Global analysis of community-associated methicillin-resistant Staphylococcus aureus exoproteins reveals molecules produced in vitro and during infection

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is a threat to human health worldwide. Although progress has been made, mechanisms of CA-MRSA pathogenesis are poorly understood and a comprehensive analysis of CA-MRSA exoproteins has not been conducted. To address that deficiency, we used proteomics to identify exoproteins made by MW2 (USA400) and LAC (USA300) during growth in vitro. Two hundred and fifty unique exoproteins were identified by 2-dimensional gel electrophoresis coupled with automated direct infusion-tandem mass spectrometry (ADI-MS/MS) analysis. Eleven known virulence-related exoproteins differed in abundance between the strains, including alpha-haemolysin (Hla), collagen adhesin (Cna), staphylokinase (Sak), coagulase (Coa), lipase (Lip), enterotoxin C3 (Sec3), enterotoxin Q (Seq), V8 protease (SspA) and cysteine protease (SspB). Mice infected with MW2 or LAC produced antibodies specific for known or putative virulence factors, such as autolysin (Atl), Cna, Ear, ferritin (Ftn), Lip, 1-phosphatidylinositol phosphodiesterase (Plc), Sak, Sec3 and SspB, indicating the exoproteins are made during infection in vivo. We used confocal microscopy to demonstrate aureolysin (Aur), Hla, SspA and SspB are produced following phagocytosis by human neutrophils, thereby linking exoprotein production in vitro with that during host–pathogen interaction. We conclude that the exoproteins identified herein likely account in part for the success of CA-MRSA as a human pathogen

Supplementary Material for: The Role of Innate Immunity in Promoting SaeR/S-Mediated Virulence in Staphylococcus aureus

The ability of <i>Staphylococcus aureus</i> to infect tissues is dependent on precise control of virulence through gene-regulatory systems. While the SaeR/S two-component system has been shown to be a major regulator of <i>S. aureus</i> virulence, the influence of the host environment on SaeR/S-regulated genes (<i>saeR/S </i>targets) remains incompletely defined. Using QuantiGene 2.0 transcriptional assays, we examined expression of genes with the SaeR binding site in USA300 exposed to human and mouse neutrophils and host-derived peptides and during subcutaneous skin infection. We found that only some of the <i>saeR/S </i>targets, as opposed to the entire SaeR/S virulon, were activated within 5 and 10 min of interacting with human neutrophils as well as α-defensin. Furthermore, mouse neutrophils promoted transcription of <i>saeR/S</i> targets despite lacking α-defensin, and the murine skin environment elicited a distinctive expression profile of <i>saeR/S</i> targets. These findings indicate that <i>saeR/S</i>-mediated transcription is unique to and dependent on specific host stimuli. By using isogenic USA300Δ<i>saeR/S</i> and USA300Δ<i>agr</i> knockout strains, we also determined that SaeR/S is the major regulator of virulence factors, while Agr, a quorum-sensing two-component system, has moderate influence on transcription of the <i>saeR/S </i>targets under the tested physiological conditions

Nasal carriage of staphylococcus aureus, including community-associated methicillin-resistant strains, in Queensland adults

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections are emerging in southeast Queensland, Australia, but the incidence of carriage of CA-MRSA strains is unknown. The aim of this study was to assess the nasal carriage rate of S. aureus, including CA-MRSA strains, in the general adult population of southeast Queensland. 396 patients presenting to general practices in two Brisbane suburbs and 303 volunteers randomly selected from the electoral rolls in the same suburbs completed a medical questionnaire and had nasal swabs performed for S. aureus. All isolates of S. aureus underwent antibiotic susceptibility testing and single-nucleotide polymorphism (SNP) and binary typing, including determination of Panton–Valentine leukocidin (PVL). The nasal carriage rate of methicillin-susceptible S. aureus (MSSA) was 202/699 (28%), a rate similar to that found in other community-based nasal carriage studies. According to multivariate analysis, nasal carriage of S. aureus was associated with male sex, young adult age group and Caucasian ethnicity. Only two study isolates (one MSSA and one CA-MRSA) carried PVL. The nasal carriage rate of MRSA was low, at 5/699 (0.7%), and only two study participants (0.3%) had CA-MRSA strains. CA-MRSA is an emerging cause of infection in southeast Queensland, but as yet the incidence of carriage of CA-MRSA in the general community is low