Examination of a New Virulence Factor in a Select Strain of Methicillin Resistant Staphylococcus aureus

In this dissertation I address the topic of a newly discovered virulence factor in a select strain of methicillin resistant Staphylococcus aureus (MRSA) JKD6159. S. aureus has been able to acquire resistance to a wide variety of antibiotics including methicillin and vancomycin. While this antibiotic resistance is a growing concern, S. aureus also encodes an assortment of virulence factors. In this dissertation, we examine the emergence of a streptolysin S like gene cluster (sag) gene cluster that has appeared in a select strain of MRSA. The sag (Streptolysin S-associated gene) cluster is a major virulence factor in Streptococcus pyogenes, as it is responsible for the biosynthesis of the historical hemolysin toxin known as Streptolysin S. Using bioinformatic analysis, we have identified the presence of the sag gene cluster in the MRSA strain JKD6159, a recent MRSA isolate from Australia. To examine the function of the Sag-associated gene cluster in the MRSA JKD6159 isolate, we took several experimental approaches to gain a deeper understanding of the mechanistic role of this gene cluster in the JKD6159 isolate. We generated an isogenic mutant in which one of the genes responsible for the biosynthesis of the putative SLS-like toxin was inactivated (JKD::ΔsagB) in order to more precisely determine the role of the presence of this gene cluster in the MRSA JKD6159 strain. Disruption of the sag gene cluster in JKD6159 did not result in significant differences in hemolysis. Bacterial growth and biofilm formation was also not found to be affected between wt and the ΔsagB mutant. Conditioned media from the wt and ΔsagB strain do not result in any growth inhibition of S. pyogenes, E. coli, or S. epidermidis. During co-culture experiments with JDK6159 and E. coli, differential morphology of the E. coli can be observed between the wt JKD6159 and the ΔsagB strain. Eukaryotic cell infections with wt and ΔsagB JKD6159 did not result in differential cytotoxicity or differential cytokine expression. In vivo mice infections revealed no significant difference in either lesion size or recovered CFUs between the wt, ΔsagB or ΔsagB+sagB strains. Analysis of in vitro generated toxin revealed no evidence of the hypothesized post translational modifications. 2D proteomic studies were also undertaken to examine if the sag cluster played a role in the regulation of expressed proteins in JKD6159. Metabolic protein differences were observed between the wt and ΔsagB strains including an increase in carbamate kinase and threonine synthase expression and a decrease in the catabolite control protein A and MraW in wt vs. SagB mutants. Although we have obtained evidence 1. that E. coli during co-culture exhibits differential morphology between wt and ΔsagB 2. that the Sag-like gene associates with the production of different S. aureus proteins during in vitro growth, the exact role of the Sag-like gene cluster in the MRSA strain JKD6159 remains to be determined.