Graduation Year

2024

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Lindsey N. Shaw, Ph.D.

Committee Member

Wenqi Yu, Ph.D.

Committee Member

Sophie Darch, Ph.D.

Keywords

MRSA, regulation of virulence, Staphylococcus aureus, TetR family regulator

Abstract

Point-mutation variants of the TetR family regulator, FarR, have emerged across Staphylococcus aureus sequence types as conferring antibiotic and fatty acid resistance, particularly in Hospital-Associated Methicillin Resistant S. aureus (HA-MRSA) strains. FarR also exhibits an additional largely uncharacterized relationship to virulence factor production, seemingly through the global repressor of toxins—Rot, which functions to regulate S. aureus’ ability to colonize and persist in the host via a complex cascade of signaling. While Rot protein is post-transcriptionally regulated by the highly characterized Agr system, there is little known about its transcriptional regulation. Herein, we explored these dynamics by initially characterizing virulence phenotypes in a farR transposon mutant of strain USA300 LAC. Specifically, the farR mutant displayed a decrease in proteolysis, cytolysis, and hemolysis, accompanied by an increase in biofilm production. The mutant also exhibited a decreased ability to escape from—but increased ability to survive within—human neutrophils, mirroring a phenotypic relationship characteristic of low virulence mutants. Using Western blot analysis, we determined that the farR mutant had increased abundance of surface protein A (Spa), whilst diminished production of both the Panton-Valentine leukocidin (PVL) and alpha-hemolysin (Hla). A subsequent transcriptional analysis highlighted decreased expression of myriad secreted virulence factors in the mutant strain, as well as identified potential regulators of Rot. These findings explicate the role of FarR as an indirect global regulator of pathogenicity in conjunction with the transcriptional regulation of Rot. Site-directed alanine mutagenesis and protein modeling shed light on how specific residues may contribute to FarR’s mechanism of action. As such, further investigation of FarR structure and function are ongoing to explore exactly how FarR fulfills such a role.

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