Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Lindsey Shaw

Keywords

Cell Wall Damage, DNA Damage, Pathogenesis, Purine Biosynthesis, Regulator

Abstract

Previously our laboratory had identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function ó factor, óS, involved in stress response and disease causation. Here we present additional characterization of óS, demonstrating a role for it in protection against DNA damage, cell wall disruption and interaction with components of the innate immune system. Promoter mapping reveals the existence of four unique sigS start sites, one of which appears to be subject to auto-regulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild-types, but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate (MMS) and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Ex vivo transcriptional analysis reveals that significant expression of sigS can be induced upon phagocytosis by RAW 264.7 murine macrophage-like cells. Regulation of óS appears to be unique, as the downstream encoded protein, SACOL1828, seemingly acts as a positive activator, rather than as an expected anti-sigma factor. Using a global transposon screen we have elucidated additional genes implicated in the regulation of sigS, including those involved in cell wall stability, cellular detoxification, virulence and DNA base excision repair. Phenotypically, óS mutants display sensitivity to a broad range of DNA damaging agents, such as ultraviolet light, MMS and ethidium bromide. These effects are seemingly mediated via regulation of the purine biosynthesis pathway, as microarray, proteomic and qRT-PCR analysis of óS mutants reveal decreased transcription of all genes involved. Enzymatic profiling of PurA involved in adenine biosynthesis, demonstrates decreased activity in the óS mutant. Finally, we provide further evidence for the role of óS in S. aureus pathogenesis, revealing that sigS mutants display decreased ability to cause localized infections and are impaired in their interactions with components of the human innate immune system. Collectively, our data argues for the important, and perhaps novel, role of óS in the stress and virulence responses of S. aureus.

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