Mechanistic Investigations of Helicobacter pylori Chemotaxis

背景：幽门螺杆菌存在于高酸性胃中，感染可导致胃癌。为了让幽门螺旋杆菌在这种环境中生存，它必须检测到诸如酸之类的化学线索。这些线索将幽门螺旋杆菌引向胃上皮。我们和其他人已经证明，化学受体TlpB负责检测HCl。我们已经确定了TlpB的质周区域的原子结构，并惊奇地发现尿素是结合的。幽门螺旋杆菌利用尿素中和环境，在胃酸中生存。我们发现尿素稳定TlpB，可能是幽门螺杆菌感知酸的机制的一部分。我们还发现，TlpB可以检测细菌群体感应分子自诱导剂2 (AI-2)，我们怀疑它可以促进幽门螺杆菌在整个胃中的扩散。TlpB不直接与AI-2相互作用，然而，我们已经确定了五个候选的质周结合蛋白。在哈维弧菌中也发现了类似的AI-2检测机制，其中一种质周蛋白结合AI-2，然后与一种膜结合的化学受体相互作用，类似于TlpB。确定幽门螺杆菌对酸和AI-2趋化作用的机制，有助于设计治疗幽门螺杆菌感染的新型抗生素，降低胃癌的发病率。

Background: Helicobacter pylori inhabit the highly acidic stomach and infection can lead to gastric cancer. In order for H. pylori to survive in this environment, it must detect chemical cues such as acid. These cues direct H. pylori to the stomach epithelium. We and others have shown that the chemoreceptor TlpB is responsible for detecting HCl. We have determined the atomic structure of the periplasmic domain of TlpB and found, to our surprise, that urea is bound. H. pylori uses urea to neutralize its environment and survive in stomach acid. We have found that urea stabilizes TlpB and may be part of the mechanism by which H. pylori senses acid. We have also shown that TlpB detects the bacterial quorum sensing molecule autoinducer 2 (AI-2), that we suspects promotes H. pylori dispersal throughout the stomach. TlpB does not interact directly with AI-2, however, we have identified five candidate periplasmic binding proteins that may. A similar AI-2 detection mechanism has been shown in Vibrio Harveyi where a periplasmic protein binds AI-2 and then interacts with a membrane bound chemoreceptor, similar to TlpB. Determining the mechanisms of H. pylori chemotaxis to acid and AI-2 will aid in designing novel antibiotics to treat H. pylori infection and decrease the incidence of gastric cancer. Objective/Hypothesis: Previous studies indicate that the chemoreceptor TlpB is responsible for H. pylori chemotaxes away from acid and AI-2. We propose to determine the mechanisms by which TlpB detects and responds to both acid and AI-2. Specific Aims: (1) We will test the hypothesis that TlpB responds to acid by binding urea in a pH sensitive manner; (2) we will test the hypothesis that TlpB responds to AI-2 through interactions with a periplasmic binding protein that binds AI-2. Study Design: First, we will determine if urea binds and stabilizes TlpB in a pH sensitive manner. We will then identify mutations in TlpB that disrupt urea binding and prevent proper acid chemotaxis. These TlpB mutants will be used in crystallography studies to identify conformation changes in the presence or absence of urea. To determine TlpB's role in sensing AI-2, we have identified candidate periplasmic binding proteins that may bind AI-2 and, when deleted in H. pylori, may be defective in AI-2 chemotaxis. We will then determine whether the candidate proteins bind AI-2 and/or TlpB directly. The structures of the periplasmic binding proteins of interest with AI-2 and TlpB will be pursued. Cancer Relevance: These studies will contribute to our understanding of how H. pylori senses chemical cues within the environment of the stomach. The detection of these cues by H. pylori is vital for its ability to infect the stomach and promote gastric cancer. More knowledge of the mechanisms of acid and AI-2 chemotaxis will provide clear targets for drug design and ultimately decrease the prevalence of gastric cancer.