Methicillin-resistant Staphylococcus aureus (MRSA) remains a major human pathogen. Traditionally, MRSA infections occurred exclusively in hospitals and were limited to immunocompromised patients or individuals with predisposing risk factors. However, recently there has been an alarming epidemic caused by community-associated (CA)-MRSA strains, which can cause severe infections that can result in necrotizing fasciitis or even death in otherwise healthy adults outside of healthcare settings. In the US, CA-MRSA is now the cause of the majority of infections that result in trips to the emergency room. It is unclear what makes CA-MRSA strains more successful in causing human disease compared with their hospital-associated counterparts. Here we describe a class of secreted staphylococcal peptides that have a remarkable ability to recruit, activate and subsequently lyse human neutrophils, thus eliminating the main cellular defense against S. aureus infection. These peptides are produced at high concentrations by standard CA-MRSA strains and contribute significantly to the strains' ability to cause disease in animal models of infection. Our study reveals a previously uncharacterized set of S. aureus virulence factors that account at least in part for the enhanced virulence of CA-MRSA.

SUMMARY Virulence of the emerging Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) and other highly pathogenic S. aureus depends on the recently discovered phenol-soluble modulin (PSM) peptide toxins, which combine the capacities to attract and lyse neutrophils. The molecular basis of PSM-stimulated neutrophil recruitment has remained unknown. We demonstrate that the human formyl peptide receptor 2 (FPR2/ALX), which has previously been implicated in control of endogenous inflammatory processes, senses PSMs at nanomolar concentrations and initiates proinflammatory neutrophil responses to CA-MRSA. Specific blocking of FPR2/ALX or deletion of PSM genes in CA-MRSA led to severely diminished capacities of neutrophils to detect CA-MRSA. A specific inhibitor of FPR2/ALX and its functional mouse counterpart blocked PSM-mediated leukocyte infiltration in vivo in a mouse model. Thus, the innate immune system uses a new FPR2/ALX-dependent mechanism to sense bacterial peptide toxins and detect highly virulent bacterial pathogens. FPR2/ALX represents an attractive target for new anti-infective or anti-inflammatory strategies.

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.

Our study highlights the roles of surfactant-like molecules in bacterial inflammation with important implications for the prevention and therapy of inflammatory disorders. It describes a potential pathway for the transfer of hydrophobic bacterial lipoproteins, the major TLR2 agonists, from the cytoplasmic membrane of Gram-positive bacteria to the TLR2 receptor at the surface of host cells. Moreover, our study reveals a molecular mechanism that explains how cytoplasmic and membrane-embedded bacterial proteins can be released by bacterial cells without using any of the typical protein secretion routes, thereby contributing to our understanding of the processes used by bacteria to communicate with host organisms and the environment.

Phenol-soluble modulins (PSMs) are a family of peptides with multiple functions in staphylococcal pathogenesis. To gain insight into the structural features affecting PSM functions, we analyzed an alanine substitution library of PSMα3, a strongly cytolytic and proinflammatory PSM of Staphylococcus aureus with a significant contribution to S. aureus virulence. Lysine residues were essential for both receptor-dependent proinflammatory and receptor-independent cytolytic activities. Both phenotypes also required additional structural features, with the C terminus being crucial for receptor activation. Biofilm formation was affected mostly by hydrophobic amino acid positions, suggesting that the capacity to disrupt hydrophobic interactions is responsible for the effect of PSMs on biofilm structure. Antimicrobial activity, absent from natural PSMα3, could be created by the exchange of large hydrophobic side chains, indicating that PSMα3 has evolved to exhibit cytolytic rather than antimicrobial activity. In addition to gaining insight into the structure-function relationship in PSMs, our study identifies nontoxic PSMα3 derivatives for active vaccination strategies and lays the foundation for future efforts aimed to understand the biological role of PSM recognition by innate host defense.

Sepsis caused by Gram-positive bacterial pathogens is a major fatal disease but its molecular basis remains elusive. Toll-like receptor 2 (TLR2) has been implicated in the orchestration of inflammation and sepsis but its role appears to vary for different pathogen species and clones. Accordingly, Staphylococcus aureus clinical isolates differ substantially in their capacity to activate TLR2. Here we show that strong TLR2 stimulation depends on high-level production of phenol-soluble modulin (PSM) peptides in response to the global virulence activator Agr. PSMs are required for mobilizing lipoproteins, the TLR2 agonists, from the staphylococcal cytoplasmic membrane. Notably, the course of sepsis caused by PSM-deficient S. aureus is similar in wild-type and TLR2-deficient mice, but TLR2 is required for protection of mice against PSM-producing S. aureus. Thus, a crucial role of TLR2 depends on agonist release by bacterial surfactants. Modulation of this process may lead to new therapeutic strategies against Gram-positive infections.

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Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are the cause of a severe pandemic consisting primarily of skin and soft tissue infections. The underlying pathomechanisms have not been fully understood and we report here a mechanism that plays an important role for the elevated virulence of CA-MRSA. Surprisingly, skin abscess induction in an animal model was correlated with the amount of a major cell wall component of S. aureus, termed wall teichoic acid (WTA). CA-MRSA exhibited increased cell-wall-associated WTA content (WTA) and thus were more active in inducing abscess formation via a WTA-dependent and T-cell-mediated mechanism than S. aureus strains with a WTA phenotype. We show here that WTA is directly involved in S. aureus strain-specific virulence and provide insight into the underlying molecular mechanisms that could guide the development of novel anti-infective strategies.