Date of Award

2019

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

Abstract

Staphylococcus aureus infections are a global burden on human health. The wide rangeof infectious diseases caused by S. aureus stem from the production of myriad virulence factors that manipulate host immune responses and promote bacterial survival. These virulence traits, coupled with the rise in antibiotic resistance, demonstrate a need for novel therapeutics to control infection. Many current therapeutic initiatives focus on targeting the virulence factors produced by S. aureus or the regulatory systems that control their expression such as the Accessory gene regulatory system, Agr.Initial characterization of a putative S. aureus peptidase MroQ indicates a major role in regulation of the Agr system. A DmroQ mutant phenocopies a Dagr mutant for decreases in global secreted proteins, Agr-regulated secreted factors, and AIP production. Furthermore, DmroQ and Dagr mutants are attenuated for bacterial burden and exhibit reduced gross pathology during murine skin and soft tissue infection at 120 hours. From these results, I hypothesized that MroQ promotes the maturation and transport of Agr AIP. The first goal of my thesis work was to explore the role of MroQ in regulating Agr function. By assessing the ability of WT S. aureus condition medium to activate DmroQ, DmroQ DagrB, and DagrB transcriptional reporter strains, I determined that MroQ is involved in the processing and export of AIP and does not affect the AgrC/A two-component system. I also found that a DmroQ mutant has a reduced ability to process AgrD when AgrD is constitutively expressed, suggesting that MroQ is required for efficient AIP processing.Bioinformatic analysis suggests that MroQ is a putative Type-II CAAX protease containing 7 transmembrane a-helices and two conserved catalytic motifs, EEXXXR andFXXXH. The second goal of my thesis was to interrogate the role of these conserved catalytic motifs in the function of MroQ and subsequent activation of the Agr system. To assess if conserved amino acids E141, E142, or H180 are required for MroQ function, I generated three strains each harboring one site-directed amino acid substitution (E141A, E142A, and H180A) followed by characterizing exoprotein profiles, AIP production, and in vitro or in vivo pathogenic functions. From these experiments, I concluded that all three catalytic amino acids were important for MroQ function and its regulation of Agr activity. Through use of N-terminally tagged AgrD expression constructs, I was able to assess the direct effect of MroQ and its active site mutants on AgrD processing. DmroQ, DmroQ + mroQ(E142A), and DmroQ + mroQ(H180A) strains fail to process AgrD and cannot secrete mature peptide leading to accumulation of processing intermediates in the bacterial cytosol. Interestingly, a DmroQ + mroQ(E141A) strain does not generate stable AIP or any of its processing intermediates, suggesting a role for E141 in conferring AgrD stability. From these experiments I concluded that a WT allele of MroQ is required for efficient AIP maturation (either processing or transport), resulting in optimal Agr activity and S. aureus pathogenesis.The final goal of my thesis was to investigate the role of MroQ in the processing of AgrD variants. I generated congenic strains containing allelic variants of the Agr system by transducing Agr Type I-IV into a Dagr and DmroQ Dagr and evaluated protein secretion and hemolytic activity, two markers of Agr activation. The results of these experiments indicated that MroQ is required for Agr Type I, II, and IV activity, but not for Agr Type III activity. These studies further our understanding of how S. aureus regulates Agr activity and identify an Agr Type that has diverged in regard to Agr regulation.

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Available for download on Monday, August 12, 2024

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