Staphylococcus epidermidis strategies to avoid killing by human neutrophils

Abstract

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.

Figure 1. S. epidermidis and S. aureus PSMs.

All known S. aureus (S. a.) and S. epidermidis (S. e.) 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.

Figure 2. Neutrophil lysis by S. epidermidis culture filtrates.

Neutrophil (PMN) lysis by undiluted S. epidermidis 18-h culture filtrates was determined by measuring release of LDH (incubation time, 1 h). Culture filtrate from S. aureus LAC (18-h culture) was used as a comparison.

Figure 3. Secondary structure of S. epidermidis PSM peptides.

Secondary structure of S. epidermidis 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, http://heliquest.ipmc.cnrs.fr).

Figure 4. Neutrophil lysis by S. epidermidis PSM peptides and culture filtrates of PSMδ-expression strains.

(A) Neutrophil (PMN) lysis by synthetic, N-formylated PSM peptides at 10 µg/ml was determined by measuring release of LDH (incubation time, 1 h). PSMα3 (S. aureus) was used as a comparison at the same concentration. (B) Neutrophil lysis using supernatants (18-h cultures) of a PSMδ-over-expressing agr-negative (lacking intrinsic PSM production) and corresponding control strains (incubation time, 6 h). pTXpsmδ, pTX construct expressing PSMδ; pTX16, control plasmid. Strains were grown in TSB with 0.5% xylose and 12.5 µg/ml tetracycline. **, p<0.01, paired t-tests. Blue bars, PSMδ concentration in the culture filtrates relative to that in the 1457 wild-type (set to 100%).

Figure 5. Hemolysis by S. epidermidis culture filtrates and PSM peptides.

Hemolysis was determined by assays using sheep blood. (A) Hemolysis by synthetic, N-formylated PSMs of S. epidermidis. Negative control, DPBS. (B) Hemolysis by S. epidermidis culture filtrates (undiluted) and S. aureus 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.

Figure 6. PSM concentrations in S. epidermidis culture filtrates.

PSM concentrations in 18-h S. epidermidis and S. aureus 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 agr mutants (O47, 1457 agr). Relative PSM composition (α-type, δ-toxin, β-type) is shown at the right for S. aureus LAC and S. epidermidis 1457. Relative compositions were similar to that of 1457 in the other S. epidermidis strains (except in agr-negative O47 and 1457 agr).

Figure 7. IL-8 release by neutrophils stimulated by S. epidermidis PSMs and culture filtrates.

PMNs were incubated with synthetic, N-formylated PSMs (10 µg/ml) (A) or 18-h culture filtrates (diluted 1∶100) (B) and release of the cytokine IL-8 was measured by ELISA (for culture filtrates with further 1∶2 dilution). LPS, lipopolysaccharide, 10 ng/ml; LTA, S. aureus lipoteichoic acid, 1 µg/ml.

Figure 8. Survival of aps and sepA deletion mutants in human neutrophils.

Survival of S. epidermidis 1457 and S. aureus 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.