33) Since low pH induced SCVs and/or non-replicating S. aureus and medium pH neutralization resulted in bacterial regrowth, we treated infected host cells with lysosomotropic alkalinizing agents. Chloroquine, bafilomycin A1 or ammonium chloride all neutralized the phagolysosomal pH. Host cells treated with lysosomotropic alkalinizing agents exhibited significantly lower percentages of SCVs seven days after infection (FIG. 3). No differences in total colony counts between control and treated cells were observed. Lysosomotropic alkalinizing agents did not inhibit bacterial growth at the concentrations used. No significant differences in host cell viability were observed after treatment with the alkalinizing agents.

(34) 1.7 Growth Resumption of Intra-Phagolysosomal Persisting S. aureus by Chloroquine Treatment Resulting in Reduction of SCV Percentages in Cells and in Mice

(35) Growth resumption of S. aureus persisters through treatment of host cells with chloroquine was assessed. Fluorescence microscopy revealed that S. aureus localized within phagolysosomes in both, control and chloroquine-treated host cells, three days post-infection. We observed no dividing bacteria in infected host cells without chloroquine treatment. However, chloroquine facilitated bacterial cell division as assessed by transmission electron microscopy (TEM).

(36) Mice infected with S. aureus Cowan were treated with flucloxacillin alone (control), or in combination with chloroquine (FIG. 5a). Chloroquine treatment significantly reduced the frequency of SCVs in mice in various organs (FIG. 5b) and compartments (FIG. 5c). Absolute bacterial numbers were comparable, independent of chloroquine treatment

(37) 1.8 Discussion

(38) This study showed that low pH, as found in abscesses and within lysosomes, induced the persisting S. aureus subpopulations SCVs and non-replicating persisters. Raising pH in the culture medium or within the phagolysosomes using alkalinizing agents reverted S. aureus to normal growth. SCV formation was shown to be triggered by antibiotic pressure. In addition, extreme environmental stresses such as prolonged exposure to low temperature, very acidic or alkaline environments, or osmotic stress may trigger SCV and/or persister formation in S. aureus and coagulase-negative staphylococci. These observations, together with our new findings, show how multiple stimuli lead to S. aureus persister formation. Localization within the host cell shields S. aureus from commonly used antibiotics such as the extracellularly active beta-lactams with poor cell penetration In addition, the low intraphagolysosomal pH renders antibiotics with intracellular activity such as clindamycin and fluoroquinolones less active. We found that the addition of lysosomotropic alkalinizing agents to the usually prescribed antibiotics such as flucloxacillin reduced the frequency of S. aureus SCVs in vitro as well as in vivo. We thus identified a simple strategy to circumvent the host dependent component of S.aureus persister formation. In clinical settings, the presence of SCVs in osteomyelitis and device-related infections has been associated with increased relapse rates, despite administration of antibiotics. Bacteria adapt to antibiotic stress by SCV and/or persister formation. We now showed that S. aureus SCVs and non-replicating persisters retained the ability to revert to a highly virulent and fast-growing form. The capacity to revert to fast growth (phenotype switching) results in relapsing infection. In addition, it renders identification of SCVs difficult. Further aggravating the SCV problem in clinics is underestimation of SCVs in clinical microbiology laboratories, since they form tiny and thus difficult to detect colonies which are easily overgrown by their fast growing counterparts. We postulate that the addition of alkalinizing agents to the usually prescribed antibiotics will reduce the frequency of SCVs and could therefore reduce recurrence rates in the future. Persisting bacteria are not unique to S. aureus but have also been described to occur in various other human pathogens, such as Salmonella spp., Pseudomonas aeruginosa, Escherichia coli and Mycobacterium tuberculosis. In addition to low pH, bacterial persisters can arise due to mechanisms that include the toxin-antitoxin systems. Accordingly, activation of a SOS response (ppGpp) in response to DNA damage due to oxidative stress results in decreased ATP levels. This leads to the shutdown of metabolism resulting in reduced growth. Various toxin-antitoxin modules are activated by acidification and/or nutrient starvation in Salmonella, causing formation of persisters. In accordance with our findings, Salmonella persister formation has been reported in macrophages triggered by the acidic and nutritionally poor environment of the Salmonella-containing vacuole that was reversible by addition of bafilomycin A1 44. In contrast to bafilomycin A1, chloroquine is routinely used in patients to treat malaria as well as some rheumatic diseases. Phagolysosomal pH neutralization with chloroquine may therefore provide a novel therapeutic eradication strategy against intracellular persisting staphylococcal reservoirs.

Example 2

(39) Effective treatment of Staphylococcus Aureus Persisters by Phagolysosomal Alkalinization Combined with a Bactericidal Agent Comprising a Protein Transduction Domain.

(40) The bactericidal agents according to the invention with a protein transduction domain for efficient delivery into the infected host cell with a sequence selected from the group consisting of SEQ ID NO: 30-47 are used for combination with phagolysosomal pH neutralization for treatment of intracellular S. aureus infection in vitro and in vivo. The treatment results in effective treatment of the intracellular S. aureus infection, with some variety in the efficiency depending on the specific bactericidal agent according to the invention used.

Example 3

(41) Effective Treatment of Intracellular Infection with Staphylococcus aureus.

(42) The bactericidal agents according to the invention with a protein transduction domain for efficient delivery into the infected host cell with a sequence selected from the group consisting of SEQ ID NO: 27-47 were used for treatment of intracellular S. aureus infection, either combined or not with phagolysosomal pH neutralization.

(43) Method Intracellular S. aureus Killing Assay.

(44) S. aureus was grown in LB broth at 37° C., shaking at 220 rpm, overnight. The overnight culture was diluted in fresh LB (1:10) and grown further for 2 h. Then, the bacteria were centrifuged, the pellet was washed with PBS and the culture was re-suspended in PBS with the OD600 adjusted to 0.4 (c.a. 2×10.sup.8 CFU/mL). Bacterial cells were sonicated prior to infection (SONOPULS HD 2070) for 1 minute with 1 second pulses at 40% of the power. MG-63 osteosarcoma cells were grown in 12-well dishes with the amount of 5×10.sup.5 cells/well in 1 mL EMEM culture media with 10% fetal bovine serum (FBS) for 24 h prior infection. Then, the cells were infected with S. aureus Newman and Cowan at the following conditions: (A) S. aureus Newman at MOI of 0.1 for 3 h, (B) S. aureus Newman at MOI of 1.0 for 24 h, and (C) S. aureus Cowan at MOI of 1.0 for 72 h. The plates were centrifuged at 1200 rpm for 5 min and incubated at 37° C. with the flush of CO2. After invasion, eukaryotic cells were washed 3× with PBS to remove remaining extracellular S. aureus and exposed to floxacillin (1 mg/mL) for 2 h to kill any left non-internalized bacteria. For each experiment (A, B, and C) one part of the samples was exposed to chloroquine treatment (20 μM) to increase lysosomal pH and evaluate its effect on further treatment with endolysins. The supernatant from the antibiotic-treated cells was plated to check for floxacillin treatment efficiency. Then, the eukaryotic cells were again washed with PBS (3×) to remove dead bacteria and subjected to endolysin treatment. The composition of applied endolysin preparations is summarized in Table 2. Eukaryotic cells were treated with 1 mL of 1 μM endolysin preparation (diluted in EMEM supplemented with 1 mg/mL floxacillin and +/−20 μM chloroquine) for (A) 1 h and 4 h, (B and C) 4 h. The control was treated with 1 mL of 1 mg/mL floxacillin, +/−20 μM chloroquine in EMEM. The cultures were then washed 3× with PBS and examined under microscope to determine if there had been osteoblast cell lysis. Next, they were trypsinized (Trypsin-EDTA 0.25%, Gibco®) and lysed with 800 μL 0.1% Triton X-100. The cell lysate was subjected to serial dilution plating on LB and overnight incubation at 37° C.

(45) TABLE-US-00002 TABLE 2 Composition of the endolysin mixtures used for intracellular eradication of S. aureus. Concentration of each Components of the endolysin mixture Ratio component CHAP-CBD + M23-CBD 1:1 500 nM:500 nM CHAP-CBD-TAT + M23-CBD-TAT 1:1 500 nM:500 nM CHAP-CBD-R9 + M23-CBD-R9 1:1 500 nM:500 nM CHAP-CBD-Penetratin + M23-CBD- 1:1 500 nM:500 nM Penetratin CHAP-TAT + M23-TAT + Ami-TAT 1:1:1 333 nM:333 nM:333 nM CHAP-R9 + M23-R9 + Ami-R9 1:1:1 333 nM:333 nM:333 nM CHAP-Penetratin + M23- 1:1:1 333 nM:333 nM:333 nM Penetratin + Ami-Penetratin
Results

(46) Successful expression and purification of all protein constructs was achieved. The summary of all expressed endolysin constructs with the corresponding molecular weights and concentrations is shown in Table 3. Relatively high concentrations were obtained for most of the endolysin constructs, implying that a correct protein expression and purification strategy was used.

(47) TABLE-US-00003 TABLE 3 Summary of all expressed endolysin constructs with the corresponding molecular weight and concentration SEQ ID Mol. Weight Concentration Concentration NO: Protein (KDa) (mg/ml) (μM) 27 M23-CBD 29.973 3.82 127.4 28 CHAP-CBD 33.088 2.36 71.33 29 Ami-CBD 35.385 3.30 93.26 44 M23-CBD-TAT 31.916 1.13 34.30 38 CHAP-CBD-TAT 35.031 1.85 52.81 32 Ami-CBD-TAT 37.328 0.40 10.71 43 M23-CBD-R9 31.620 0.45 14.23 37 CHAP-CBD-R9 34.736 1.05 30.20 31 Ami-CBD-R9 37.033 0.30 8.10 42 M23-CBD- 32.444 0.56 17.26 Penetratin 36 CHAP-CBD- 35.559 2.03 57.08 Penetratin 30 Ami-CBD- 37.856 0.41 10.82 Penetratin 47 M23-TAT 17.485 1.26 72.00 41 CHAP-TAT 20.600 1.44 69.90 35 Ami-TAT 22.897 0.83 36.25 46 M23-R9 17.189 2.51 146.00 40 CHAP-R9 20.305 0.83 40.88 34 Ami-R9 22.601 0.76 33.63 45 M23-Penetratin 18.012 0.80 44.41 39 CHAP-Penetratin 21.128 0.73 34.55 33 Ami-Penetratin 23.424 0.96 41.00

(48) All expressed endolysins were effective in killing S. aureus Cowan in plate assay and in time-killing assay.

(49) Intracellular S. aureus Killing Assay

(50) The mixtures of endolysin constructs as listed in Table 2 were further tested for their bacterial killing efficacy in cell tissue cultures.

(51) In our model MG-63 osteosarcoma cells were used to mimic the condition of osteomyelitis. Cells were first infected for a certain time with the pathogen S. aureus Newman or Cowan and then treated with the endolysin mixture in presence or absence of chloroquine. We expected that increasing intracellular pH by application of chloroquine would create more favorable conditions for endolysin activity.

(52) Osteosarcoma cells were exposed to S. aureus Newman and Cowan for 3 h followed by 2 h floxacillin treatment to inactivate any non-internalized bacteria. Then, the tissue cells were treated for 1 h and 4 h with 1 μM of endolysin mixture with and without chloroquine. The results are shown in FIG. 5. FIG. 5A shows the result of treatment of S. aureus Newman by endolysin mixtures exclusively and FIG. 5B shows the result of a combination treatment with chloroquine. Clearly, 4 h treatment was more effective in both experimental settings. Moreover, endolysin treatment in the presence of chloroquine resulted in greater decrease in bacterial counts. Interestingly, mixture of the CPP-free endolysins showed a very good killing efficacy, which was not expected. It is, however, possible that the vast amount of positive charge associated with CBD attracts the enzyme to the negatively charged cell membrane and induces its intracellular translocation. Such a putative mechanism for CPP-free endolysin translocation is the key of the translocation of CPPs. All endolysin mixtures displayed similar killing properties in this assay, even though their activity differed substantially depending on the presence of CBD and type of CPP tag. It is likely that different constructs have different penetrating properties, depending on the CPP, EAD and CBD presence. For instance, EAD-CBD mixture has very high activity but might not be transported via the cell membrane as efficient as any CPP-containing variants. Whereas, EAD-CPP constructs do not have as high activity but are smaller and contain the transduction domain, which make their intracellular transport more efficient and their diffusivity better due to the lack of immobilization of the enzyme on the cell surface mediated by CBD binding. As a result, enzymes with different properties might have given similar results eventually. FIG. 5C and FIG. 5D show the results of the same experiment performed with a different strain of the pathogen: S. aureus Cowan. In this case, the treatment did not seem to be effective. For both samples, without chloroquine (FIG. 5C) and with chloroquine (FIG. 5D), the decrease in the viable counts is negligible. Only treatment with the mixture of CHAP-CBD-R9+M23-CBD-R9 in presence of chloroquine resulted in a significant eradication of the pathogen. It is known that S. aureus Cowan resides intracellularly in the lysosomes. It is very likely that intracellular localization of S. aureus Newman is different—it may just reside in the cytoplasm, hence the results of the two experiments are different. Moreover, the intracellular fate of CPP-tagged endolysin constructs is unknown at this point, therefore one may only speculate that endolysin constructs fused with the R9 tag are the only ones that accumulate in the lysosomes, hence they showed good killing properties against S. aureus Cowan.

(53) To simulate a real-life infection osteosarcoma tissue cells were infected with S. aureus for 24 h and 72 h before the treatment. Such a long infection time allowed the bacteria to settle intracellularly in their final destinations. Again, the cells were treated with the mixtures of endolysin constructs in presence and in absence of chloroquine. FIG. 6A and FIG. 6B show the results of the treatment of osteosarcoma cells infected for 24 h with S. aureus Newman in absence and in presence of chloroquine, respectively. FIG. 6C and FIG. 6D correspond to the results of treated cells infected for 72 h with S. aureus Cowan. In both cases treatment with endolysin mixtures seems effective, especially with the addition of chloroquine. The best result was obtained for the treatment of S. aureus Newman with EAD-CBD-R9 mixture in the presence of chloroquine, where ca. 60% of the pathogen was eradicated (FIG. 6B). Equally good results were achieved for S. aureus Cowan treated with the mixtures of EAD-CBD, EAD-CBD-TAT, EAD-CBD-R9 and EAD-CBD-Penetratin, where around 50% of the pathogen was killed. In general, CBD-containing endolysin constructs displayed better killing efficacy, including EAD-CBD mixture, not fused with the transduction domain.

(54) Altogether it was demonstrated that treatment of intracellular S. aureus by engineered endolysins together with chloroquine was more effective that without chloroquine. Application of such alkalizing agents likely enhances the activity of lytic enzymes intracellularly and the combinatorial treatment of endolysins with chloroquine could be a good alternative to conventional antibiotic therapy.

(55) In conclusion, this study showed the potential of CPP-fused endolysins in treatment of intracellular and extracellular S. aureus. Such a method of therapy could be used to treat patients with conditions like e.g. osteomyelitis, endocarditis, bacteremia or sepsis, as well as bovine mastitis in dairy cattle.

Example 4

(56) Several bactericidal agents according to the invention comprising a functional enzymatic domain from a cell wall lytic enzyme and further comprising a protein transduction domain on the N-terminal side of the molecule were prepared and analyzed according to methods as described elsewhere herein (R9-CHAP-CBD, R9-M23-CBD, TAT-Ami-CBD, TAT-CHAP-CBD, and TAT-M23-CBD). All data were collected in turbidity reduction assays on S. aureus SA113 substrate cells and at 100 nM protein concentrations (except Ami-CBD constructs where 1 μM was used) according as described previously in WO2013/169104. FIG. 7 depicts the performance of the constructs. As can be observed from FIG. 7, in general, the N-terminal CPP tagged proteins are active, but perform less compared to un-tagged or C-terminal tagged variants when analyzed on S. aureus cells. To analyze the efficiency of intracellular killing, the constructs should be tested in an intracellular S. aureus killing assay as in example 3.

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