Effect of Helicobacter pylori cell shape on pathogenesis

描述（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%的人类胃。幽门螺杆菌是一种细胞外病原体，在靠近胃粘膜上皮细胞的地方发现6,7。幽门螺杆菌在其表面表达极性鞭毛，鞭毛的运动性和趋化性都是胃粘膜感染所必需的13-16。螺旋细胞的形状被认为增强了幽门螺杆菌的鞭毛为基础的运动性，通过粘性黏液层，从而允许有效的定植7。我们的实验室发现了几个促进幽门螺杆菌正常螺旋细胞形状的基因，其中一些基因是小鼠感染模型中胃粘膜有效定植所必需的17。这些基因单独或串联的缺失会通过肽聚糖（PG）交联的改变影响细菌的弯曲和扭曲，从而产生不同的非螺旋棒形状（即弯曲的、“c”形的和直的棒）。我们的初步研究表明，细胞形状在幽门螺旋杆菌的游泳速度或游泳行为的其他方面起作用。我们假设螺旋细胞形状对幽门螺杆菌的运动很重要，并且通过影响速度和/或化学感觉方向转换，细胞形状影响幽门螺杆菌在胃粘膜不同生态位中的定位。目的1将描述细胞形状对运动的影响。我们将通过比较不同细胞形状突变体和野生型螺旋菌来测试螺旋细胞形状是否能提高幽门螺杆菌的速度和游泳行为。使用实时视频显微镜，我们将记录细菌的游泳行为，并确定纯化胃黏液中细菌的方向开关频率，胃黏液是胃中幽门螺杆菌的相关聚合物（目的1A）。我们还将通过测量低pH下胃黏液凝胶固定细菌细胞的鞭毛和细胞体旋转来直接探讨细胞形状对平移运动的影响（aim 1B）。目的2将确定细菌细胞形状对幽门螺杆菌在胃内特定胃位定位的影响。目的3将研究PG片段递送到宿主胃上皮细胞并调节先天免疫检测的机制。幽门螺杆菌通过cagT4SS （cagT4SS）将PG片段传递给宿主胃上皮细胞，通过细胞内模式识别分子NOD118激活一条主要的促炎途径。表达NOD1的细胞可以对从幽门螺杆菌细胞释放的消化PG做出反应，这些PG积累了NOD1激动剂muro-tripeptides18。我们的一些细胞形状突变体在PG小囊中积累了高水平的这种激动剂。因此，驱动细胞形状改变的细胞壁修饰事件可能影响PG片段的合成和释放，PG片段被递送到宿主胃上皮细胞。我们将产生过表达细胞壁修饰酶的菌株，以确定PG降解的扰动是否会通过cagT4SS或通过cagT4SS独立机制影响宿主细胞的递送（目的3A）。我们还将研究PG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).