Graduation Year

2021

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 N. Shaw, Ph.D.

Committee Member

Prahathees Eswara, Ph.D.

Committee Member

Wenqi Yu, Ph.D.

Committee Member

Xingmin Sun, Ph.D.

Keywords

Agr, G5 proteins, MeoW, MroQ, Bacterial Two-Hybrid

Abstract

Virulence factors and the bacterial cell envelope are two important components in S. aureus pathogenesis and survival. More importantly, understanding the regulation of these cellular processes is crucial to further understanding and combating this successful pathogen. To date, numerous factors have been identified as playing a role in the regulation of Agr activity in S. aureus, including transcription factors, antisense RNAs, and host elements. Herein we investigate the product of SAUSA300_1984 (termed MroQ), a transmembrane G5 peptidase family protein, as a novel effector of this system. Using a USA300 mroQ mutant we observed a drastic reduction in proteolysis, hemolysis, and pigmentation that was fully complementable. This appears to result from diminished agr activity, as we observed significant decreases in expression of both RNAII and RNAIII in the mroQ mutant. Such effects appear to be direct, rather than indirect, as known agr effectors demonstrated limited alterations in their activity upon mroQ disruption. A comparison of RNA-sequencing datasets for both mroQ and agr mutants reveal a profound overlap in their regulomes, with the majority of factors affected being known virulence determinants. Importantly, the preponderance of alterations in expression were more striking in the agr mutant, indicating that MroQ is necessary, but not sufficient, for Agr function. Mechanism profiling revealed that putative residues for metalloprotease activity within MroQ are required for its Agr controlling effect, and protein-protein interaction studies indicate that MroQ directly binds to AgrB. Towards this latter point, it appears that the proteolytic activity of MroQ, as well as the C-terminal end of AgrB, is required for such engagement. Finally, virulence assessment demonstrated that mroQ and agr mutants both exhibited increased formation of renal abscesses, but decreased skin abscess formation, alongside diminished dermonecrosis.

Further to this, we investigated the function of a non-peptidase homolog of MroQ, termed MeoW, as a major regulator of cell envelope biogenesis. A USA300 meoW mutant demonstrated minor alterations in hemolytic capacity, however no change in proteolysis or pigmentation was observed. Importantly, the disruption of meoW resulted in significant changes in susceptibility to various cell wall targeting agents, with the most notable being a decrease in susceptibility to lysostaphin and vancomycin, but an increase in susceptibility to penicillin G. Further, a significant decrease in susceptibility to Congo red was observed in a meoW mutant, as well as decreased uptake of positively charged dyes, suggesting alterations of cell surface charge or cell wall composition. Following proteomic and western blot analysis, a substantial decrease in the abundance of teichoic acids was noted, potentially providing an explanation for the cell envelope phenotypes documented. This was corroborated by fluorescence and transmission electron microcopy, which revealed an altered cell envelope, and a thickened cell wall. Finally, the meoW mutant demonstrated an increased capacity to survive in whole human blood, suggesting that MeoW plays an important role in virulence, perhaps as a result of its direct role in cell envelope biogenesis.

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