Project description:Group A Streptococcus (GAS) is one of the world’s most successful pathogens, causing a multitude of common infections such as pharyngitis, cellulitis, and impetigo. It is also responsible for more severe diseases such as rheumatic fever, necrotizing fasciitis, and toxic shock syndrome. Group A Streptococcus produces a powerful peptide toxin known as streptolysin S (SLS). Although recent advances have begun to uncover the structure of SLS, many questions remain as to its route of synthesis, export and function. A fundamental yet unanswered question about SLS is the mechanism employed by GAS to resist the effects of its own toxin. To address this question, we developed a unique microarray-based approach aimed at identifying bacterial genes involved in SLS immunity. We measured changes in gene expression in a non-SLS producing GAS strain (∆SLS) after exposure to active SLS, as well as to an inactive SLS isoform. Initial exposure of a non-SLS producing GAS to the native SLS toxin resulted in significant upregulation of several gene candidates. Proteins encoded by these gene candidates were produced and tested for their ability to neutralize SLS toxin activity in vitro. The protein encoded by the gene Spy_0787 decreased the cytolytic activity of wt SLS by 50%. Bacterial immunity is still a relatively unknown and unexplained phenomena. Insights into how toxin-producing microorganisms achieve resistance against the effects of their own toxin could uncover important therapeutic targets to neutralize virulence factors such as SLS, as well as other related peptide toxins. The microarray technique described here can be leveraged to other microbial systems to understand how microorganisms in general react to antibacterial and cytotoxic compounds for which the mechanism of action is unknown. Supernatants containing wt and S39A forms of SLS secreted by GAS strains were collected from 50 ml of overnight cultures of each strain at 37 degrees C in Todd Hewett broth supplemented with 10 mg/ml of bovine serum albumin fraction V (Sigma chemicals) in order to stabilize the toxin. The supernatant was then isolated and subjected to filter sterilization. For SLS exposure conditions, bacterial pellets containing GAS ∆SagA mutants were incubated with 1. supernatants containing wtSLS; 2. supernatants containing SLS S39A isoform; or 3. sterile TH media with 10mg/ml of BSA. The bacteria pellets were quickly resuspended after which the bacterial pellets were recollected for RNA isolation. A total of three exposure timepoints were used: 0h (immediately after exposure to SLS-containing supernatants), 3h post-exposure, and 6h post-exposure. The pellets were frozen at -80C until used for RNA extraction. Total RNA extraction and purification from the bacterial pellets were performed using the RNeasy isolation kit per manufacturer's instructions (Qiagen). RNA samples were processed following standard Roche NimbleGen gene expression analysis protocols. Double-stranded cDNA was synthesized from 10 μg of total RNA using random hexamer primers with the SuperScript cDNA synthesis kit (Invitrogen). One μg of cDNA was labeled with Cy3-random nonamers (Roche NimbleGen) for microarray hybridization.

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