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

描述(申请人提供)：侵袭性胃肠炎和肠热病是世界范围内死亡和发病率的主要来源。这些综合征发病的最初步骤包括毒力细菌破坏胃肠上皮屏障，在肠道组织中定植和生长，在某些情况下，随后通过淋巴管或血流向其他器官扩散。其中几种病原体的上皮细胞侵袭受体已经被定义。然而，许多这些进入的受体并不是在肠腔一侧发现的，而是上皮细胞基侧膜的丰富成分。这意味着病原体必须首先打破上皮紧密连接，或者找到替代机制来穿过上皮屏障，然后才能到达它们的受体以进行上皮入侵。我们提出了一种新的单核细胞增多性李斯特菌肠道侵袭和定植模型。我们发现，肠绒毛的顶端是一个特殊的部位，正常的细胞挤出过程允许基侧受体暴露在管腔表面。单核细胞增多性李斯特菌在这些部位发现其进入受体E-钙粘素，并侵入绒毛末端的胃肠上皮。我们建议建立一种豚鼠胃肠道李斯特氏菌感染的口腔模型，通过表征肠道侵袭和定植的部位和动力学来检验该模型。我们将检验这一假设，即细胞挤出区的入侵会导致李斯特菌在胃肠道中无症状携带，并研究这种入侵模式与系统性疾病之间的关系。由于在豚鼠身上发现的技术限制，我们还建议测试入侵李斯特菌INLA的基因改变是否可以改善其与小鼠E-钙粘附素的相互作用，以建立一种小鼠肠道李斯特氏病模型。在体内实验的同时，我们将使用细胞培养实验来确定内吞作用参与李斯特菌在细胞排出区内化的机制。我们将利用极化的MDCK细胞单层来测试李斯特菌在细胞挤出带入侵过程中利用连接重塑的假设。在美国，单核细胞增多性李斯特菌已经成为食源性疾病死亡的一个重要原因，因为它导致了27.6%的肠道感染死亡。其他入侵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、肠道侵袭性大肠杆菌和弯曲杆菌。这些生物是如何突破胃肠道上皮屏障的，目前还不清楚。我们建议研究一种新的范式，解释单核细胞增多性李斯特菌，也许还有其他侵袭性肠道病原体，如何突破肠道屏障。

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.