Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Molecular Biosciences

Major Professor

Lindsey Shaw, Ph.D.

Committee Member

Prahathees Eswara, Ph.D.

Committee Member

Wenqi Yu, Ph.D.

Committee Member

Sophie Darch, Ph.D.

Keywords

clinical isolates, proteases, Staphylococcus aureus, transcriptional regulation, USA300

Abstract

Staphylococcus aureus is a Gram-positive opportunistic pathogen capable of causing myriad diseases, ranging from skin infections to pneumonia, endocarditis, and sepsis. It does this via the production of an array of virulence factors including toxins, adhesins, immunoglobulin-binding proteins, and proteases. Towards these latter elements, S. aureus produces ten secreted proteases that play a dual role in infection: firstly, by modulating the stability and abundance of other virulence factors, and secondly, by cleaving host factors to facilitate infection. Given their importance, we set out to explore the natural variability of protease production in a collection of clinical isolates from Tampa General Hospital. These strains first underwent phenotypic characterization followed by whole genome sequencing (WGS). The goal of this project was to explore proteolytic variation and connect that with genomic alterations, so as to mechanistically define factors critical for proteolysis in clinical isolates. Upon analysis, we observed a spectrum of protease activity, of which mutations in the scp promoter, aur promoter, agr promoter, and genes for Aureolysin, AgrC, MroQ, SarT, SaeS, SarX, and Rot appear to contribute to the proteolytic variation observed across USA300. One of these USA300 isolate, TGH337, was of interest as it exhibited profoundly enhanced proteolysis. As highly proteolytic S. aureus strains are typically avirulent, we endeavored to determine the regulatory basis for this unique observation. WGS and transcriptomics were performed, revealing the proteases Aureolysin (68.7-fold), Staphopain B (4.9-fold), and V8 (4.1-fold) displayed increased transcription. No known protease regulators displayed altered expression in TGH337, however thirteen other regulators were changed in abundance. Of these, increased SarZ and AhrC was found to be causative of the hyper-proteolysis of TGH337, as disruption of either sarZ or ahrC restored wild-type proteolysis levels. We then performed a screen of transcriptional reporters, to identify the factor responsible for the upregulation of SarZ and AhrC in TGH337. This screen identified novel regulators of both loci, however none of the identified regulators of AhrC were altered in TGH337 in a manner which would explain its increased abundance. None of the identified regulators of SarZ were altered in a causative manner, until we identified a non-synonymous single nucleotide polymorphism (nSNP) in TGH337 in the major virulence regulator Rot (Repressor of Toxins), which we also found to be a repressor of sarZ. Complementation studies performed with wild-type and nSNP-containing Rot revealed the T69M mutation in TGH337 inhibits the ability of Rot to repress members of its regulon, including sarZ, aur, and sspAB. Thus, we reveal a novel circuit of protease regulation whereby inactive Rot contributes to increased abundance of SarZ, resulting in hyper-proteolysis in TGH337. Given that this work highlights gaps present in our understanding of protease regulation – particularly in clinical strains – we next developed a novel, high-throughput screen to explore the multifaceted regulation of the proteases. This works by coupling the use of luciferase-based reporter gene fusions with the miniaturization of transduction to a 96-well plate format. We then employed this screen to further investigate the spl and scp loci, as they are understudied in comparison to the aur and ssp loci. In so doing, we identified thirty-seven novel regulators of the spl promoter and six novel regulators of the scp promoter. Collectively, this study expands our comprehension of the highly sophisticated regulatory network controlling secreted protease production. This also shines new light on virulence factor control and suggests that regulatory circuits may be strikingly different in strains from infected patients, highlighting the importance of utilizing clinical isolates.

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