Graduation Date

Fall 12-19-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Vinai C. Thomas

Abstract

Staphylococcus aureus is a versatile pathogen capable of colonizing and infecting diverse host environments. A key factor in its success is a strong antioxidant system that protects it from host imposed oxidative stress. In this dissertation, we examine how the amino acid cysteine (L-Cys) contributes to the antioxidant response of S. aureus.

L-Cys uptake and biosynthesis are tightly regulated by the transcriptional repressor CymR, yet the benefit of maintaining low intracellular L-Cys levels and the conditions under which its repression is relieved remain unclear. We focus on two oxidative stress conditions encountered during interactions with immune effectors such as neutrophils and macrophages. The first involves disulfide stress, where L-Cys accumulation becomes toxic, and the second involves hypochlorite stress, where L-Cys accumulation is protective.

S. aureus experiences disulfide stress when it gets exposed to hypothiocyanous acid produced by myeloperoxidase. To model this stress, we used diamide, a well characterized inducer of disulfide bonds in cellular thiols. We show that S. aureus overcomes disulfide stress by activating the Spx regulon through oxidation of its redox switch. Spx activation promotes thiol disulfide exchange and increases bacillithiol (BSH) biosynthesis which helps S. aureus maintain a reduced intracellular environment and prevents the need for L-Cys uptake. In mutants unable to activate the Spx redox switch, CymR dependent upregulation of L-Cys transport causes excessive cysteine accumulation. Although increased L-Cys levels help preserve the reduced intracellular environment of S. aureus, it impairs growth due to L-Cys toxicity. Further, we demonstrate that L-Cys toxicity results from depletion of the labile iron pool, reducing iron dependent metabolism and revealing a tradeoff between redox buffering and metal homeostasis.

In contrast, exposure to hypochlorous acid, a potent oxidant that generates chloramines and induces irreversible thiol oxidation, reveals a protective role for cysteine accumulation. We show that increased L-Cys import is central to hypochlorite resistance. Loss of the cystine transporters TcyABC and TcyP lead to increased oxidation of BSH, the major low molecular weight thiol in S. aureus, and extensive protein thiol oxidation. Our findings support a model in which high intracellular L-Cys, rather than BSH, serves as the primary thiol sink for hypochlorous acid.

Finally, we show that regulation of L-Cys uptake is essential for S. aureus survival within human neutrophils. Together, our work reveals how S. aureus fine-tunes intracellular L-Cys availability to avoid toxicity while maximizing its value as a redox buffer. These insights uncover new vulnerabilities in L-Cys and redox homeostasis that may be exploited to enhance immune clearance.

Comments

2025 Copyright, the authors

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Available for download on Saturday, December 11, 2027

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