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, 70 (5), 1453-65

Evolutionarily Distinct Bacteriophage Endolysins Featuring Conserved Peptidoglycan Cleavage Sites Protect Mice From MRSA Infection

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Evolutionarily Distinct Bacteriophage Endolysins Featuring Conserved Peptidoglycan Cleavage Sites Protect Mice From MRSA Infection

Mathias Schmelcher et al. J Antimicrob Chemother.

Abstract

Objectives: In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs.

Methods: PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model.

Results: Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment.

Conclusions: Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites.

Keywords: antibiotic resistance; antimicrobial; biofilm; peptidoglycan hydrolase.

Figures

Figure 1.
Figure 1.
Modular organization, purification and staphylolytic activity of nine different staphylococcal SH3b domain-containing PGHs. (a) Schematic representation of nine PGHs representing different homology groups reported previously. EADs are depicted as hatched and white/dotted bars and SH3b CBDs as black bars. The scale indicates amino acid (AA) positions. M23, M23 endopeptidase domain; SH3b, bacterial SH3 domain (CBD). All proteins harbour a C-terminal 6 × His-tag (not represented). (b) SDS–PAGE of 6 × His-tagged PGHs purified by immobilized metal ion affinity chromatography; 5 μg of protein was loaded in each lane. Expected molecular weights: 80α, 54.9 kDa; phi11, 55.1 kDa; LysK, 55.8 kDa; P68, 29.6 kDa; lysostaphin, 28.2 kDa; 2638A, 56.6 kDa; Twort, 54.3 kDa; phiSH2, 57.4 kDa; and WMY, 55.0 kDa. Note that C-terminally 6 × His-tagged 2638A purification yielded a double band (for an explanation see the text). (c) Zymogram with the enzymes shown in (b) and S. aureus Newman cells embedded in the gel. The same amounts of protein as in SDS–PAGE were loaded. (d) Specific activity of the nine PGHs against S. aureus Newman determined by turbidity reduction assays performed with 0.2 μM enzyme in buffer containing 200 mM NaCl. Error bars indicate standard deviations from three independent experiments.
Figure 2.
Figure 2.
Comparison of lytic activity of nine PGHs against live S. aureus cells at varying ionic strength. Specific activity was determined by turbidity reduction assays using 0.2 μM enzyme against S. aureus Newman cells suspended in Tris buffer, pH 7.5, with NaCl concentrations ranging from 0 to 600 mM. All assays were performed in triplicate. For graphs of individual enzymes with error bars, see Figure S2.
Figure 3.
Figure 3.
ESI–MS analysis of PGH cleavage sites. (a) ESI–MS spectra (m/z range 400–1000) of S. aureus Newman PG digested with four different dual lytic domain endolysins. All spectra contain an identical dominant peak at m/z = 702. (b) Mass spectra (m/z range 650–750) of S. aureus SA113 ΔtagO PG after single digestion with full-length PGHs or double digestion with two different enzymes. Data were normalized to the highest peak (=100%) within the shown m/z range. Note that the numerous peaks in the P68 + CHAP-K spectrum merely represent amplified background noise. (c) Portion of the S. aureus PG (top) with dotted and cross-hatched arrows indicating cleavage sites of the amidase and CHAP/M23 endopeptidase domains, respectively (cross-hatched arrows indicate that both the M23 domain of 2638A and the CHAP domains of the other seven endolysins exhibit d-Ala-Gly endopeptidase activity). Domains are depicted with corresponding patterns in the schematics (bottom). Simultaneous digestion with both catalytic domains yields the boxed fragment (A2QKG5) of m/z = 702, corresponding to the dominant peak in (a).
Figure 3.
Figure 3.
ESI–MS analysis of PGH cleavage sites. (a) ESI–MS spectra (m/z range 400–1000) of S. aureus Newman PG digested with four different dual lytic domain endolysins. All spectra contain an identical dominant peak at m/z = 702. (b) Mass spectra (m/z range 650–750) of S. aureus SA113 ΔtagO PG after single digestion with full-length PGHs or double digestion with two different enzymes. Data were normalized to the highest peak (=100%) within the shown m/z range. Note that the numerous peaks in the P68 + CHAP-K spectrum merely represent amplified background noise. (c) Portion of the S. aureus PG (top) with dotted and cross-hatched arrows indicating cleavage sites of the amidase and CHAP/M23 endopeptidase domains, respectively (cross-hatched arrows indicate that both the M23 domain of 2638A and the CHAP domains of the other seven endolysins exhibit d-Ala-Gly endopeptidase activity). Domains are depicted with corresponding patterns in the schematics (bottom). Simultaneous digestion with both catalytic domains yields the boxed fragment (A2QKG5) of m/z = 702, corresponding to the dominant peak in (a).
Figure 4.
Figure 4.
Biofilm-disrupting activity of staphylococcal PGHs. Biofilms of S. aureus SA113 were grown in polystyrene 96-well plates for 24 h at 37°C. After washing and incubation for 2.5 h at 37°C with various concentrations of C-terminally His-tagged PGHs in elution buffer or elution buffer only as a control, wells were stained with crystal violet.
Figure 5.
Figure 5.
Efficacy of PGHs and antibiotics in a mouse model of systemic MRSA infection. (a) Survival of mice infected intraperitoneally with MRSA and subjected to different treatment regimens. Treatment groups listed on the right and corresponding survival curves are displayed in matching colours. Numbers of animals in each group (N) are given in parentheses. Antibiotics are shown in italics and PGHs are underlined. Differences in survival rates between PBS-treated control mice and animals treated with 80α, phi11, LysK, lysostaphin, 2638A, WMY or vancomycin were statistically significant (P < 0.02; Fisher's exact test). (b) Average septicaemia scores of mice recorded for 48 h following infection. Scores for animals in different treatment groups listed on the right (displayed in matching colours) correspond to the matrix shown in (c). (c) Composite matrix of septicaemia rating the disease state of infected animals characterized by defined clinical signs.

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