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状杆状细菌，感染50%的人类胃部。幽门螺杆菌是一种细胞外病原体，与胃粘膜上皮细胞紧密相连。幽门螺杆菌在其表面表达极鞭毛，感染胃粘膜需要基于鞭毛的运动和趋化作用。螺旋细胞的形状被认为通过粘性粘液层增强了幽门螺杆菌基于鞭毛的运动性，从而允许有效的定植。我们的实验室在幽门螺杆菌中发现了几个促进正常螺旋细胞形状的基因，其中一些基因是在小鼠感染模型中高效定植胃粘膜所必需的。这些基因的单独或串联缺失通过肽聚糖(PG)交联17的改变影响细菌的曲率和扭曲，导致远端非螺旋杆形状(即弯曲的、“C”形和直杆)。我们的初步研究表明，细胞形状对幽门螺杆菌的游泳速度或游泳行为的其他方面起着作用。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/或化学感觉方向切换，细胞形状影响幽门螺杆菌在胃不同粘膜生态位中的定位。目标1将描述细胞形状对运动性的影响。我们将通过将不同细胞形状的突变体与野生型螺旋细菌进行比较，来测试螺旋细胞形状是否提高了幽门螺杆菌的速度和游泳行为。使用实时视频显微镜，我们将记录游泳行为，并确定纯化的胃粘蛋白中细菌的定向切换频率(目标1A)。我们还将通过测量固定在低pH值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讨细胞形状对平移运动的影响(目标1B)。目的2将确定细菌细胞形状对幽门螺杆菌定位于胃内特定胃壁龛的影响。目的3研究PG片段转运到宿主胃上皮细胞并调节天然免疫检测的机制。幽门螺杆菌通过CAG-IV型分泌器(CagT4SS)将PG片段递送到宿主胃上皮细胞，通过细胞内模式识别分子NOD118激活主要的促炎途径。表达Nod1的细胞可以对幽门螺杆菌细胞释放的PG产生反应，这些PG积聚了NOD1激动剂Muro-tritices18。我们的一些细胞形状突变体在PG球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会影响运送到宿主胃上皮细胞的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降解的扰动是否会影响通过cagT4SS或通过cagT4SS非依赖机制(AIM 3A)向宿主细胞的输送。我们还将研究PG循环突变导致促炎细胞因子释放增加的机制(AIM 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).