Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis

Abstract

Staphylococcus aureus community-acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA-MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA-MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α-type and β-type PSMs on neutrophil killing after phagocytosis. Using fluorescently labeled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time-lapse microscopy. Phagocytosis of the CA-MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increased neutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.

The psm α Locus Regulates Production of Staphylococcus aureus Alpha-Toxin during Infection

“…However, at stationary phase, psm␣1 mRNA expressed by the ⌬psm␣ pTx ⌬ -psm␣ strain accumulated to levels indistinguishable from those of WT bacteria (see Fig. S1B in the supplemental material), providing a rationale for the ability to complement the psm␣ defect in previous studies using stationaryphase cultures (28,29,41). In order to provide additional confirmation that an unanticipated secondary mutation was not responsible for the early psm␣-mediated defect in Hla secretion, this phenotype observed for the USA300 S. aureus strain was confirmed by using isogenic ⌬psm mutants of CA-MRSA of the USA400 lineage (MW2) (Fig.…”

“…2C). Previous studies successfully complemented the ⌬psm defect in neutrophil killing by expressing psm loci under the control of the xylose promoter in which the repressor element had been genetically deleted (28,29,41). Our initial attempts to complement the Hla secretion defect were unsuccessful using this system (see Fig.…”

“…Secreted from staphylococci via a dedicated secretion system (26), the effects of PSMs on host immune cells are mediated by toxin binding to formyl peptide receptor 2 (FPR2) (27). At micromolar concentrations, PSMs cause the lysis of human neutrophils, with the PSM␣ peptides displaying the most potent cytolytic capacity (28,29). At submicromolar concentrations, the peptides are proinflammatory, stimulating neutrophil chemotaxis by signaling through FPR2 (27,30).…”

“…At submicromolar concentrations, the peptides are proinflammatory, stimulating neutrophil chemotaxis by signaling through FPR2 (27,30). A growing body of evidence has implicated PSM␣ peptides in particular as a major contributor to disease pathogenesis, especially in highly virulent CA-MRSA strains (25,(27)(28)(29)31). Deletion of the psm␣ locus leads to an attenuation of virulence in multiple animal models of disease, including models of dermonecrotic skin infection and lethal sepsis (21,28).…”

The Bicomponent Pore-Forming Leucocidins of Staphylococcus aureus

“…S. aureus lukAB (lukHG) deletion mutants escape less efficiently from phagocytosed PMNs, leading to reduced bacterial rebound after phagocytosis (234,235). Similar escape defects are observed with the cytolytic phenol-soluble modulins (236). Such findings again highlight that the redundant effects imparted by cytotoxic molecules produced by S. aureus (in this case, PSMs and LukAB) are likely to synergize to promote optimal bacterial escape after PMN phagocytosis.…”