Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Lindsey Shaw, Ph.D.

Committee Member

James Riordan, Ph.D.

Committee Member

Prahathees Eswara, Ph.D.

Committee Member

Wenqi Yu, Ph.D.

Keywords

gene regulation, proteases, Staphylococcus aureus, transcriptional regulation, virulence factors, xdrA

Abstract

Staphylococcus aureus is a highly successful pathogen capable of producing a wealth of virulence factors in the human host. Of note, ten extracellular proteases are produced alongside these virulence factors and play a multifaceted role during infection. They not only cleave host proteins to promote bacterial invasion, immune evasion and survival, but also control disease progression by modulating the stability of self-derived pathogenic determinants. The importance of the secreted proteases modulating virulence factor stability is evidenced by our groups previous finding that a protease-null strain has a substantially increased infectious capacity in a murine model of sepsis; resulting from the unchecked accumulation of virulence factors. However, as the pathogenic potential was only determined for a strain genetically lacking all 10 secreted proteases, the importance of the individual proteases has yet to be determined. As such, we constructed and assessed the pathogenic potential of a combinatorial protease mutant library that revealed the enhanced killing observed for the complete protease-null strain during sepsis appears to be driven by the absence of only Aureolysin and Staphopain A. Further, using a proteomics approach, we identified a number of secreted factors increased in abundance in both the aur/scpA and protease-null mutant, but not in the parent, including LukA, Sbi, and PSMα4. Using our murine model of sepsis, we created an aur/scpA/lukA mutant and found that, although it still exhibited hypervirulence, the progression of infection was substantially delayed in comparison to the aur/scpA mutant. Thus, it appears that LukA is necessary, but not sufficient, for the hypervirulence observed, and that other secreted virulence factors seemingly also contribute to this phenotype. Further to this, given the complex regulatory roles of the secreted proteases, tight regulation of their production is obviously required. Whilst this process has been well studied, a major oversight has been the consideration of proteases as a single entity, rather than 10 enzymes produced from 4 different promoters. As such, we next fully characterized the regulation of each protease gene by known major regulators, discovering vast differences in the way each protease operon is controlled. Additionally, we deepened our understanding of protease regulation using a global screen to identify novel loci controlling protease activity, identifying 7 new elements that strongly altered protease activity, including XdrA, Rbf and Rex. We find that each of the novel regulators, other than ArgR2, appears to influence protease expression though the well studied primary network of protease regulation. Using the data generated from our study, we were able to generate of a comprehensive protease regulatory map that further emphasizes the complexity of their regulation. Lastly, in effort to continue to understand the network controlling virulence factor production, we further characterized XdrA and its effects on S. aureus gene expression. Using an unbiased global analysis, we find that XdrA has a broad impact on the transcriptome, influencing the expression of several important virulence determinants, and factors involved in gene regulation. When assessing the role of XdrA in virulence, we find that an xdrA mutant has an increased ability to survive in whole human blood, mediated in part by increased survival within neutrophils. Furthermore, the increased survival within neutrophils appears to result from an upregulation in expression of sodM, recA, and sae, all of which assist bacterial cells in combating the effects of oxidative stress. In addition to these changes, we find that the xdrA mutant has a decreased abundance of cytolytic toxins, likely resulting from changes in agr and sae activity. We suggest that the broad impact of XdrA on the expression of genes involved in immune evasion, DNA damage, and oxidative stress tolerance, collectively result in a survival advantage, allowing for the increased ability to causes disease in vivo, when xdrA is disrupted. Collectively, this study sheds light onto the role of secreted proteases in the infectious process, generates a comprehensive regulatory map of their regulation, and presents characterization of a novel protease regulator and its unique influence on the pathogenic potential of S. aureus.

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