Toxin Mediates Sepsis Caused by Methicillin-Resistant <i>Staphylococcus epidermidis</i>

<div><p>Bacterial sepsis is a major killer in hospitalized patients. Coagulase-negative staphylococci (CNS) with the leading species <i>Staphylococcus epidermidis</i> are the most frequent causes of nosocomial sepsis, with most infectious isolates being methicillin-resistant. However, which bacterial factors underlie the pathogenesis of CNS sepsis is unknown. While it has been commonly believed that invariant structures on the surface of CNS trigger sepsis by causing an over-reaction of the immune system, we show here that sepsis caused by methicillin-resistant <i>S</i>. <i>epidermidis</i> is to a large extent mediated by the methicillin resistance island-encoded peptide toxin, PSM-mec. PSM-mec contributed to bacterial survival in whole human blood and resistance to neutrophil-mediated killing, and caused significantly increased mortality and cytokine expression in a mouse sepsis model. Furthermore, we show that the PSM-mec peptide itself, rather than the regulatory RNA in which its gene is embedded, is responsible for the observed virulence phenotype. This finding is of particular importance given the contrasting roles of the <i>psm-mec</i> locus that have been reported in <i>S</i>. <i>aureus</i> strains, inasmuch as our findings suggest that the <i>psm-mec</i> locus may exert effects in the background of <i>S</i>. <i>aureus</i> strains that differ from its original role in the CNS environment due to originally “unintended” interferences. Notably, while toxins have never been clearly implied in CNS infections, our tissue culture and mouse infection model data indicate that an important type of infection caused by the predominant CNS species is mediated to a large extent by a toxin. These findings suggest that CNS infections may be amenable to virulence-targeted drug development approaches.</p></div

Bacterial survival during incubation with human neutrophils and in whole human blood.

<p>(<b>A,B</b>) Survival in whole, heparinized human blood. (<b>C,D</b>) Survival during incubation with human neutrophils (MOI 10:1). (<b>E,F</b>) Killing of human neutrophils (MOI 100:1). Error bars show ±SEM. *, <i>P</i> <0.05, **, <i>P</i> <0.01. ***, <i>P</i> <0.001. ****, <i>P</i> <0.0001 (unpaired t-tests for RP62A; 1-way ANOVA with Bonferroni post-tests for SE620). Δ<i>psm-mec</i>, isogenic <i>psm-mec</i> deletion mutant; <i>psm-mec</i>*, <i>psm-mec</i> gene start codon mutant. In (A) and (C), no comparisons between SE620Δ<i>psm-mec</i> and SE620<i>psm-mec</i>* were statistically significant.</p

PSM production in <i>S</i>. <i>epidermidis</i> strains and isogenic <i>psm-mec</i> deletion and <i>psm-mec</i> start codon mutants.

<p>PSMs were measured in stationary phase (16 h) cultures using RP-HPLC/ESI-MS in triplicate. Error bars show ±SEM. $, Residual PSM-mec production in the SE620 <i>psm-mec</i> start codon mutant is likely due to strong gene expression and usage of a non-canonical start codon, as we previously found also in <i>S</i>. <i>aureus psm-mec</i> start codon mutants [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006153#ppat.1006153.ref014" target="_blank">14</a>].</p

Time-dependent expression of <i>psm-mec</i>.

<p>Expression of the <i>psm-mec</i> RNA relative to that of 16S RNA in <i>S</i>. <i>epidermidis</i> RP62A during growth in TSB. The experiment was performed in triplicate. Error bars show ±SEM.</p

Mouse sepsis model.

<p>Female, 6–10 weeks old, C57BL/6NCRl mice (n = 5 for all groups except SE620Δ<i>psm-mec</i> and SE620<i>psm-mec</i>*, n = 10) were injected via the tail vein with 5 x 10⁸ CFU of the indicated bacterial strains and monitored for disease development every 8 h for up to 120 h. (<b>A,B</b>) Survival curves; (<b>C,D</b>) CFU in blood at 12 h; (<b>E,F</b>) CFU in kidneys at 12 h. Statistical analysis is by Log-rank (Mantel-Cox) tests for survival curves, otherwise using 1-way ANOVA with Bonferroni post-tests. Error bars show ±SEM. N.S., not significant. Δ<i>psm-mec</i>, isogenic <i>psm-mec</i> deletion mutant; <i>psm-mec</i>*, <i>psm-mec</i> gene start codon mutant.</p

Impact of <i>psm-mec</i> on biofilm formation in <i>S. epidermidis</i>.

<p>Biofilm formation by <i>S</i>. <i>epidermidis</i> strains and isogenic <i>psm-mec</i> deletion and <i>psm-mec</i> start codon mutants. Biofilm formation was measured using a semi-quantitative microtiter plate assay. 24 wells per group were measured. ****, P<0.0001 (1-way ANOVA, Bonferroni post tests vs. wild-type strain); N.S., not significant. Error bars show ±SEM. Δ<i>psm-mec</i>, isogenic <i>psm-mec</i> deletion mutant; <i>psm-mec</i>*, <i>psm-mec</i> gene start codon mutant.</p

Microarray results, RP62A¹.

<p>Microarray results, RP62A<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006153#t001fn001" target="_blank">¹</a>.</p

Mouse sepsis model, cytokine concentrations, strain SE620.

<p>Cytokine concentrations (<b>A</b>, CXCL1, <b>B</b>, IL-1β, <b>C</b>, TNF-α) were determined at 12 h post infection in the mouse sepsis model using commercial ELISA kits. Statistical analysis is by 1-way ANOVA with Bonferroni post-tests. Error bars show ±SEM. ****, <i>P</i><0.0001. Δ<i>psm-mec</i>, isogenic <i>psm-mec</i> deletion mutant; <i>psm-mec</i>*, <i>psm-mec</i> gene start codon mutant.</p

Microarray results, SE620¹.

<p>Microarray results, SE620<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006153#t002fn001" target="_blank">¹</a>.</p