Role of ATP-Binding Cassette Transporters in Innate Intestinal Defense

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. <P>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. <P>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. <P>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. <P>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. <P> 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.