Author ORCID Identifier

0000-0002-2565-5607

Date of Award

Fall 12-8-2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Cynthia N. Cornelissen, PhD

Second Advisor

Zehava Eichenbaum, PhD

Third Advisor

Timothy L. Denning, PhD

Abstract

Neisseria gonorrhoeae and its obligate human host require transition metals for biological functions including cell signaling and metabolism, gene regulation, enzymatic processing, and oxidative stress resistance. During infection, the host employs two innate immunity mechanisms: either starve the pathogen of metals or overload the pathogen with intoxicating amounts of metals, either resulting in bacterial growth inhibition. The mechanisms by which the host starves or overloads the pathogen with metal nutrients are termed nutritional immunity and metal overload, respectively. In response to nutritional immunity and metal overload, N. gonorrhoeae differentially expresses metal transport systems that allow it to maintain homeostatic metal concentrations within the cytoplasm. Such transporters include the high-affinity zinc importer, ZnuABC, which is regulated by the zinc uptake regulator Zur, and the manganese exporter, MntX. As the gonococcus acquires and maintains antibiotic resistance mechanisms, the necessity to develop novel therapeutics and treatments becomes more urgent. Metal transporters are attractive therapeutic targets as they are often required for survival and virulence. However, the precise mechanism by which zinc and manganese are transported, sensed by Zur, and impact the transcriptional response to maintain metal homeostasis have not yet been elucidated. Investigating the mechanism by which zinc and manganese are transported and sensed is crucial to characterizing gonococcal metal homeostasis in the face of host-employed nutritional immunity and metal overload. In this work, I hypothesized that Zur mounts zinc- and manganese-dependent transcriptional responses to metal limitation and metal overload and that this response maintains internal metal concentrations at homeostatic levels. RNA-sequencing, RT-qPCR showed that Zur is a zinc-dependent regulator of the genes encoding ZnuABC and that manganese-dependent regulation by Zur is strain-specific. ICP-MS, growth assays, and transporter complementation experiments showed that internal homeostatic manganese levels differ between gonococcal strains and that this difference can be attributed to the manganese exporter, MntX. Therefore, novel treatment strategies that target metal transporters as a means of starving or overloading and subsequently killing N. gonorrhoeae should be informed by the metal environment sensed by N. gonorrhoeae and the intracellular metal pools maintained by different gonococcal strains.

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