Infective endocarditis and kidney infections are serious complications of Staphylococcus aureus sepsis. We investigated the role of superantigens (SAgs) in the development of lethal sepsis, infective endocarditis, and kidney infections. SAgs cause toxic shock syndrome, but it is unclear if SAgs contribute to infective endocarditis and kidney infections secondary to sepsis. We show in the methicillin-resistant S. aureus strain MW2 that lethal sepsis, infective endocarditis, and kidney infections in rabbits are critically dependent on high-level SAgs. In contrast, the isogenic strain lacking staphylococcal enterotoxin C (SEC), the major SAg in this strain, is attenuated in virulence, while complementation restores disease production. SAgs’ role in infective endocarditis appears to be both superantigenicity and direct endothelial cell stimulation. Maintenance of elevated blood pressure by fluid therapy significantly protects from infective endocarditis, possibly through preventing bacterial accumulation on valves and increased SAg elimination. These data should facilitate better methods to manage these serious illnesses. IMPORTANCE The Centers for Disease Control and Prevention reported in 2007 that Staphylococcus aureus is the most significant cause of serious infectious diseases in the United States (R. M. Klevens, M. A. Morrison, J. Nadle, S. Petit, K. Gershman, et al., JAMA 298:1763–1771, 2007). Among these infections are sepsis, infective endocarditis, and acute kidney injury. Infective endocarditis occurs in 30 to 60% of patients with S. aureus bacteremia and carries a mortality rate of 40 to 50%. Over the past decades, infective endocarditis outcomes have not improved, and infection rates are steadily increasing (D. H. Bor, S. Woolhandler, R. Nardin, J. Brusch, D. U. Himmelstein, PLoS One 8:e60033, 2013). There is little understanding of the S. aureus virulence factors that are key for infective endocarditis development and kidney abscess formation. We demonstrate that superantigens are critical in the causation of all three infections. We show that their association results from both superantigenicity and direct toxic effects on endothelial cells, the latter likely contributing to delayed endothelium healing. Our studies contribute significantly to understanding the development of these illnesses and are expected to lead to development of important therapies to treat such illnesses.

The vaginal microbiota can impact the susceptibility of women to bacterial vaginosis (BV) and sexually transmitted infections (STIs). BV is characterized by depletion of Lactobacillus spp., an overgrowth of anaerobes (often dominated by Gardnerella vaginalis) and a pH > 4.5. BV is associated with an increased risk of acquiring STIs such as chlamydia and gonorrhea. While these associations have been identified, the molecular mechanism(s) driving the risk of infections are unknown. An ex vivo porcine vaginal mucosal model (PVM) was developed to explore the mechanistic role of Lactobacillus spp. in affecting colonization by G. vaginalis and Neisseria gonorrhoeae. The data presented here demonstrate that all organisms tested can colonize and grow on PVM to clinically relevant densities. Additionally, G. vaginalis and N. gonorrhoeae form biofilms on PVM. It was observed that lactic acid, acetic acid, and hydrochloric acid inhibit the growth of G. vaginalis on PVM in a pH-dependent manner. N. gonorrhoeae grows best in the presence of lactic acid at pH 5.5, but did not grow well at this pH in the presence of acetic acid. Finally, a clinical Lactobacillus crispatus isolate (24-9-7) produces lactic acid and inhibits growth of both G. vaginalis and N. gonorrhoeae on PVM. These data reveal differences in the effects of pH, various acids and L. crispatus on the growth of G. vaginalis and N. gonorrhoeae on a live vaginal mucosal surface. The PVM is a useful model for studying the interactions of commensal vaginal microbes with pathogens and the mechanisms of biofilm formation on the vaginal mucosa.

Staphylococcal superantigens (SAgs), such as toxic shock syndrome toxin-1 (TSST-1), are the main cause of toxic shock syndrome (TSS). SAgs deregulate the host immune system after penetrating epithelial barriers such as the vaginal mucosa. In response to TSST-1, human vaginal epithelial cells (HVECs) produce cytokines and undergo morphological changes. The epithelial signaling mechanisms employed by SAgs remain largely unknown and are the focus of the work presented here. Analysis of published microarray data identified a network of genes up-regulated by HVECs in response to TSST-1 that includes the sheddase, a disintegrin and metalloproteinase 17 (ADAM17). Investigation revealed that the ADAM17 proteolytic targets, amphiregulin (AREG), transforming growth factor α (TGFα), syndecan-1 (SDC1), and tumor necrosis factor receptor 1 (TNFR1), are shed from HVECs in response to TSST-1. TAPI-1 (an ADAM inhibitor) completely abrogates all observed shedding and the production of the cytokine interleukin-8 (IL-8). Knock-down studies show that ADAM17, but not the closely related ADAM10, is required for AREG, TGFα, and TNFR1 shedding. Both ADAM10 and ADAM17 contribute to SDC1 shedding and IL-8 production by HVECs in response to TSST-1. EGFR signaling is critical for up-regulation of IL-8 at the transcriptional level in response to TSST-1 and is also necessary for AREG, TGFα, and TNFR1 shedding. A model is proposed describing the interactions of TSST-1, ADAMs, and the EGFR that lead to establishment of a proinflammatory positive feedback loop in epithelial cells and demonstrate a role for SAgs in the initial stages of disease.

Biofilms complicate treatment of Staphylococcus aureus (SA) wound infections. Previously, we determined alpha-toxin (AT)-promoted SA biofilm formation on mucosal tissue. Therefore, we evaluated SA wound isolates for AT production and biofilm formation on epithelium and assessed the role of AT in biofilm formation. Thirty-eight wound isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (ST), and spa typing. We measured biofilm formation of these SA isolates in vitro and ex vivo and quantified ex vivo AT production. We also investigated the effect of an anti-AT monoclonal antibody (MEDI4893*) on ex vivo biofilm formation by methicillin-resistant SA (USA 300 LAC) and tested whether purified AT rescued the biofilm defect of hla mutant SA strains. The predominant PFGE/ST combinations were USA100/ST5 (50%) and USA300/ST8 (33%) for methicillin-resistant SA (MRSA, n = 18), and USA200/ST30 (20%) for methicillin-susceptible SA (MSSA, n = 20). Ex vivo AT production correlated significantly with ex vivo SA wound isolate biofilm formation. Anti-alpha-toxin monoclonal antibody (MEDI4893*) prevented ex vivo biofilm formation by MRSA USA300 strain LAC. Wild-type AT rescued the ex vivo biofilm defect of non-AT producing SA strains. These findings provide evidence that AT plays a role in SA biofilm formation on epithelial surfaces and suggest that neutralization of AT may be useful in preventing and treating SA infections.

Secreted factors of Staphylococcus aureus can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an ex vivo porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live S. aureus. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an S. aureus exotoxin-mediated disease and may represent an attractive host target for therapeutics.

MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical Factin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.