The role of the H. pylori virulence protein CagA in host intestinal epithelial pr

描述（由申请人提供）：人体胃肠道内有数千种常驻微生物，称为肠道微生物群，对发育至关重要，在健康个体中，促进正常生理机能。然而，一个新兴的想法是，病原体可以改变微生物群落，导致人类病理学。人类病原体幽门螺杆菌导致胃部病变，如炎症和细胞增殖，可导致胃癌。这些病理至少部分是由于易位的毒力蛋白CagA的作用，并可能通过改变肠道微生物群。在这里，我们建议使用两个转基因模型，果蝇和斑马鱼，表达CagA在肠上皮细胞内的CagA表达的细胞自主的影响和非细胞自主的影响，这种表达对微生物群。这些模型将用于研究病原体衍生因子（如CagA）如何影响肠道微生物生态，并影响肠道上皮细胞信号通路，以促进胃肠道癌症等疾病。在这些实验中获得的知识将提供有价值的见解，细菌病原体和居民之间的相互作用，有助于人类疾病的机制，并将提供新的思路，预防和治疗疾病的感染性病因。假设：H.幽门螺杆菌蛋白CagA以细胞自主方式促进肠细胞增殖，并且还改变肠道微生物群组成，这促进非细胞自主细胞增殖。具体目标：目标1：我将确定CagA表达是否诱导肠上皮细胞自主增殖。目的2：我将测试的假设，CagA在肠道上皮细胞的表达改变宿主微生物群，并导致果蝇细胞增殖。目标3：我将测试CagA转基因斑马鱼携带改变的微生物群的假设，这是必要的，足以导致CagA相关的肠道病变，如增生。研究设计：为了研究CagA对肠上皮细胞的细胞自主作用，我将在果蝇和斑马鱼中使用马赛克方法。这种方法允许我直接测量表达CagA的细胞中的细胞增殖速率，并确定这些细胞是否仅经历JNK或Wnt途径激活。此外，使用报告细胞系，我将确定是否CagA的细胞自主表达改变免疫应答诱导细胞自主表达的抗菌肽。为了确定CagA的表达是否改变了肠道微生物群，以及这是否足以诱导果蝇肠道中的细胞增殖，我将过表达抗菌肽，并确定这是否是必要的或足以改变微生物群。此外，使用整个果蝇微生物群的移植或通过单一关联实验，我将确定改变的微生物群是否足以诱导细胞增殖，并确定负责这种效应的单一细菌物种。最后，我将测试CagA在斑马鱼肠上皮中的表达是否足以改变微生物群和免疫功能。通过移植实验，我还将确定微生物群是否是诱导斑马鱼肠道细胞增殖所必需或足够的。这里概述的方法提供了一个独特的机会，调查保守的作用，H。pylori毒力因子CagA对肠上皮细胞的影响。两个独立的模型系统的使用将提供有价值的洞察这种常见的微生物的保护和病理性质。这些数据对于了解病原体来源的癌症的病因至关重要，并为治疗和预防的潜在途径提供有价值的见解。

DESCRIPTION (provided by applicant): The human gastrointestinal tract houses thousands of resident microbes, termed the gut microbiota, which are critical for development and, in healthy individuals, promote normal physiology. However, an emerging idea is that pathogens can alter microbial communities, contributing to human pathologies. The human pathogen, Helicobacter pylori causes stomach pathologies, such as inflammation and cell proliferation, that can lead to gastric cancer. These pathologies are due at least in part to the action of a translocated virulence protein CagA, and potentially by an altered gut microbiota. Here, we propose to use two transgenic models, Drosophila and zebrafish, expressing CagA in the intestinal epithelium to investigate the cell autonomous effects of CagA expression within the gut epithelium and the non-cell autonomous effects of this expression on the microbiota. Together these models will be used to investigate how pathogen derived factors, such as CagA, influence intestinal microbial ecology and affect intestinal epithelial signaling pathways to promote diseases such as gastrointestinal cancers. Knowledge gained in these experiments will provide valuable insights into the mechanisms by which interactions between bacterial pathogens and resident commensals contribute to human disease, and will provide new ideas for preventing and treating diseases with an infectious etiology. Hypothesis: The H. pylori protein, CagA, promotes intestinal cell proliferation in a cell autonomous fashion and also alters gut microbiota composition, which promotes non-cell autonomous cell proliferation. Specific Aims: Aim1: I will determine if CagA expression induces cell-autonomous proliferation of the gut epithelium. Aim2: I will test the hypothesis that CagA expression in the gut epithelium changes the host microbiota and causes cell proliferation in Drosophila. Aim3: I will test the hypothesis that CagA transgenic zebrafish harbor an altered microbiota, which is necessary and sufficient to cause CagA-associated gut pathologies, such a hyperplasia. Research design: To examine the cell-autonomous effects of CagA on the intestinal epithelium I will use a mosaic approach in both Drosophila and zebrafish. This approach allows me to directly measure rates of cell proliferation in CagA expressing cells and determine whether these cells exclusively undergo JNK or Wnt pathway activation. Additionally, using reporter lines I will determine whether cell autonomous expression of CagA alter the immune response by inducing cell autonomous expression of antimicrobial peptides. To determine whether expression of CagA alters the gut microbiota and whether this is sufficient to induce cell proliferation in the Drosophila gut, I will overexpress antimicrobial peptides and determine if this is necessary or sufficient to alter the microbiota. Additionally, using transplantation of the entire Drosophila microbiota or through mono association experiments I will determine if the altered microbiota is sufficient to induce cell proliferation and identify the single bacterial species responsible for this effect. Finally, I wil test whether expression of CagA in the zebrafish intestinal epithelium is sufficient to alter the microbiota and immune function. Using transplantation experiments I will also determine whether the microbiota is necessary or sufficient to induce cell proliferation in the zebrafish intestine. The approach outlined here provides a unique opportunity to investigate the conserved role of the H. pylori virulence factory CagA on the intestinal epithelium. The use of two independent model systems will provide valuable insight into the conservation and pathologic nature of this common microbe. This data will be vital for understanding the etiology of pathogen-derived cancers and provide valuable insight into potential avenues for treatment and prevention.