Many microbes use chemotaxis as a system for thriving and colonizing their various environmental niches. Chemotaxis is the ability of organisms to swim toward favorable environments and away from toxic environments. Helicobacter pylori is a chemotactic pathogen that infects over half the world’s population and successfully colonizes the gastric mucosa of humans. Chemotaxis is an important virulence factor that helps H. pylori effectively colonize the human stomach. The chemotaxis complex system consists of various proteins: chemoreceptors, coupling proteins, and the CheA kinase. Chemoreceptors sense the environment and transmit the response to the CheA kinase through coupling proteins. Coupling proteins of the CheW or CheV type physically link chemoreceptors to the CheA kinase. H. pylori has a unique chemotaxis system that contains multiple coupling proteins, 1 CheW and 3 CheV proteins—CheV1, CheV2, and CheV3 respectively that all play a role in chemotaxis. It is unknown why some chemotaxis systems contain multiple types of coupling proteins. In this thesis, the role and function of the coupling proteins CheV1 and CheW was investigated, as well as the stoichiometry of the chemotaxis proteins that make up the chemoreceptor-CheA kinase complex in H. pylori. CheV1 and CheW had similar protein interaction networks in vivo and in vitro with the chemoreceptors, the CheA kinase, and themselves. Both proteins promoted CheA autophosphorylation activity with CheW having slightly better CheA activation than CheV1. CheV1 and CheW were also both needed to retain the CheA kinase at the cell membrane where the chemotaxis complex is found. Cellular localization of the chemoreceptors and CheA kinase required the presence of both CheV1 and CheW. Ultimately, these results indicate that CheV1 and CheW work together to promote super chemotaxis cluster formation at cell poles which is important for eliciting a chemotaxis response. Also reported here are the concentration and ratio of the chemotaxis proteins in H. pylori. H. pylori possessed significantly higher concentrations of chemoreceptors, coupling proteins, and the CheA kinase in comparison to E. coli and B. subtilis. Despite these differences, a chemoreceptor trimer of dimers to one CheA dimer was maintained in H. pylori. There was also a conserved ratio of 1.8 CheW to 1 CheA dimer like B. subtilis and E. coli. The stoichiometry of the chemotaxis proteins was also analyzed in cheV1 and cheW mutants. cheW mutants had approximately half the amount of CheA kinase as wildtype similar to a receptorless mutant. Addition of a protease inhibitor restored CheA amounts in mutants indicating that protein interactions within the chemotaxis complex protect proteins from degradation thus ultimately maintaining optimal chemotaxis. Finally, we found that cheV1 mutants rapidly accumulate suppressor mutants in a soft agar assay.

The primary agent responsible for both gastric ulcers and cancers is the human pathogen Helicobacter pylori. The aim of this dissertation is to enhance our understanding of how this bacterium can cause disease in humans and persist in the host over many years. We have identified a novel H. pylori virulence factor, called ImaA, which dampens H. pylori caused inflammation and promotes long-term colonization in the host. ImaA is an outer membrane protein that H. pylori expresses at exceptionally high levels in the stomach and is critical for the bacterium's ability to survive.

The first part of this dissertation expands on the discovery that imaA is expressed at higher levels in the mouse stomach, when compared to laboratory conditions. We went on to demonstrate that an imaA isogenic mutant exhibits a colonization defect in the mouse and is greatly outcompeted for colonization in a co-infection with wild-type H. pylori. We furthermore performed localization assays using an ImaA-targeted antibody to verify that the ImaA protein resides in the outer membrane. We identified low pH to be the signal for imaA's upregulation in the stomach and demonstrated that this upregulation is dependent on the ArsRS two-component regulatory system. Concluding the first part of this dissertation, we revealed that the imaA mutant evokes a stronger immune response in gastric epithelial cells, compared to that of wild-type H. pylori.

The second part of this dissertation focuses on ImaA's interactions with the host. The primary mechanism for H. pylori caused inflammation is through the secretion of the effector molecule, CagA, into host cells. We show here that in an imaA mutant infection, there are more CagA molecules delivered into gastric epithelial cells, than in a wild-type infection. We went on to show that the imaA mutant induces elevated levels of inflammation, independently of CagA, as an imaA cagA double mutant causes more inflammation than a cagA single mutant. This result directed us to the cagPAI T4SS, the surface-exposed unit that delivers CagA into the host cell. Through flow cytometry experiments, we showed that imaA mutants bind higher levels of the host receptor for the cagPAI T4SS, @alpha;5β1 integrin. Finally, using the gerbil mode for infection, we discovered that ImaA is dispensable for the very initial stages of host colonization, but that the protein becomes essential very rapidly, as imaA mutants are cleared from the stomach within a week, post-infection.

Taken together, this dissertation characterizes the novel H. pylori virulence factor, ImaA, and demonstrates that this protein is critical for the bacterium's ability to control inflammation and persist in the host stomach.

Helicobacter pylori translates environmental cues into a swimming response using its chemotactic signaling system. Generally, chemotactic signaling in H. pylori follows the enteric paradigm. The H. pylori system, however, has three additional proteins called CheVs that are hybrid proteins combining a CheW domain with a response receiver (REC) domain. Studies on H. pylori have shown that each CheV participates in wild-type chemotaxis although the exact role of each protein in the chemotactic signaling system is unclear. The goal of this study is to better understand the function of CheVs in the pathway, by specifically testing spatial localization of the CheV proteins and whether they prefer specific chemoreceptors. We carried out subcelluar fractionation followed by western analysis to determine whether any of the H. pylori CheV proteins prefer the TlpA or TlpB chemoreceptors. Additionally, immunofluorescence was applied to observe the localization of each CheV in wild-type H. pylori, in isogenic strains having only TlpA or TlpB, and in isogenic strains lacking one CheV or CheW. We found that all three CheV proteins are detected in membrane fractions with either TlpA or TlpB, and surprisingly, even in the absence of all receptors. This outcome suggested that CheV proteins associate with the membrane independently of the chemoreceptors. In wild type, we found that the CheVs are primarily localized at the cell poles. In contrast, strains lacking chemoreceptors had CheV proteins diffused throughout the cytoplasm. Strains bearing a single chemoreceptor had CheVs both at the poles and diffused within the cell. In the absence of CheV1, both CheV2 and CheV3 where diffused; on the contrary, loss of either CheV2 or CheV3 did not affect the localization of the remaining CheV proteins. Immunofluorescence studies also suggested that both CheV1 and CheW are involved in localizing the chemoreceptors to the cell pole. These results suggest that either TlpA or TlpB can guide the localization of CheVs to the cell pole. In a cell with only a single chemoreceptor, the quantity of chemoreceptor complex formed by TlpA or TlpB is altered and appears to affect CheV localization. These studies suggest that the H. pylori CheV proteins do not prefer TlpA or TlpB, and instead interact with all chemoreceptors equally. Unlike the other CheV proteins, CheV1 showed a significant effect on the polar localization of chemoreceptors, and concomitantly, CheV2 and CheV3. CheW similarly was required for polar chemoreceptors. These studies thus show that the H. pylori CheV1 and CheW proteins have a major role in the spatial localization of chemotaxis transduction proteins.

Helicobacter pylori is a human stomach pathogen and class 1 carcinogen responsible for the development of peptic ulcers and gastric cancer. H. pylori infects around half of the global population and is a high priority target for the development of new eradication therapies by the WHO. Exploring alternative treatment options for H. pylori is imperative with increasing failure rates for antibiotic treatment. Here we explore the influence of factors that affect antibiotic efficacy at both the therapeutic and bacterial resistance mechanism levels. In the first study, we explore the effect of the proton pump inhibitor omeprazole on the growth and redistribution of H. pylori strain SS1 in murine gastric glands. Using the bacterial localization in gastric glands (BLIG) method to enumerate intraglandular H. pylori populations, we find that omeprazole promotes the growth and redistribution of bacterial populations in gastric glands in mice. We also find that this effect is dose- and region-dependent. Lower doses of omeprazole promote growth and redistribution as a measure of gland population and gland occupancy in both the gastric antrum and gastric corpus, while higher doses of omeprazole diminish growth and redistribution. We also find that the intraglandular population of H. pylori grows and redistributes in the gastric antrum more robustly than observed in the gastric corpus. Taken together, these results indicate that omeprazole influences gland-resident populations of H. pylori in previously unreported ways. Our second study partially characterizes a putative efflux pump discovered during an RNAseq screen of biofilm-forming H. pylori. Using Phyre2 and the NCBI database, we found that the putative efflux pump, HP0759, was most similar to NorM-like efflux pumps. We also report that an HP0759 knockout is more susceptible to rifampicin, but not other antibiotics, as measured by E-test. Both studies present new findings on alternative mechanisms of drug response and survival in the stomach by H. pylori.