Browsing by Subject "Cytolysins"
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Item Anti-virulence paradigm for development of staphylococcal therapies(2010-02) Lin, Ying-ChiStaphylococcus aureus is a major human pathogen capable of causing various diseases, from skin infections to life-threatening pneumonia and toxic shock syndrome (TSS). S. aureus exoproteins contribute significantly to S. aureus pathogenesis via causing inflammation, tissue disruption, and immune evasion. Antibiotics treat S. aureus disease by eliminating bacteria, but provide no protection from S. aureus exoproteins, once released. With the emergence of antibiotic-resistant S. aureus, new therapeutic options to treat/prevent S. aureus-associated disease are critical. Given most S. aureus diseases initiate locally on mucosal surfaces or the skin, it was hypothesized that S. aureus exoproteins that have pro-inflammatory and/or cytotoxic effects on epithelial cells, contributing directly to S. aureus pathogenesis. Therefore, anti-staphylococcal therapies that inhibit toxin production and/or prevent toxin effects on host cells could reduce or prevent S. aureus infections. A global approach was taken to characterize pro-inflammatory properties of exoproteins from two genetically close TSS S. aureus isolates, a pulmonary TSS isolate (MNPE) and a menstrual TSS isolate (CDC587), on epithelial cells. Cytolysins, alpha- and gamma-toxins, superantigens, and staphopain (protease) were determined as the most pro-inflammatory (via interleukin-8) to epithelial cells. MNPE, originating from skin, produced large amounts of alpha-toxin and SAgs, but little other virulence factors, whereas CDC587, a mucosal strain, produced gamma-toxin, small amounts of alpha-toxin, and large numbers of secreted virulence factors. These findings implied that pro-inflammatory S. aureus exoproteins play key roles in environmental selection and disease severity. As proof of principle, glycerol monolaurate (GML), a lauric acid monoester known to inhibit S. aureus exoprotein production and to have anti-inflammatory effects, was compared with its monoether, dodecylglycerol (DDG), as local anti-virulence agents to prevent S. aureus disease using a rabbit wiffleball abscess/TSS model. GML, but not DDG, significantly decreased TSST-1 and local inflammation (via tumor necrosis factor-alpha in the wiffleball and prevented rabbit death from TSS. In summary, these studies identified key exoproteins important in S. aureus mucosal pathogenesis and determined the potential for anti-toxin agents, such as GML, to treat and/or prevent S. aureus diseases. These studies also suggest the addition of anti-toxin components will improve the effectiveness of antistaphylococcal vaccines and immunotherapies.Item Interactions of Staphylococcal and Streptococcal exotoxins with vaginal epithelium(2009-05) Brosnahan, Amanda JoThe 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.