Effect of Helicobacter pylori cell shape on pathogenesis

描述（由申请人提供）：幽门螺杆菌是一种革兰氏阴性螺旋杆状细菌，感染50%的人的胃。H.幽门螺杆菌是一种细胞外病原体，发现其非常接近胃粘膜上皮细胞6，7。幽门螺杆菌在其表面上表达极性鞭毛，并且胃粘膜的感染需要基于鞭毛的运动性和趋化性13-16。螺旋细胞形状被认为是增强H。幽门螺杆菌的鞭毛为基础的运动通过粘性粘液层，以允许有效的殖民7。我们的实验室发现了几个基因，促进正常的螺旋细胞形状在H。幽门螺杆菌和这些基因中的一些是在小鼠感染模型中胃粘膜的有效定殖所需的17。这些基因的单独或串联缺失通过改变肽聚糖（PG）交联17影响细菌的曲率和扭曲，从而导致明显的非螺旋杆形状（即弯曲、“c”形和直杆）。我们的初步研究表明，细胞形状在H。pylori的游泳速度或游泳行为的其他方面。我们假设螺旋细胞的形状对H. pylori运动性，并通过影响速度和/或化学感觉方向转换，细胞形状影响H. pylori在胃的不同胃粘膜龛内的定位。目的1将表征细胞形状对运动性的影响。我们将测试螺旋细胞形状是否增强H。pylori的速度和游泳行为，通过比较不同细胞形状的突变体与野生型螺旋细菌。利用实时视频显微镜，我们将记录游泳行为，并确定细菌在纯化的胃粘蛋白中的方向转换频率，胃粘蛋白是H。胃中的幽门螺杆菌（目的1A）。我们还将通过在低pH下测量固定在胃粘蛋白凝胶中的细菌细胞的鞭毛和细胞体旋转来直接探测细胞形状对平移运动的影响（目的1B）。目的2探讨细菌细胞形态对H. pylori与胃内的特定胃龛。目的3探讨PG片段进入宿主胃上皮细胞并调节天然免疫检测的机制。H.幽门螺杆菌通过cag-type IV分泌器（cagT 4SS）将PG片段递送至宿主胃上皮细胞，以通过细胞内模式识别分子NOD 118激活主要的促炎途径。表达NOD 1的细胞可以对H.幽门螺杆菌细胞，积累muro-三肽18，NOD 1激动剂。我们的一些细胞形状突变体在PG球囊中积累高水平的这种激动剂。因此，驱动细胞形状变化的细胞壁修饰事件可能影响递送至宿主胃上皮细胞的PG片段的合成和释放。我们将产生过表达细胞壁修饰酶的菌株，以确定PG降解的扰动是否影响通过cagT 4SS或通过cagT 4SS非依赖性机制递送至宿主细胞（目的3A）。我们还将研究PG再循环突变体引起促炎细胞因子释放升高的机制（目的3B）。

DESCRIPTION (provided by applicant): Helicobacter pylori is a Gram-negative helical rod-shaped bacterium that infects the stomach of 50% of all humans. H. pylori is an extracellular pathogen that is found in close proximity to the mucosal epithelial cells lining the stomach 6,7. H pylori express polar flagella on its surface, and both flagellar-based motility and chemotaxis are required for infection of the gastric mucosa 13-16. Helical cell shape is thought to enhance H. pylori's flagellar based-motility through the viscous mucus layer to allow for efficient colonization7. Our lab discovered several genes that promote normal helical cell shape in H. pylori and some of these genes are required for efficient colonization of the gastric mucosa in a mouse infection model17. Deletion of these genes individually or in tandem affect the bacteria's curvature and twist through alterations in peptidoglycan (PG) cross-linking17, resulting in distinc non-helical rod shapes (i.e. curved, "c"-shaped, and straight rods). Our preliminary studies suggest that cell shape plays a role in H. pylori's swimming velocity or some other aspect of swimming behavior. We hypothesize that helical cell shape is important for H. pylori motility and by affecting velocity and/or chemosensory directional switching, cell shape impacts H. pylori's localization within different gastric mucosal niches of the stomach. Aim 1 will characterize the effect of cell shape on motility. We will test whether helical cell shape enhances H. pylori's velocity and swimming behavior by comparing the different cell shape mutants to the wild-type helical bacteria. Using live video microscopy, we will record the swimming behavior and determine the directional switching frequency of the bacteria in purified gastric mucin, the relevant polymer for H. pylori in the stomach (aim 1A). We will also directly probe the effect of cell shape on translational motion by measuring flagellar and cell body rotation for bacterial cell immobilized in gastric mucin gel at low pH (aim 1B). Aim 2 will determine the effect of bacterial cell shape on localization of H. pylori to specific gastric niches within the stomach. Aim 3 will investigate the mechanisms by which PG fragments are delivered to host gastric epithelial cells and modulate innate immune detection. H. pylori delivers PG fragments to host gastric epithelial cells via the cag-type IV secretion apparatus (cagT4SS) to activate a major pro- inflammatory pathway through the intracellular pattern recognition molecule NOD118. NOD1-expressing cells can respond to digested PG released from H. pylori cells that accumulate muro-tripeptides18, a NOD1 agonist. Some of our cell shape mutants accumulate high levels of this agonist in the PG sacculus. Thus, the cell wall modification events that drive cell shape changes may impact the synthesis and release of PG fragments that are delivered to host gastric epithelial cells. We will generate strains that overexpress cell wall modifications enzymes to determine if perturbations in PG degradation affect delivery to host cells via the cagT4SS or by cagT4SS-independent mechanisms (aim 3A). We will also investigate the mechanisms by which PG recycling mutants cause elevated pro-inflammatory cytokine release (aim 3B).