THE ROLE OF CELL SHAPE AND CELL WALL IN HELICOBACTER PYLORI PATHOGENESIS

描述(申请人提供)：幽门螺杆菌的名字很明显，它是一种螺旋细菌，它定植于人的胃中，导致从轻度胃炎到消化性溃疡和胃癌的各种临床结果。对幽门螺杆菌毒力的描述包括一个被广泛接受的假设，即细菌的螺旋形状增强了其游过粘稠的上皮粘液层并成功在胃内定植的能力。然而，这一理论从未被检验过，幽门螺杆菌形状的分子决定因素仍然未知。利用转座子突变体文库的可视屏幕，我们鉴定了显示两种改变细胞形状的克隆：直杆和弧形弧菌。遗传分析揭示了三个染色体位点，其中一个至少有三个基因都影响细胞形状。生化分析表明，其中一些蛋白质改变了细胞壁的肽聚糖(PG)结构。所有被测试的突变体在软琼脂中的光晕形成中都显示出轻微的、但可重现的缺陷，表明游泳或趋化性受到影响。一个类弧菌和两个杆状突变体被测试了它们在小鼠胃中定植的能力，与野生型或互补菌株相比，它们都表现出了不同的表型。有趣的是，杆状突变体的定殖率高于螺旋突变体，而弧菌突变体的定殖率较低。因此，已确定的螺旋形状决定基因会影响胃的定植，但如何以及为什么仍有待阐明。我们对目前的拨款提案有两个目标。目标1的目标是了解在我们的螺旋丧失筛查中确定的基因产物如何改变细胞形状。细胞壁的作用是什么？在形状类内或形状类之间识别的基因产物是否相互作用？它们是否与已知的PG生物合成和降解酶或细菌细胞骨架元件相互作用？目标2的目标是了解这些蛋白质在胃定植过程中的功能。定植表型是否如先前假设的那样是由游泳改变引起的，还是趋化性改变了？已确定的细胞壁修饰对于细菌在胃中遇到的压力(如渗透压、酸度和接触抗菌肽的波动)的生存能力是否重要？细胞壁的改变是否会影响宿主免疫系统的PG信号和/或检测？我们相信，这项工作将在细胞生物学和发病机制方面产生重大进展。微生物细胞生物学中一个悬而未决的问题是如何以及为什么保持螺旋形状。从细胞生物学和发病机制的角度来看，这些蛋白中的一些修饰PG的发现也具有重要意义。幽门螺杆菌的PG通过细胞内病原体识别分子Nod1激活一条主要的促炎途径。上皮细胞对特定PG组分的识别对于细菌与宿主的共生和致病相互作用都是重要的。总之，实验目的将探索幽门螺杆菌如何以及为什么保持其螺旋形状及其在发病机制中的作用。与公共卫生相关：从名字上看，幽门螺杆菌是一种螺旋细菌，它定植在人类胃中，导致从轻度胃炎到消化性溃疡和胃癌的一系列临床结果。我们发现这种细菌促进螺旋形状的基因可以改变其细胞壁。研究这些细胞壁修饰如何促进螺旋形状和细菌在其独特的利基环境中的生存，将有助于我们更好地了解这种细菌是如何致病的，并发现抑制感染的新方法。

DESCRIPTION (provided by applicant): Evident in its name, Helicobacter pylori is a helical bacterium that colonizes the human stomach causing clinical outcomes ranging from mild gastritis to peptic ulcer and gastric cancer. Descriptions of H. pylori virulence include a widely accepted hypothesis that the bacterium's spiral shape enhances its ability to swim through the viscous epithelial mucus layer and successfully colonize the stomach. This theory, however, has never been tested and the molecular determinants of H. pylori's shape remain unknown. Using a visual screen of a transposon mutant library, we identified clones displaying two classes of altered cell shape: straight rod and curved vibrioid. Genetic analyses revealed three chromosomal loci, one with at least three genes all affecting cell shape. Biochemical analyses indicate that some of these proteins modify the cell wall peptidoglycan (PG) structure. All mutants tested show slight, but reproducible defects in halo formation in soft agar suggesting compromised swimming or chemotaxis. One vibrioid and two rod-shaped mutants were tested for their ability to colonize the mouse stomach and all showed an altered phenotype compared to wild type or complemented strains. Interestingly the rod-shaped mutants colonized better than their spiral counter parts while the vibrioid mutant showed lower colonization. Thus the identified spiral shape-determining genes influence stomach colonization, but how and why remains to be elucidated. We have two goals with the current grant proposal. The goal of Aim 1 is to understand how the gene products identified in our loss of spirality screen alter cell shape. What is the role of the cell wall? Do the gene products identified within or between shape classes interact? Do they interact with known PG biosynthetic and degrading enzymes or with bacterial cytoskeletal elements? The goal of Aim 2 is to understand how these proteins function during stomach colonization. Are the colonization phenotypes due to altered swimming as previously hypothesized, or is chemotaxis altered? Are the identified cell wall modifications important for the bacteria's ability to survive stresses encountered in the stomach such as fluctuations in osmolarity, acidity and exposure to antimicrobial peptides? Do the cell wall alterations influence PG signaling and/or detection by the host immune system? We believe this work will yield significant advancements in both cell biology and pathogenesis. An outstanding question in microbial cell biology is how and why spiral shape is maintained. The discovery that some of these proteins modify PG is also significant from both cell biology and pathogenesis perspectives. H. pylori's PG activates a major pro-inflammatory pathway via the intracellular pathogen recognition molecule Nod1. Recognition of specific PG components by epithelial cells has been shown to be important for both symbiotic and pathogenic interactions of bacteria and their host. Altogether the experimental aims will probe how and why H. pylori maintains its spiral shape and its role in pathogenesis. PUBLIC HEALTH RELEVANCE: Evident in its name, Helicobacter pylori is a helical bacterium that colonizes the human stomach causing clinical outcomes that range from mild gastritis to peptic ulcer and gastric cancer. We discovered genes that promote spiral shape by this bacterium can alter its cell wall. Study of how these cell wall modifications promote spiral shape and bacterial survival in its unique niche will help us better understand how this bacteria causes disease and discover new ways to inhibit infection.