Microbial Invasion of the Intestine at the Cell Extrusion Zone of the Villus Tip

描述（由申请人提供）：侵袭性胃肠炎和肠道热是世界范围内死亡率和发病率的主要来源。这些综合征发病的最初步骤涉及毒性细菌破坏胃肠道上皮屏障，在肠组织中定植和生长，在某些情况下，随后通过淋巴或血流全身扩散到其他器官。上皮细胞侵袭的受体已经被定义为这些病原体中的几种。然而，许多这些进入受体并不存在于肠腔侧，而是上皮细胞基底外膜的丰富成分。这意味着病原体必须首先破坏上皮紧密连接，或者在到达受体进行上皮入侵之前找到穿越上皮屏障的替代机制。我们提出了一个新的模型，肠道入侵和定植病原体单核增生李斯特菌。我们发现，肠绒毛的尖端是一个特殊的部位，在这里，正常的细胞挤压过程允许基底外侧受体暴露在管腔表面。单核增生李斯特菌在这些部位找到e -钙粘蛋白，它的进入受体，并侵入绒毛尖端的胃肠道上皮。我们建议建立豚鼠口腔李斯特菌胃肠道感染模型，通过表征肠道入侵和定植的部位和动力学来验证该模型。我们将验证侵袭细胞挤压区导致胃肠道无症状携带李斯特菌的假设，并研究这种侵袭模式与全身性疾病之间的关系。由于在豚鼠中发现的技术限制，我们还提议测试李斯特菌入侵蛋白InlA的遗传改变是否可以改善其与小鼠e -钙粘蛋白的相互作用，以建立小鼠肠道李斯特菌病模型。与体内实验并行，我们将使用细胞培养实验来确定细胞挤压区李斯特菌内化的内吞作用机制。我们将利用极化的MDCK细胞单层来验证李斯特菌在侵袭细胞挤压区时利用连接重塑的假设。在美国，单核细胞增生李斯特菌已成为食源性疾病导致死亡的重要原因，因为27.6%的肠道感染死亡是由李斯特菌造成的。其他侵袭上皮的重要病原体包括轮状病毒、沙门氏菌、志贺氏菌、耶尔森氏菌、肠侵入性大肠杆菌和弯曲杆菌。这些微生物如何破坏胃肠道上皮屏障尚不清楚。我们建议研究一种新的范式来解释单核增生李斯特菌以及其他侵入性肠病原体是如何突破肠道屏障的。

DESCRIPTION (provided by applicant): Invasive gastroenteritis and enteric fever are major sources of mortality and morbidity worldwide. The initial steps in the pathogenesis of these syndromes involve a breach of the gastrointestinal epithelial barrier by virulent bacteria, colonization and growth in the intestinal tissue, and in some cases subsequent systemic spread to other organs via the lymphatics or blood stream. The receptors for epithelial cell invasion have been defined for several of these pathogens. However, many of these receptors for entry are not found on the luminal side of the intestine, but are instead abundant components of the basolateral membranes of epithelial cells. This implies that pathogens must first breach the epithelial tight junctions, or find alternative mechanisms to cross the epithelial barrier before reaching their receptors for epithelial invasion. We have proposed a novel model of intestinal invasion and colonization by the pathogen Listeria monocytogenes. We found that the tips of intestinal villi are a specialized site where the normal cell extrusion process allows basolateral receptors to be exposed at the luminal surface. Listeria monocytogenes finds E-cadherin, its receptor for entry, at these sites and invades the gastrointestinal epithelium of the villus tip. We propose to establish an oral model of Listeria gastrointestinal infection in Guinea pigs to test this model by characterizing the sites and kinetics of intestinal invasion and colonization. We will test the hypothesis that invasion of the cell extrusion zone results in asymptomatic carriage of Listeria in the gastrointestinal tract and study the relationship between this mode of invasion and systemic disease. Because of technical limitations found in Guinea pigs, we also propose to test whether genetic alterations in the Listeria invasin InlA can improve its interaction with murine E-cadherin, in order to develop a murine model of intestinal Listeriosis. In parallel with in vivo experiments, we will use cell culture experiments to define the mechanisms of endocytosis involved in internalization of Listeria at the cell extrusion zone. We will utilize polarized MDCK cell monolayers to test the hypothesis that Listeria take advantage of junction remodeling during invasion of the cell extrusion zone.Listeria monocytogenes has emerged as a significant cause of mortality due to food-borne illness in the United States, since it was responsible for 27.6% of deaths from enteric infection. Other important pathogens that invade the epithelium include rotavirus, Salmonella, Shigella, Yersinia, enteroinvasive E. coli, and Campylobacter. How these organisms breach the gastrointestinal epithelial barrier is not clearly understood. We propose to study a new paradigm that explains how Listeria monocytogenes, and perhaps also other invasive enteropathogens, breach the intestinal barrier.