Helicobacter pylori is a ubiquitous bacterial pathogen, colonizing half of the world’s population. Stomach colonization leads to inflammation development, called gastritis. Depending on the severity of gastritis, more serious diseases can develop, such as peptic ulcers or gastric cancer. To successfully colonize the gastric niche, H. pylori uses chemotaxis. Through this system, proteins called chemoreceptors sense a variety of beneficial and harmful conditions, regulating the localization and fitness of H. pylori in the gastric niche. The H. pylori chemoreceptor TlpA alters host inflammation through an unknown mechanism. To better understand this phenotype, this thesis characterizes 1) the sensing profile of TlpA and 2) how TlpA affects the host immune response and the localization of H. pylori in vivo. TlpA has a dCache_1 periplasmic sensing domain and previous studies have identified two signals sensed by this chemoreceptor. It has become appreciated that dCache_1 sensor domains can bind diverse sets of ligands, and ligand binding can occur through either the membrane-proximal or -distal subdomain. To better understand what signals TlpA may be responsible for sensing in vivo, we performed an extensive screen for novel TlpA ligands using ligand binding arrays and surface plasmon resonance. TlpA-dependent chemotactic responses towards hits from this screen were characterized using a temporal, live-cell tracking assay. Novel TlpA chemoattractants were identified, and their binding was characterized using in silico molecular docking experiments and in vitro SPR assays with TlpA membrane-proximal and -distal subdomain point mutants. Roles in ligand binding were identified for each subdomain. Additionally, an antagonistic TlpA ligand was identified, which blocked the normal chemotaxis response to TlpA chemoattractants. Antagonistic ligands are an emerging theme in signal transduction and may play a role in regulating chemotactic responses in vivo. In total, these studies provide a clearer understanding of the signals sensed by TlpA, the role of dCache_1 subdomains in ligand binding, and identifies a potential mechanism for regulating chemotactic responses.
To better understand how TlpA affects host inflammation, we performed detailed, longitudinal colonization studies in an in vivo murine model over 8-months. Throughout this period, we determined total colonization loads, assessed gland colonization, measured inflammation via histology, and performed flow cytometry to analyze the innate and effector CD4+ T-cell populations recruited to the stomach during infection. We found that TlpA attenuates inflammation in the corpus of the stomach at 5-month post-infection, which is associated with a decreased Th17 frequency. In addition, TlpA altered the timing of transient increases in inflammation. Also, in agreement with other reports, TlpA is not required for WT colonization levels overall in the stomach. However, TlpA regulates the localization of H. pylori within the gastric glands of the corpus and antrum. This regulation is most prominent during the first month of infection. From this work, we propose a possible link between TlpA regulated gland colonization and inflammation development.
The regulation of host inflammation is critical for controlling H. pylori disease outcomes. In total, this work furthers our understanding of this process by characterizing the sensing capabilities of TlpA and providing a detailed understanding of the localization and immune response affected by TlpA during colonization. This work lays the groundwork for future studies aiming to understand how the chemotaxis system of H. pylori alters disease outcomes and could potentially lead to the development of novel chemotaxis-related therapeutics.
