Date Awarded

2022

Document Type

Thesis

Degree Name

Master of Science (M.Sc.)

Department

Biology

Advisor

Mark Forsyth

Abstract

Helicobacter pylori colonizes the human gastric mucosa and causes various gastroduodenal diseases from ulcers to gastric cancer. Colonization relies heavily on H. pylori two-component systems (TCSs) to respond to the surrounding host environment. We evaluated gene regulation mediated by the CrdRS TCS of H. pylori 26695. Only a few studies have evaluated this TCS leaving the signal(s) yet to be exhaustively determined and the need for new regulon screening to be completed. We performed RNA sequencing (RNA-Seq) on three isogenic H. pylori 26695 mutants: a control, the deletion of the histidine kinase sensory component crdS (ΔcrdS), and the deletion of the response regulator component crdR (ΔcrdR). Three biological replicates of RNA-Seq using these mutants established a 40 gene regulon putatively controlled by the CrdRS TCS. Quantitative real-time PCR (RT-qPCR) was used to validate RNA-Seq data and to determine the relative quantity of mRNA for 13 of the identified regulon genes. The expression level of 9 of the 13 chosen regulon members was confirmed in this manner. We further investigated a subset of genes of this regulon that were both significantly (p<0.05) in RNA-Seq experiments and ¬>2-fold dysregulated in RT-qPCR assays. These 7 genes were evaluated in H. pylori 26695 CrdR D53A and CrdS H173A phospho-incompetent missense mutants to confirm the dysregulation seen in RNA-Seq and RT-qPCR experiments using the ΔcrdS and ΔcrdR deletion mutants was not due to polar effects of the large deletions present in these mutants. As Hung et al. demonstrated a role for nitric oxide (NO) in CrdRS signaling, the control and missense crdS and crdR mutants were thus evaluated under 10 µM nitric oxide conditions. This revealed 4 novel genes (ureA, acxA, hofD, HP1440) whose regulation is controlled by the CrdRS TCS via nitric oxide, but never previously reported to be controlled by this nitrosative stress. These genes play an important role in urease, acetone carboxylase, and outer membrane protein function, some of which have been shown as essential to H. pylori colonization and survival. Further experiments evaluating the role of nitric oxide on regulon member expression and other potential signals such as acetone will strengthen the understanding of the CrdRS TCS and its role in H. pylori survival and colonization.

DOI

https://dx.doi.org/10.21220/s2-w4aj-z371

Rights

© The Author

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