The vaginal environment is comprised of stratified squamous epithelium, with intracellular lipids to create a permeability barrier. The vaginal mucosa can be colonized by a variety of bacteria, including commensal organisms such as lactobacilli and potentially pathogenic organisms such as Staphylococcus aureus, Streptococcus pyogenes, and Neisseria gonorrhoeae. Both S. aureus and Streptococcus pyogenes secrete toxins known as superantigens that are responsible for causing toxic shock syndrome (TSS). Vaginally, S. aureus can cause menstrual TSS through the production of toxic shock syndrome toxin-1 (TSST-1). In this thesis it is demonstrated that another group of bacterial toxins known as cytolysins can augment penetration of superantigens across porcine vaginal epithelium in an ex vivo model. The staphyloccal cytolysin α toxin induced a proinflammatory cytokine response from human vaginal epithelial cells (HVECs), which is thought to enhance permeability of the epithelium thereby allowing TSST-1 to better traverse the mucosal barrier. The streptococcal cytolysin streptolysin O (SLO), on the other hand, directly damaged the cells of the epithelium, creating holes in the barrier to allow streptococcal pyrogenic exotoxin A (SPE A) to penetrate. SLO also enhanced penetration of Streptococcus pyogenes across ex vivo porcine vaginal epithelium, whereas α toxin did not enhance S. aureus penetration.
Both superantigens are capable of inducing a proinflammatory immune response from HVECs, which is thought to contribute to their penetration of the mucosa and subsequent ability to induce TSS. A dodecapeptide region (12 amino acids) found in all superantigens has been implicated in epithelial interactions. This region is distinct from those residues required for superantigenicity. Alanine mutants were generated along this region for TSST-1 and SPE A, and mutant toxins were tested for their ability to induce IL-8 production from HVECs. Multiple toxin mutants led to lower IL-8 production when incubated with HVECs compared to wild type toxins. All toxin mutants maintained superantigenicity when incubated with peripheral blood mononuclear cells. Select low IL-8 activity mutants were tested in vivo in two rabbit models of TSS. All toxin mutants but one were lethal IV, whereas most low IL-8 activity mutants showed delayed progression to TSS when administered vaginally. Two mutants, D130A TSST-1 and K137A SPE A, were incapable of causing TSS vaginally.
HVECs were further tested for their proinflammatory response to multiple vaginal organisms. While a commensal organism, Lactobacillus, and a latex bead control did not induce IL-8 from HVECs, potentially pathogenic organisms induced a wide range of IL-8 responses from the cells. The fatty acid monoester glycerol monolaurate (GML) blocked all IL-8 responses from HVECs. When incubated simultaneously on HVECs, lactobacilli also blocked all responses to pathogenic organisms. This led to pursuit of a possible anti-inflammatory factor made by lactobacilli. Using transwells, it was shown that Lactobacillus crispatus 01026 secretes a factor responsible for inhibiting the IL-8 response to TSST-1. Supernate collected from an overnight culture of L. crispatus also inhibited T cell proliferation due to TSST-1. Further studies will need to be done to characterize this inhibitory factor. It is possible that both GML and the L. crispatus inhibitory factor will prove to be useful for controlling or preventing inflammatory infections that initiate at the vaginal epithelium.
University of Minnesota Ph.D. dissertation. May 2009. Major: Microbiology, Immunology and Cancer Biology. Advisor: Patrick M. Schlievert. 1 computer file (PDF); xiii, 177 pages.
Brosnahan, Amanda Jo.
Interactions of Staphylococcal and Streptococcal exotoxins with vaginal epithelium.
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