Direct and synergistic hemolysis caused by Staphylococcus phenol-soluble modulins: implications for diagnosis and pathogenesis

Abstract

Phenol-soluble modulins are secreted staphylococcal peptides with an amphipathic α-helical structure. Some PSMs are strongly cytolytic toward human neutrophils and represent major virulence determinants during Staphylococcus aureus skin and blood infection. However, capacities of PSMs to lyse human erythrocytes have not been investigated. Here, we demonstrate that many S. aureus and Staphylococcus epidermidis PSMs lyse human erythrocytes. Furthermore, synergism with S. aureus β-toxin considerably increased the hemolytic capacities of several PSMs. This synergism may be of key importance in PSM and β-toxin-producing S. aureus or in mixed-strain or -species infections with PSM and β-toxin producers. Of specific interest, several PSMs, in particular PSMα peptides, contributed to a considerable extent to synergistic hemolysis with β-toxin or when using the β-toxin-producing strain RN4220 in CAMP assays. Thus, CAMP-type assays should not be used to detect or quantify S. aureus δ-toxin production, but may be used for an overall assessment of Agr functionality. Our study suggests an additional role of PSMs in staphylococcal pathogenesis and demonstrates that the repertoire of staphylococcal hemolysins is not limited to S. aureus and is much larger and diverse than previously thought.

Fig. 1. Lysis of human erythrocytes by S. aureus and S. epidermidis PSMs

PSMs at the given concentrations were incubated with a 2% solution of defibrinated human blood for 1 h at 37°C. Hemolysis was measured by determining OD_540nm in an ELISA reader. S. aureus PSMs are grouped on the left, S. epidermidis PSMs on the right. PSM-mec is produced by some methicillin-resistant strains of both species. PSMα1 ΔMG and PSMα2 ΔMG are naturally occurring processed PSMα1, or PSMα2, respectively, in which the two N-terminal amino acids (MG) have been removed.

Fig. 2. Synergistic in comparison to direct lysis of human and sheep erythrocytes by S. aureus and S. epidermidis PSMs

PSMs at final concentrations of 0.1 or 1 µg/ml were incubated with a 2% solution of defibrinated human blood for 30 min at 37°C. Hemolysis was measured by determining OD_540nm in an ELISA reader. S. aureus PSMs are grouped on the left, S. epidermidis PSMs on the right. PSM-mec is produced by some methicillin-resistant strains of both species. PSMα1 ΔMG and PSMα2 ΔMG are naturally occurring processed PSMα1, or PSMα2, respectively, in which the two N-terminal amino acids (MG) have been removed. Dashed lines depict the level of OD_540nm in control samples with only buffer or only β-toxin, which showed no hemolytic capacity.

Fig. 3. CAMP assay with S. aureus RN4220 and pure PSM peptides of S. aureus and S. epidermidis

PSMs were applied to filter disks (15 µl, 1 mg/ml), dried, and filters were placed next to streaked RN4220 bacteria on sheep blood agar plates. Plates were incubated for 24 h and photographed. Note that both direct and synergistic hemolysis is seen with S. aureus PSMα3 (arrows).

Fig. 4. CAMP reaction of different S. aureus strains and MW2 psm mutants

Cultures of different S. aureus strains (A) or MW2 wild-type or psm mutant strains (B) were spotted next to filters with β-toxin solution. Plates were incubated for 24 h and photographed. Note direct and synergistic hemolysis zones, as shown for strain MW2 as example (arrows). (C) MW2 and MW2 psm mutants were cross-streaked with RN4220 for analysis of synergistic hemolysis.

Fig. 5

Hemolysis of culture filtrates of LAC psm mutants on sheep blood agar plates. 24-h culture filtrates of strain S. aureus LAC (USA300) (LAC wt) and isogenic psm mutants were applied to filter disks (15 µl, undiluted). Plates were incubated for 24 h and photographed.

Fig. 6. PSM production in strains of the NCTC8325 lineage

PSM production was measured by RPHLC/ESI-MS in stationary phase (8-h) cultures of strains of the NCTC8325 (8325) lineage and other strains for comparison. Values for PSMα3 (left y-axis) and δ-toxin (right y-axis) are shown; differences between strains regarding PSMα1, α2, and α4 values were similar to those seen with PSMα3.