Date of Award
Master of Science (MS)
Microbiology and Immunology
Staphylococcus aureus is an opportunistic Gram-positive pathogen that transiently colonizes 30% of the population at any given time. Upon breaching host skin or mucosal barriers, S. aureus can infect and cause disease in nearly all host tissues. Once inside the host, S. aureus expresses a plethora of virulence factors, mostly regulated by the Accessory Gene Regulatory (Agr) quorum sensing system, which facilitates the evasion of immune responses or directly promotes pathogenesis. The S. aureus Agr system relies on the production of a small peptide known as an auto-inducing peptide (AIP) to control virulence gene expression in response to cell density. AIP is generated from a precursor peptide that is cleaved at its C-terminus by a protease known as AgrB. Recent work from our lab and others identified an additional protease known as the Membrane Regulator of Agr Quorum Sensing (MroQ) that was recently shown to be a critical component for Agr system activity. MroQ cleaves the N-terminal leader peptide of the AIP precursor, resulting in the full maturation of AIP. Despite the critical role MroQ plays in S. aureus pathogenesis and quorum sensing, much of MroQ and its biochemical properties remain elusive. To facilitate our study of MroQ, I explored its topology in the S. aureus membrane. Using the substituted cysteine accessibility method (SCAM), I labeled engineered cysteines that were introduced at several positions along the length of the MroQ protein in order to assign locations of individual residues to the extracellular or intracellular face of the membrane. My research findings provided insight into the overall topology of MroQ, where I have shown that the C-terminus of the MroQ is positioned on the extracellular side of the cell, and identified residues at several periodic positions across the length of the protein that are also positioned on the extracellular face of the cell. My experimental results largely align with one of the two computational models of MroQ topology. Altogether, my data refine our knowledge of the MroQ structure and support predicted topology that includes seven transmembrane passes with the N-terminus of the protein located in the intracellular compartment and C-terminus located in the extracellular region. These results refine our overall understanding of MroQ protein and its function as well as its possible role in other cellular functions.
Bhusal, Sagar, "Investigating Structural and Biochemical Characteristics of a Peptidase Required for Staphylococcus Aureus Virulence" (2023). Master's Theses. 4471.
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Available for download on Monday, July 21, 2025