Contribution of Spx to Staphylococcus aureus physiology and virulence

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PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a potent human pathogen due to its large repertoire of virulence factors, allowing this bacterium to infect any part of the human body. Our long-term goal is to understand the mechanisms by which S. aureus modulates virulence factor expression and activity to cause disease. This includes uncovering novel pathways contributing to virulence factor production and responses to environmental changes. The Spx protein in used by Gram-positive bacteria to regulate gene expression in response to disulfide and oxidative stress. Most of what is known about Spx stems from studies in Bacillus and Streptococcus and only a few studies have examined the importance of Spx in S. aureus. The interpretation of two of those studies are hampered due to secondary site mutations that were later discovered. Recently, we published a study that included an spx mutant in a clinically relevant MRSA strain of the USA300 lineage. In this background, Spx contributes to survival under diamide stress and pigment production. Spx is essential in S. aureus but can be circumvented by controlled expression of thioredoxin, a technique we employ. Despite thioredoxin production, the spx mutants have a growth defect. We also have new preliminary data that the spx mutant has altered activity of key hemolytic secreted proteins. The reason for the growth deficiency and altered virulence factor production has not been elucidated and is the focus of this application. Based on our preliminary data, we hypothesize that Spx modulates metabolism and key regulatory networks that control virulence factor expression. To test this, three aims are proposed. Aim 1 uses RNAseq ChIPseq, and targeted metabolomics to define the Spx regulon and impact of Spx on metabolism. Aim 2 builds on our preliminary data showing reduced hemolysin activity to identify the mechanism behind this phenotype. This examines key regulatory networks believed to be involved using a combination of mutants, reporters, and quantitative assays. Aim 3 tests for the first time the contribution of Spx to S. aureus virulence using a murine skin infection model. Furthermore, it uses live animal imaging to examine virulence factor expression in vivo. Completion of these studies will provide insight into virulence factor regulation in this important pathogen. In addition, it will be the first to define the Spx regulon in S. aureus and determine the contribution of Spx to S. aureus virulence.