Secreted virulence factors in lethal illness due to Staphylococcus aureus

Adam Russell Spaulding

doi:10.17077/etd.dx8depdd

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. Rabbit cardiac physiology is considered similar to humans, and rabbits exhibit susceptibility to S. aureus superantigens and cytolysins. As such, rabbits are an excellent model for studying pneumonia, infective endocarditis, and sepsis, We examined the ability of USA200, USA300 and USA400 strains to cause vegetations and lethal sepsis in rabbits. USA200, TSST-1+ strains that produce only low amounts of Α-toxin, exhibited modest LD50 in sepsis (1x108-5x108) colony-forming units (CFUs), and 3/4 caused significant IE. USA200 strain MNPE, which produces high levels of Α-toxin, was both highly lethal (LD50 5x106 CFUs) and effective in causing IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD50s 1 x 106 and 5 x 107 CFUs) but were minimally capable of causing IE. USA400 strains were both highly lethal (LD50s of 1 x 107 and 5 x 107 CFUs) and highly effective causes of IE. Additional studies investigated the role of phenol soluble modulins in infection. We showed that PSMs are important for the ability of S. aureus to cause sepsis but not infective endocarditis. Additionally, immunization against PSMs did not protect rabbits from lethal infection. Our studies show that clonal groups of S. aureus differ in abilities to cause infective endocarditis and lethal sepsis and suggest that secreted virulence factors, including superantigens and cytolysins, account for some of these differences. This thesis also investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (Β-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin Α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins Β-toxin and Γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. Additionally, a heptavalent vaccine composed of the pentavalent units plus SEB and SE-l X protected rabbits from lethal pneumonia caused by USA100 strain 209. Passive immunization using pooled sera protects previously non-immunized rabbits from lethal pneumonia due to MNPE. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.

Secreted virulence factors in lethal illness due to Staphylococcus aureus

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