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

描述（由申请人提供）：人类胃肠道容纳了数千种常驻微生物，称为肠道微生物群，它们对发育至关重要，并且在健康个体中促进正常生理。然而，一个新兴的想法是病原体可以改变微生物群落，导致人类疾病。人类病原体幽门螺杆菌会引起胃部病变，如炎症和细胞增殖，从而导致胃癌。这些病理至少部分是由于易位的毒力蛋白CagA的作用，并且可能是由于肠道微生物群的改变。本研究拟利用果蝇和斑马鱼两种在肠上皮中表达CagA的转基因模型，研究CagA在肠上皮内表达的细胞自主效应及其对微生物群的非细胞自主效应。这些模型将共同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上皮信号通路，从而促进胃肠道癌症等疾病。在这些实验中获得的知识将为了解细菌病原体和常驻共栖生物之间的相互作用对人类疾病的影响机制提供有价值的见解，并将为预防和治疗感染性病因疾病提供新的思路。假设：幽门螺杆菌蛋白（CagA）以细胞自主的方式促进肠道细胞增殖，并改变肠道微生物群的组成，从而促进非细胞自主的细胞增殖。目的1：我将确定CagA表达是否诱导肠上皮细胞自主增殖。目的2：我将在果蝇中验证肠道上皮中CagA表达改变宿主微生物群并导致细胞增殖的假设。目的3：我将验证一个假设，即CagA转基因斑马鱼体内的微生物群发生了改变，这是引起CagA相关肠道病变（如增生）所必需和充分的。研究设计：为了研究CagA对肠上皮的细胞自主作用，我将在果蝇和斑马鱼中使用马赛克方法。这种方法使我能够直接测量表达CagA细胞的细胞增殖率，并确定这些细胞是否只经历JNK或Wnt途径激活。此外，使用报告细胞系，我将确定CagA的细胞自主表达是否会通过诱导细胞自主表达抗菌肽来改变免疫反应。为了确定CagA的表达是否会改变肠道微生物群，以及这是否足以诱导果蝇肠道中的细胞增殖，我将过度表达抗菌肽，并确定这是否必要或足以改变微生物群。此外，通过移植整个果蝇微生物群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，并确定导致这种效果的单一细菌种类。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诱导斑马鱼肠道细胞增殖的必要条件还是充分条件。本文概述的方法提供了一个独特的机会来研究幽门螺杆菌毒力工厂CagA在肠上皮上的保守作用。使用两个独立的模型系统将为这种常见微生物的保护和病理性质提供有价值的见解。这些数据对于了解病原体衍生癌症的病因学至关重要，并为潜在的治疗和预防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.