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

描述（由申请方提供）：侵袭性胃肠炎和肠热病是全球死亡率和发病率的主要来源。这些综合征发病机制的最初步骤涉及有毒细菌破坏胃肠上皮屏障，在肠组织中定植和生长，以及在某些情况下随后通过消化道或血流全身扩散到其他器官。上皮细胞侵袭的受体已被确定为这些病原体中的几种。然而，这些受体中的许多并不存在于肠腔侧，而是上皮细胞基底外侧膜的丰富组分。这意味着病原体必须首先破坏上皮紧密连接，或者在到达它们的受体进行上皮入侵之前找到替代机制来穿过上皮屏障。我们提出了一种新的模型肠道入侵和定居的病原体李斯特菌单核细胞增多症。我们发现，小肠绒毛的尖端是一个特殊的网站，正常的细胞挤出过程允许基底外侧受体暴露在管腔表面。单核细胞增生李斯特菌在这些部位发现其受体E-钙粘蛋白，并侵入绒毛尖端的胃肠道上皮。我们建议建立一个豚鼠李斯特菌胃肠道感染的口腔模型，通过表征肠道侵袭和定植的部位和动力学来测试该模型。我们将测试的假设，即细胞挤压区的入侵导致在胃肠道李斯特菌的无症状携带，并研究这种模式的入侵和全身性疾病之间的关系。由于在豚鼠中发现的技术限制，我们还提出测试李斯特菌侵袭素InlA的遗传改变是否可以改善其与鼠E-钙粘蛋白的相互作用，以开发肠道李斯特菌病的鼠模型。在体内实验的同时，我们将使用细胞培养实验来确定李斯特菌在细胞挤出区内化所涉及的内吞作用机制。我们将利用极化的MDCK细胞单层来检验李斯特菌在细胞挤出区的入侵过程中利用连接重塑的假设，单核细胞增生李斯特菌已成为美国食源性疾病死亡的重要原因，因为它是肠道感染死亡的27.6%。其他重要的侵入上皮的病原体包括轮状病毒、沙门氏菌、志贺氏菌、耶尔森氏菌、肠侵袭性大肠杆菌。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，解释单核细胞增生李斯特菌，也许还有其他侵入性肠道病原体，突破肠道屏障。

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.