ROLE OF ATP-BINDING CASSETTE TRANSPORTERS IN INNATE INTESTINAL DEFENSE

描述（由申请人提供）：制定针对食源性病原体的保护策略对处于危险中的人群至关重要。食源性单核细胞增生李斯特菌造成了相当大的发病率和死亡率。这项研究的长期目标是阐明先天防御机制对肠道病原体的重要保护作用。我们的总体假设是，人类atp依赖的膜转运蛋白p -糖蛋白通过修饰入侵所需的宿主细胞骨架蛋白，在对抗单核细胞增生乳杆菌的先天防御机制中起作用。我们的假设基于以下初步数据：1)在体内和体外模型中，p-糖蛋白的表达程度和功能与单核增生乳杆菌的侵袭程度呈负相关；2)p-糖蛋白的转运活性在侵袭过程中迅速上调；3)p-糖蛋白的表达水平在基线时影响¿-catenin的细胞定位以及感染期间的动员。因此，本研究旨在研究p -糖蛋白如何通过关注病原体入侵所需的宿主蛋白来保护宿主免受单核增生乳杆菌的侵害。特异性目的1：确定p -糖蛋白是否影响单核增生李斯特菌进入细胞所需的宿主蛋白的定位、表达水平或功能。为了实现这一目标，我们将采用p -糖蛋白ON/OFF细胞系，并使用共聚焦显微镜和Western免疫印迹法来确定细胞定位或细胞骨架蛋白的表达水平是否受到p -糖蛋白的影响。我们还将确定p -糖蛋白是否通过两个关键的宿主受体改变信号传导。特异性目的2：确定p -糖蛋白是否影响单核细胞增生李斯特菌进入细胞时宿主蛋白的募集。为了实现这一目标，我们将使用p -糖蛋白ON/OFF细胞系和共聚焦显微镜来确定p -糖蛋白表达水平是否影响入侵过程中宿主蛋白的正常募集或聚集。

DESCRIPTION (provided by applicant): The development of protective strategies against foodborne pathogens is critical for populations at risk. The food borne bacterium Listeria monocytogenes is responsible for considerable morbidity and mortality. The long-term goal of the proposed research is to elucidate innate defense mechanisms important for protection against enteric pathogens. Our global hypothesis is that the human ATP-dependent membrane transporter P-glycoprotein functions an innate defense mechanism against L. monocytogenes by modifying host cytoskeleton proteins required for invasion. We base this hypothesis on preliminary data demonstrating that 1) the extent of P-glycoprotein expression and function negatively correlate with the degree of L. monocytogenes invasion using in vivo and in vitro models, 2) P-glycoprotein transport activity is rapidly up-regulated during the invasion process and 3) the level of P-glycoprotein expression influences ¿-catenin cellular localization at baseline as well as mobilization during infection. Therefore, studies described in the current proposal are designed to investigate how P-glycoprotein protects the host against L. monocytogenes by focusing on host proteins required by the pathogen for invasion. SPECIFIC AIM 1: Determine if P-glycoprotein influences the localization, expression level or function of host proteins required for Listeria monocytogenes entry into cells. To accomplish this aim, we will employ the P-glycoprotein ON/OFF cell line and use confocal microscopy and Western immunoblotting to determine if the cellular localization or level of expression of cytoskeletal proteins is influenced by P-glycoprotein. We will also determine if P-glycoprotein alters signaling through two key host receptors. SPECIFIC AIM 2: Determine if P-glycoprotein influences the recruitment of host proteins during Listeria monocytogenes entry into cells. To accomplish this aim, we will employ the P-glycoprotein ON/OFF cell line and confocal microscopy to determine if the level of P-glycoprotein expression affects the normal recruitment or clustering of host proteins during invasion. In order to protect the public from foodborne pathogens, an understanding of innate defense mechanisms is required. Once this is accomplished, we can use this information to develop protective strategies that exploit these preexisting barriers to L. monocytogenes and potentially other pathogens.