Mechanistic Investigations of Helicobacter pylori Chemotaxis

描述（由申请人提供）：幽门螺杆菌栖息在高度酸性的胃中，感染可导致胃癌。为了使H.幽门螺杆菌要在这种环境中生存，它必须检测酸等化学信号。这些线索引导H。pylori到胃上皮。我们和其他人已经表明，化学受体TlpB负责检测HCl。我们已经确定了TlpB的周质结构域的原子结构，并发现，令我们惊讶的是，尿素结合。H.幽门螺杆菌利用尿素来中和它的环境，并在胃酸中生存。我们已经发现，尿素稳定TlpB，可能是H.幽门感觉酸。我们还发现TlpB检测到细菌群体感应分子自诱导物2（AI-2），我们怀疑它促进了H。幽门螺旋杆菌在胃中的扩散。TlpB不直接与AI-2相互作用，然而，我们已经鉴定了五种可能的候选周质结合蛋白。类似的AI-2检测机制已在哈维氏弧菌中显示，其中周质蛋白结合AI-2，然后与膜结合的化学感受器相互作用，类似于TlpB。确定了H.幽门螺杆菌对酸和AI-2的趋化性将有助于设计新的抗生素来治疗H. pylori感染，降低胃癌的发病率。目的/假设：以往的研究表明，化学感受器TlpB负责H。pylori化学转运蛋白远离胃酸和AI-2。我们建议确定TlpB检测和响应酸和AI-2的机制。具体目标：（1）我们将检验TlpB通过以pH敏感的方式结合尿素来响应酸的假设;（2）我们将检验TlpB通过与结合AI-2的周质结合蛋白相互作用来响应AI-2的假设。研究设计：首先，我们将确定尿素是否以pH敏感的方式结合并稳定TlpB。然后，我们将鉴定TlpB中破坏尿素结合并阻止适当的酸趋化性的突变。这些TlpB突变体将用于晶体学研究，以确定尿素存在或不存在下的构象变化。为了确定TlpB在感知AI-2中的作用，我们已经鉴定了可能结合AI-2的候选周质结合蛋白，并且当在H. pylori感染可能存在AI-2趋化性缺陷。然后我们将确定候选蛋白是否直接结合AI-2和/或TlpB。将追求与AI-2和TlpB的感兴趣的周质结合蛋白的结构。癌症相关性：这些研究将有助于我们理解H。幽门螺旋杆菌在胃的环境中感知化学信号。H.幽门螺杆菌是至关重要的能力，感染胃和促进胃癌。对胃酸和AI-2趋化性机制的更多了解将为药物设计提供明确的靶点，并最终降低胃癌的患病率。

DESCRIPTION (provided by applicant): 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.