Laura M. Breshears

Superantigens Are Critical for Staphylococcus aureus Infective Endocarditis, Sepsis, and Acute Kidney Injury

ABSTRACT 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 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.

A disintegrin and metalloproteinase 17 (ADAM17) and epidermal growth factor receptor (EGFR) signaling drive the epithelial response to Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1).

Background: The staphylococcal superantigen (SAg) toxic shock syndrome toxin-1 (TSST-1) is proinflammatory to mucosal epithelial cells by an unknown mechanism. Results: Metalloproteinases and the epidermal growth factor receptor mediate the epithelial response to TSST-1. Conclusion: Inflammatory signals from the epithelium contribute to SAg-mediated disease progression. Significance: Understanding how SAgs initiate the inflammatory cascade may provide molecular targets for the development of new therapies. 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.

An unconventional myosin required for cell polarization and chemotaxis

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 F-actin, 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.

Epithelial proinflammatory response and curcumin-mediated protection from staphylococcal toxic shock syndrome toxin-1.

Staphylococcus aureus initiates infections and produces virulence factors, including superantigens (SAgs), at mucosal surfaces. The SAg, Toxic Shock Syndrome Toxin-1 (TSST-1) induces cytokine secretion from epithelial cells, antigen presenting cells (APCs) and T lymphocytes, and causes toxic shock syndrome (TSS). This study investigated the mechanism of TSST-1-induced secretion of proinflammatory cytokines from human vaginal epithelial cells (HVECs) and determined if curcumin, an anti-inflammatory agent, could reduce TSST-1-mediated pathology in a rabbit vaginal model of TSS. TSST-1 caused a significant increase in NF-κB-dependent transcription in HVECs that was associated with increased expression of TNF- α, MIP-3α, IL-6 and IL-8. Curcumin, an antagonist of NF-κB-dependent transcription, inhibited IL-8 production from ex vivo porcine vaginal explants at nontoxic doses. In a rabbit model of TSS, co-administration of curcumin with TSST-1 intravaginally reduced lethality by 60% relative to 100% lethality in rabbits receiving TSST-1 alone. In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin treated rabbits that survived. These data suggest that the inflammatory response induced at the mucosal surface by TSST-1 is NF-κB dependent. In addition, the ability of curcumin to prevent TSS in vivo by co-administration with TSST-1 intravaginally suggests that the vaginal mucosal proinflammatory response to TSST-1 is important in the progression of mTSS.

Epidermal Growth Factor Receptor Signaling Enhances the Proinflammatory Effects of Staphylococcus aureus Gamma-Toxin on the Mucosa

Staphylococcus aureus (S. aureus) produces many different exotoxins including the gamma-toxins, HlgAB and HlgCB. Gamma-toxins form pores in both leukocyte and erythrocyte membranes, resulting in cell lysis. The genes encoding gamma-toxins are present in most strains of S. aureus, and are commonly expressed in clinical isolates recovered from menstrual Toxic Shock Syndrome (mTSS) patients. This study set out to investigate the cytotoxic and proinflammatory effects of gamma-toxins on vaginal epithelial surfaces. We found that both HlgAB and HlgCB were cytotoxic to cultured human vaginal epithelial cells (HVECs) and induced cytokine production at sub-cytotoxic doses. Cytokine production induced by gamma-toxin treatment of HVECs was found to involve epidermal growth factor receptor (EGFR) signaling and mediated by shedding of EGFR ligands from the cell surface. The gamma-toxin subunits displayed differential binding to HVECs (HlgA 93%, HlgB 97% and HlgC 28%) with both components (HlgAB or HlgCB) required for maximum detectable binding and significant stimulation of cytokine production. In studies using full thickness ex vivo porcine vaginal mucosa, HlgAB or HlgCB stimulated a dose-dependent cytokine response, which was reduced significantly by inhibition of EGFR signaling. The effects of gamma-toxins on porcine vaginal tissue and cultured HVECs were validated using ex vivo human ectocervical tissue. Collectively, these studies have identified the EGFR-signaling pathway as a key component in gamma-toxin-induced proinflammatory changes at epithelial surfaces and highlight a potential therapeutic target to diminish toxigenic effects of S. aureus infections.

Motor Proteins: Tightening Your Belt with Myosin VI

New work shows that the motor protein myosin VI, acting through vinculin, plays a key role in the maturation of cadherin-based adherens junctions in epithelial cells.

Alpha-Toxin Contributes to Biofilm Formation among Staphylococcus aureus Wound Isolates

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.

Local epidermal growth factor receptor signaling mediates the systemic pathogenic effects of staphylococcus aureus toxic shock syndrome

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.

Protocol for Examining Human Vaginal Epithelial Cell Signaling in Response to Staphylococcal Superantigens.

A detailed investigation of eukaryotic signaling pathways affected by bacterial products is key to our understanding of host-pathogen interactions. Cytokine expression appears to be an important initial host cell response to many bacterial products, including the Staphylococcus aureus superantigens (SAgs). While much is understood about how SAgs signal to immune cells, very little is known about the specific cellular pathways activated by SAgs on nonimmune cells such as those of the epithelium. Here, we describe methods for analyzing SAg signaling in cultured epithelial cells, which may be extrapolated to the analysis of signaling pathways induced by other bacterial ligands on a variety of cell types.