Protein-based Tools for Studying Metabolite Signaling and Protease Activation in Gastrointestinal Pathogens

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Hatzios, Stavroula

Abstract

Certain bacterial pathogens are highly adept at infecting specific animal hosts. Such host-adapted bacterial pathogens have evolved to obtain many nutrients from the host environment, rather than synthesizing these metabolites de novo. The work presented in this dissertation primarily investigates how the host-adapted gastric pathogen Helicobacter pylori interacts with diet-derived, redox-active nutrients to enhance its fitness and stress tolerance. In Chapter 2, we describe our initial efforts to understand how small molecules may protect H. pylori from redox stress in the human stomach. Using a combination of chemical biology and microbiological approaches, we uncovered a new mechanism of microbial redox homeostasis: import of the LMW thiol ergothioneine (EGT). We structurally and functionally characterized the microbial EGT importer, EgtUV, thereby providing mechanistic insight into how the EgtU solute-binding domain (SBD) recognizes EGT. These studies may provide a roadmap for future efforts to identify additional EGT-interacting proteins with as-yet unknown influences on microbial physiology. H. pylori, like many other host-adapted bacterial pathogens, grows poorly unless cultured in complex, chemically undefined media. Therefore, understanding how such pathogens respond to specific nutritional cues has remained a longstanding challenge in the field. In Chapter 3, we describe our efforts to define EGT-specific responses in H. pylori. Our strategy was to develop the H. pylori EgtU SBD into a solid resin (“EgtU-resin”) to deplete complex growth media of EGT, enabling comparisons of H. pylori strains grown in high versus low EGT conditions and the identification of EGT-dependent phenotypes. These studies not only advance our understanding of EGT biology in a widespread human pathogen but also establish a new technology that could be adapted to control bacterial exposure to metabolites of interest. EGT can chelate metals, but the relevance of this property to biological systems has been unclear. Our studies with EgtU-resin provide initial evidence of a connection between EGT availability and nickel homeostasis, as removal of media EGT also reduced the nickel content of the media. In Chapter 4, we begin to establish a framework connecting EGT import and nickel-specific responses in H. pylori. We show that H. pylori ∆egtU strains have differential expression of nickel-responsive genes and lower intracellular nickel levels. However, this strain still retains nickel-dependent enzyme activity. Further work to define EGT-related contributions to nickel homeostasis may have implications for host—pathogen dynamics, as nickel is essential for H. pylori infectivity. Additionally, we expand connections between EgtUV and micronutrients more generally by showing that the recently discovered selenium-containing analogue of EGT called selenoneine (SEN) is imported by the microbial EGT transporter. Future work will be needed to define the sources of SEN available to bacteria during infection as well as position EGT within the context of nickel and selenium micronutrient acquisition in H. pylori. Beyond understanding nutrient-specific responses, another pressing problem in the field of microbial pathogenesis is discriminating between a bacterial pathogen and a commensal in a mixed community. Rapid and specific identification of a gastrointestinal pathogen from a patient sample containing native microbial flora can reduce time to diagnosis and prevent administration of broad-spectrum antibiotics, which are known to contribute to dysbiosis and the spread of antimicrobial resistance (AMR). To approach this problem, in Chapter 5 we describe the generation of a fluorescent biosensor for specific pathogen detection using the propeptide domain of a bacterial protease. Using the pathogen Vibrio cholerae to establish proof-of-concept, we show that the I9 propeptide domain of the V. cholerae enzyme IvaP can be functionalized with environment-sensitive dyes. Degradation of I9-biosensors by purified IvaP, V. cholerae supernatants, or V. cholerae cultures produces a measurable change in the fluorescence emission of the probe. Importantly, the I9-biosensor exhibits limited off-target activation by proteases from other common gastrointestinal pathogens or cultured intestinal cells. Altogether, we show that the specificity of proteases for degrading their cognate propeptide domains can be used to provide specificity for pathogen detection in polymicrobial communities. As secreted proteases with propeptide domains are present in many bacteria, the I9-biosensor design provides a platform for developing additional sensors for other pathogens. Altogether, this work has defined new mechanisms of microbial nutrient acquisition and established protein-based tools for studying metabolite signaling and protease activation in gastrointestinal bacterial pathogens. In pursuing questions of fundamental bacterial physiology, we have uncovered proteins with advantageous properties for use as research tools and biosensors. We anticipate that these tools will, in turn, facilitate new discoveries in bacterial pathogenesis.

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