Mechanisms and consequences of cytoskeletal control of Helicobacter pylori cell shape

项目摘要/摘要 幽门螺杆菌是一种螺旋状的革兰氏阴性病原体，其唯一已知的生态位是人类 胃。幽门螺杆菌感染了世界50%的人口，并导致慢性胃炎，可发展为 胃癌，或部分患者的溃疡[1]。幽门螺杆菌是如何导致严重疾病的，目前还不完全清楚 可以理解，但一般来说，它是通过引发炎症而发生的。细菌的螺旋细胞形状是必不可少的 对于早期感染的多个方面；幽门螺杆菌的螺旋菌株在急性(一次)期间更有力地在小鼠身上定植 周)感染比缺乏细胞形状基因的非螺旋突变体(弯曲或杆状细胞)[2-5]。螺旋状细胞形状 可能通过旋塞机制促进粘性粘液层的定植[6] 这使得幽门螺杆菌能够到达上皮细胞并在上皮表面定居[7]。此外，还增加了 来自小鼠模型的证据表明，在慢性感染(一到三个月)中，幽门螺杆菌引起 与非螺旋突变株相比，炎症和增生水平更高[5]。螺旋细胞的形状是怎样的 确定以及它如何促进胃的定植和炎症是两个基本的问题 我们对幽门螺杆菌如何致病的理解。H.螺旋细胞形态所需的多种蛋白质。 幽门螺杆菌已被发现并被提出形成一种决定螺旋细胞形状的“形状”复合体[2- 4、8]。这个项目的假设是，幽门螺杆菌的螺旋形状有助于幽门螺杆菌的穿越能力 粘液层与胃粘膜相互作用以诱导炎症，并由 细胞骨架蛋白CCMA稳定并定位于幽门螺杆菌内膜的“形状”。 以下目标将检验这一假设：目标1将描述CCMA的每个领域如何对 CCMA的聚合和适当的定位，目的2将决定是否适当的细胞内定位 CCMA受其相互作用伙伴的调制，以及这些相互作用是否受细胞形状改变的影响 CCMA中的点突变，AIM 3将调查是否通过粘膜定位于上皮细胞 幽门螺杆菌细胞螺旋参数的改变对幽门螺杆菌细胞层有影响。该项目将利用高级 显微镜和有机物培养技术帮助我们了解幽门螺杆菌中螺旋细胞的形状 确定螺旋细胞形状如何影响与胃上皮的相互作用。这项研究符合 NIAID的使命是通过提供知识来推动抗病毒疗法的发展 考虑到幽门螺杆菌的临床分离株，幽门螺杆菌感染显示出越来越多的抗生素耐药性[13-15]。 此外，这项工作将加强对螺旋细胞形状扰动如何影响致病性的理解。 并可应用于其他螺旋杆状胃肠道病原体。

PROJECT SUMMARY/ABSTRACT Helicobacter pylori is a helical-shaped Gram-negative pathogen for which the only known niche is the human stomach. H. pylori infects 50% of the world’s population and causes chronic gastritis, which can progress to gastric cancers, or ulcers in a subset of patients [1]. How exactly H. pylori causes severe disease is not fully understood, but in general it occurs through eliciting inflammation. The bacterium’s helical cell shape is essential for multiple facets of early infection; helical strains of H. pylori more robustly colonize mice during acute (one week) infections than non-helical mutants that lack cell shape genes (curved or rod cells) [2-5]. Helical cell shape may promote colonization of the viscous mucous layer of the stomach through a corkscrewing mechanism [6] which allows H. pylori to reach the epithelium and colonize the epithelial surface [7]. Furthermore, additional evidence from mouse models suggests that in chronic infections (one or three months), helical H. pylori elicit higher levels of inflammation and hyperplasia than non-helical mutant strains [5]. How helical cell shape is determined and how it promotes colonization and inflammation of the stomach are two questions essential to our understanding of how H. pylori causes disease. Multiple proteins that are required for helical cell shape in H. pylori have been identified and proposed to form a “shapesome” complex that determines helical cell shape [2- 4, 8]. The hypothesis of this project is that the helical shape of H. pylori contributes to H. pylori’s ability to traverse the mucous layer and interact with the gastric mucosa to induce inflammation and is controlled by the “shapesome” which the cytoskeletal protein CcmA stabilizes and localizes to the inner membrane of H. pylori. The following aims will test this hypothesis: Aim 1 will characterize how each domain of CcmA contributes to polymerization and proper localization of CcmA, Aim 2 will determine whether proper intracellular localization of CcmA is modulated by its interaction partners and whether these interactions are impacted by cell shape-altering point mutations in CcmA, and Aim 3 will investigate whether localization to the epithelium through the mucous layer is impacted by alterations of helical parameters of H. pylori cells. This project will leverage advanced microscopy and organoid culture techniques to inform our understanding of how helical cell shape in H. pylori is determined and how helical cell shape impacts interactions with the gastric epithelium. The research fits the mission of the NIAID by providing knowledge that could advance the development of anti-virulence therapies for H. pylori infection considering clinical isolates of H. pylori increasingly show antibiotic resistance [13-15]. Additionally, this work will enhance understanding of how perturbation of helical cell shape impacts pathogenicity of H. pylori and could be applied to other helical rod-shaped gastrointestinal pathogens.