Staphylococcus epidermidis Strategies to Avoid Killing by Human Neutrophils

Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus

Mobile Genetic Element-Encoded Cytolysin Connects Virulence to Methicillin Resistance in MRSA

Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs

Staphylococcus epidermidis surfactant peptides promote biofilm maturation and dissemination of biofilm-associated infection in mice

Biofilms are surface-attached agglomerations of microorganisms embedded in an extracellular matrix Biofilm-associated infections are difficult to eradicate and represent a significant reservoir for disseminating and recurring serious infections Infections involving biofilms frequently develop on indwelling medical devices in hospitalized patients, and Staphylococcus epidermidis is the leading cause of infection in this setting However, the molecular determinants of biofilm dissemination are unknown Here we have demonstrated that specific secreted, surfactant-like S epidermidis peptides - the beta subclass of phenol-soluble modulins (PSMs) - promote S epidermidis biofilm structuring and detachment in vitro and dissemination from colonized catheters in a mouse model of device-related infection Our study establishes in vivo significance of biofilm detachment mechanisms for the systemic spread of biofilm-associated infection and identifies the effectors of biofilm maturation and detachment in a premier biofilm-forming pathogen Furthermore, by demonstrating that antibodies against PSM beta peptides inhibited bacterial spread from indwelling medical devices, we have provided proof of principle that interfering with biofilm detachment mechanisms may prevent dissemination of biofilm-associated infectio

Hemolysis by <i>S. epidermidis</i> culture filtrates and PSM peptides.

<p>Hemolysis was determined by assays using sheep blood. (A) Hemolysis by synthetic, N-formylated PSMs of <i>S. epidermidis</i>. Negative control, DPBS. (B) Hemolysis by <i>S. epidermidis</i> culture filtrates (undiluted) and <i>S. aureus</i> LAC culture filtrate as comparison. All culture filtrates were from cultures grown for 18 h. Negative control, DPBS; positive control, 1% (v/v) Triton-X100 in DPBS.</p

Secondary structure of <i>S. epidermidis</i> PSM peptides.

<p>Secondary structure of <i>S. epidermidis</i> PSM peptides was analyzed by circular dichroism (CD) measurement. (A), molar ellipticity curves; (B) analysis of secondary structure using 3 different algorithms. (C) All PSM peptides have an amphipathic α-helix that encompasses most of the peptide for the shorter α-type and the C-terminal part of the β-type PSMs (shown as example for PSMβ1 by α-helical wheel presentation, <a href="http://heliquest.ipmc.cnrs.fr" target="_blank">http://heliquest.ipmc.cnrs.fr</a>).</p

<i>S. epidermidis</i> and <i>S. aureus</i> PSMs.

<p>All known <i>S. aureus</i> (<i>S. a.</i>) and <i>S. epidermidis</i> (<i>S. e.</i>) PSMs were aligned by a sequence comparison program (Vector NTI). Similarity on the amino acid level is depicted as a tree on the left. Aligned amino acid sequences are shown at the right, with conserved amino acids shown in blue. All PSMs contain a region with pronounced amphipathy and α-helicity, boxed in yellow.</p

Survival of <i>aps</i> and <i>sepA</i> deletion mutants in human neutrophils.

<p>Survival of <i>S. epidermidis</i> 1457 and <i>S. aureus</i> MW2 wild-type (wt) and isogenic gene deletion mutants was determined after phagocytic uptake by counting of colony forming units after 60 min incubation. Bacterial cells used for the experiment were harvested at similar points in growth at an OD_{600 nm} of ∼1.5. ***, p<0.001; **, p<0.01 versus the corresponding wild-type sample (1-way ANOVA, Dunnett's post test). Error bars represent SEM.</p

PSM concentrations in <i>S. epidermidis</i> culture filtrates.

<p>PSM concentrations in 18-h <i>S. epidermidis</i> and <i>S. aureus</i> LAC culture filtrates were determined by HPLC/MS. Peaks corresponding to N-formylated and deformylated PSM versions were measured separately and the percentage of deformylated peptides is shown as checkered bars. No PSMs were detected in the natural and constructed <i>agr</i> mutants (O47, 1457 <i>agr</i>). Relative PSM composition (α-type, δ-toxin, β-type) is shown at the right for <i>S. aureus</i> LAC and <i>S. epidermidis</i> 1457. Relative compositions were similar to that of 1457 in the other <i>S. epidermidis</i> strains (except in <i>agr</i>-negative O47 and 1457 <i>agr</i>).</p