Panton-Valentine Leukocidin (PVL) is a staphylococcal bicomponent pore-forming toxin linked to severe invasive infections. Target-cell and species specificity of PVL are poorly understood, and the mechanism of action of this toxin in Staphylococcus aureus virulence is controversial. Here, we identify the human complement receptors C5aR and C5L2 as host targets of PVL, mediating both toxin binding and cytotoxicity. Expression and interspecies variations of the C5aR determine cell and species specificity of PVL. The C5aR binding PVL component, LukS-PV, is a potent inhibitor of C5a-induced immune cell activation. These findings provide insight into leukocidin function and staphylococcal virulence and offer directions for future investigations into individual susceptibility to severe staphylococcal disease.

Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.

Evasion of the host phagocyte response by Staphylococcus aureus is crucial to successful infection with the pathogen. γ-Hemolysin AB and CB (HlgAB, HlgCB) are bicomponent pore-forming toxins present in almost all human S. aureus isolates. Cellular tropism and contribution of the toxins to S. aureus pathophysiology are poorly understood. Here, we identify the chemokine receptors CXCR1, CXCR2 and CCR2 as targets for HlgAB, and the complement receptors C5aR and C5L2 as targets for HlgCB. The receptor expression patterns allow the toxins to efficiently and differentially target phagocytic cells. Murine neutrophils are resistant to HlgAB and HlgCB. CCR2 is the sole murine receptor orthologue compatible with γ-Hemolysin. In a murine peritonitis model, HlgAB contributes to S. aureus bacteremia in a CCR2-dependent manner. HlgAB-mediated targeting of CCR2+ cells highlights the involvement of inflammatory macrophages during S. aureus infection. Functional quantification identifies HlgAB and HlgCB as major secreted staphylococcal leukocidins.

IntroductionStaphylococcus aureus is a common human pathogen that induces both community-acquired and nosocomial infections. This Grampositive bacterium is well known for its suppurative diseases such as skin-limited abscesses and boils and more seriously endocarditis, sepsis, and toxic shock syndrome. 1,2 Its invasiveness is ascribed to the production of a wide repertoire of cell surface-expressed as well as secreted virulence factors that interfere with host defense. 2,3 Superantigens constitute a large portion of the secreted arsenal of staphylococci and modulate immune responses. They trigger nonspecific activation of T lymphocytes by binding to major histocompatibility complex (MHC) class II molecules on antigenpresenting cells outside the antigen-binding cleft and V ␤ domains of T-cell receptors (TCRs). 4 We have described chemotaxis inhibitory protein of S aureus (CHIPS), an excreted virulence factor of S aureus. 5,6 CHIPS is known to inhibit formylated peptide-and complement factor C5a-induced responses in neutrophils through direct binding to the formyl peptide receptor and C5a receptor (C5aR), respectively. 7 Thereby, CHIPS inhibits the initial activation and migration of neutrophils to the site of infection; thus, it hampers clearance of S aureus by innate immune cells. Recently, the structure of CHIPS consisting of residues 31 to 121 (CHIPS ) was resolved. 8 CHIPS is composed of an ␣-helix packed onto a 4-stranded antiparallel ␤-sheet, a domain also present in the C-terminal domain of superantigens. This protein also revealed to be homologous to the C-terminal domain of staphylococcal superantigen-like 5 (SSL5) and SSL7.SSLs are a family of secreted proteins identified through sequence homology to staphylococcal and streptococcal superantigens. 9 Eleven different SSLs exist that are encoded on staphylococcal pathogenicity island 2 in a conserved order. Staphylococci contain 7 to 11 different SSLs, and their homology varies between 36% and 67%. Allelic variants show 85% to 100% homology. 10,11 Determination of the crystal structures of SSL5 12 and SSL7 13 also revealed their high structural homology to superantigens; the N-terminal oligonucleotide/oligosaccharide-binding fold and the C-terminal ␤-grasp domain characteristic for superantigens are also observed in SSLs. However, residues important for MHC class II and TCR binding of superantigens are not conserved in SSLs, which may explain their inability to display superantigenic activities. 9,10,12 Recently, Langley et al 14 described binding of complement component 5 and immunoglobulin A (IgA) by SSL7, suggesting a role for SSLs in staphylococcal defense against host immune responses. SSL7 was subsequently found to bind the C␣2/C␣3 interface of IgA Fc, which is the adhesion site for the Fc␣RI. 15 So far, no other functions have been linked to the SSLs.Neutrophil recruitment to sites of infection is a multistep process. 16 The initial tethering and rolling of neutrophils on the endothelium of vessel walls during inflammation are mediated by P-selec...

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.