Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Hatzios, Stavroula

Abstract

Helicobacter pylori is a gastric pathogen that chronically infects up to half of the global population and is the strongest risk factor for gastric cancer. Host cells infected with H. pylori are exposed to oxidative stress characterized by the continuous production of reactive oxygen species (ROS). While the correlation between H. pylori-induced chronic oxidative stress and disease has been well-documented, the molecular oxidation events that contribute to pathogenesis are largely unknown. The main intracellular targets of ROS are proteins, particularly those with redox-sensitive cysteine residues. Oxidation of reactive cysteines, termed redox signaling, can alter protein behavior and modulate important cellular processes, yet the role of cysteine oxidation in host responses to H. pylori infection, or any infection, has been underexplored. The work within this dissertation begins the exploration of how cysteine oxidation events during H. pylori infection can have significant contributions to pathogenesis. Chapter 1 provides an overview of H. pylori-induced oxidative stress and the accompanying oxidation of cellular biomolecules. While oxidation of DNA and lipids in host cells during H. pylori infection has been well established, only one study has examined host protein oxidation and reported increased levels of oxidized proteins in H. pylori-infected patients with no further investigation of their impact on patient health. Thus, this chapter highlights the need for understanding how redox signaling affects host responses to infection, and particularly how cysteine oxidation events may promote H. pylori-induced pathogenesis. Chapter 2 details depletion of the host antioxidant glutathione during H. pylori infection, a phenotype that contributes to the microbial-induced oxidative stress. While the mechanisms that drive this process are being explored in detail by my colleague in the Hatzios lab, this chapter describes the use of this phenotype for establishing infection conditions that promote oxidative stress. Using these conditions, it was possible to further study how oxidation of cysteines during infection-induced oxidative stress affects host cell biology. Chapter 3 describes the novel use of reactive cysteine profiling at the host–pathogen interface to uncover the potential sites of cysteine oxidation in H. pylori-infected cells. These data provide interesting insight into the H. pylori infection by identifying changes in host protein abundance and cysteine reactivity, and, importantly, highlighting specific cysteine residues that may be oxidized during infection. Chapter 4 focuses on the protease legumain, one of the proteins identified in Chapter 3 with reduced cysteine reactivity following infection with H. pylori. We confirmed that legumain Cys219 is oxidized during H. pylori infection, and demonstrated that legumain oxidation dysregulates intracellular legumain processing and decreases the activity of the enzyme in H. pylori-infected cells. Further, we showed that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Altogether, the findings in this dissertation establish a link between an infection-induced oxidation site and pathogenesis, particularly tumorigenesis, while underscoring the value of chemical proteomics in uncovering reactive cysteines that promote disease.

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