Staphylococcus aureus δ-toxin is an inducer of mast cell degranulation in mice and is important for promoting inflammatory skin disease. The pathogenesis of atopic dermatitis, a chronic inflammatory skin disease, is not fully understood. The condition is known to be mediated by an abnormal IgE immune response in the setting of skin barrier dysfunction and mast-cell activation. And intriguingly, in more than 90% of atopic dermatitis patients the skin lesions are colonized by Staphylococcus aureus. This study identifies a probable mechanistic link between S. aureus and allergic skin disease. Gabriel Nüñez and colleagues show that δ-toxin produced by the bacterium induces mast-cell degranulation and inflammatory skin disease in mice, and that both the IgE response and signs of dermatitis were suppressed in mast-cell-deficient mice. Atopic dermatitis is a chronic inflammatory skin disease that affects 15–30% of children and approximately 5% of adults in industrialized countries1. Although the pathogenesis of atopic dermatitis is not fully understood, the disease is mediated by an abnormal immunoglobulin-E immune response in the setting of skin barrier dysfunction2. Mast cells contribute to immunoglobulin-E-mediated allergic disorders including atopic dermatitis3. Upon activation, mast cells release their membrane-bound cytosolic granules leading to the release of several molecules that are important in the pathogenesis of atopic dermatitis and host defence4. More than 90% of patients with atopic dermatitis are colonized with Staphylococcus aureus in the lesional skin whereas most healthy individuals do not harbour the pathogen5. Several staphylococcal exotoxins can act as superantigens and/or antigens in models of atopic dermatitis6. However, the role of these staphylococcal exotoxins in disease pathogenesis remains unclear. Here we report that culture supernatants of S. aureus contain potent mast-cell degranulation activity. Biochemical analysis identified δ-toxin as the mast cell degranulation-inducing factor produced by S. aureus. Mast cell degranulation induced by δ-toxin depended on phosphoinositide 3-kinase and calcium (Ca2+) influx; however, unlike that mediated by immunoglobulin-E crosslinking, it did not require the spleen tyrosine kinase. In addition, immunoglobulin-E enhanced δ-toxin-induced mast cell degranulation in the absence of antigen. Furthermore, S. aureus isolates recovered from patients with atopic dermatitis produced large amounts of δ-toxin. Skin colonization with S. aureus, but not a mutant deficient in δ-toxin, promoted immunoglobulin-E and interleukin-4 production, as well as inflammatory skin disease. Furthermore, enhancement of immunoglobulin-E production and dermatitis by δ-toxin was abrogated in KitW-sh/W-sh mast-cell-deficient mice and restored by mast cell reconstitution. These studies identify δ-toxin as a potent inducer of mast cell degranulation and suggest a mechanistic link between S. aureus colonization and allergic skin disease.

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.

Staphylococcus aureus is a bacterial pathogen known to cause infections in epidemic waves. One such epidemic was caused by a clone known as phage-type 80/81, a penicillin-resistant strain that rose to world prominence in the late 1950s. The molecular underpinnings of the phage-type 80/81 outbreak have remained unknown for decades, nor is it understood why related S. aureus clones became epidemic in hospitals in the early 1990s. To better understand the molecular basis of these epidemics, we sequenced the genomes of eight S. aureus clinical isolates representative of the phage-type 80/81 clone, the Southwest Pacific clone [a community-associated methicillin-resistant S. aureus (MRSA) clone], and contemporary S. aureus clones, all of which are genetically related and belong to the same clonal complex (CC30). Genome sequence analysis revealed that there was coincident divergence of these clones from a recent common ancestor, a finding that resolves controversy about the evolutionary history of the lineage. Notably, we identified nonsynonymous SNPs in genes encoding accessory gene regulator C ( agrC ) and α-hemolysin ( hla )—molecules important for S. aureus virulence—that were present in virtually all contemporary CC30 hospital isolates tested. Compared with the phage-type 80/81 and Southwest Pacific clones, contemporary CC30 hospital isolates had reduced virulence in mouse infection models, the result of SNPs in agrC and hla . We conclude that agr and hla (along with penicillin resistance) were essential for world dominance of phage-type 80/81 S. aureus , whereas key SNPs in contemporary CC30 clones restrict these pathogens to hospital settings in which the host is typically compromised.

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA , and is followed by sspB , which encodes a 40.6-kDa cysteine protease, and sspC , which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.

Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells. IMPORTANCE S. aureus can infect any site of the body, including the microbicidal phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction.

Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.