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.

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.

Vaccination against Staphylococcus aureus pneumonia.

BACKGROUND: Staphylococcus aureus causes serious infections in both hospital and community settings. Attempts have been made to prevent human infection through vaccination against bacterial cell-surface antigens; thus far all have failed. Here we show that superantigens and cytolysins, when used in vaccine cocktails, provide protection from S. aureus USA100-USA400 intrapulmonary challenge. METHODS: Rabbits were actively vaccinated (wild-type toxins or toxoids) or passively immunized (hyperimmune serum) against combinations of superantigens (toxic shock syndrome toxin 1, enterotoxins B and C, and enterotoxin-like X) and cytolysins (α-, ß-, and γ-toxins) and challenged intrapulmonarily with multiple strains of S. aureus, both methicillin-sensitive and methicillin-resistant. RESULTS: Active vaccination against a cocktail containing bacterial cell-surface antigens enhanced disease severity as tested by infective endocarditis. Active vaccination against secreted superantigens and cytolysins resulted in protection of 86 of 88 rabbits when challenged intrapulmonarily with 9 different S. aureus strains, compared to only 1 of 88 nonvaccinated animals. Passive immunization studies demonstrated that production of neutralizing antibodies was an important mechanism of protection. CONCLUSIONS: The data suggest that vaccination against bacterial cell-surface antigens increases disease severity, but vaccination against secreted virulence factors provides protection against S. aureus. These results advance our understanding of S. aureus pathogenesis and have important implications in disease prevention.

Alpha-toxin promotes Staphylococcus aureus mucosal biofilm formation.

Staphylococcus aureus causes many diseases in humans, ranging from mild skin infections to serious, life-threatening, superantigen-mediated Toxic Shock Syndrome (TSS). S. aureus may be asymptomatically carried in the anterior nares or vagina or on the skin, serving as a reservoir for infection. Pulsed-field gel electrophoresis clonal type USA200 is the most widely disseminated colonizer and the leading cause of TSS. The cytolysin α-toxin (also known as α-hemolysin or Hla) is the major epithelial proinflammatory exotoxin produced by TSS S. aureus USA200 isolates. The current study aims to characterize the differences between TSS USA200 strains [high (hla(+)) and low (hla(-)) α-toxin producers] in their ability to disrupt vaginal mucosal tissue and to characterize the subsequent infection. Tissue viability post-infection and biofilm formation of TSS USA200 isolates CDC587 and MN8, which contain the α-toxin pseudogene (hla(-)), MNPE (hla(+)), and MNPE isogenic hla knockout (hlaKO), were observed via LIVE/DEAD® staining and confocal microscopy. All TSS strains grew to similar bacterial densities (1-5 × 10(8) CFU) on the mucosa and were proinflammatory over 3 days. However, MNPE formed biofilms with significant reductions in the mucosal viability whereas neither CDC587 (hla(-)), MN8 (hla(-)), nor MNPE hlaKO formed biofilms. The latter strains were also less cytotoxic than wild-type MNPE. The addition of exogenous, purified α-toxin to MNPE hlaKO restored the biofilm phenotype. We speculate that α-toxin affects S. aureus phenotypic growth on vaginal mucosa by promoting tissue disruption and biofilm formation. Further, α-toxin mutants (hla(-)) are not benign colonizers, but rather form a different type of infection, which we have termed high density pathogenic variants (HDPV).