Intestinal P-Glycoprotein: A Novel Host Defense Mechanism Against Listeria Monocytogenes

NON-TECHNICAL SUMMARY: Listeria monocytogenes is an important food-borne pathogen responsible for considerable morbidity and mortality. Although the mechanism of Listeria monocytogenes invasion into mammalian cells has been extensively studied, there is much to learn concerning host defense mechanisms at the intestinal epithelial level. Data from our laboratory indicate that the ATP-dependent efflux transporter p-glycoprotein may limit the extent of Listeria monocytogenes invasion in intestinal epithelial cells. P-glycoprotein expression in the intestine has been previously associated with decreased absorption of drugs which are substrates. Based on its purported physiological role as a cytotoxic protection mechanism, we believe that p-glycoprotein limits the entry of p60, a bacterial protein necessary for invasion. It is our hypothesis that Listeria p60 is transported out of cells by p-glycoprotein. The objective of the current application is to elucidate the role of p-glycoprotein in host defense against Listeria monocytogenes. To determine how intestinal P-glycoprotein is involved in protecting humans from foodborne infections due to Listeria monocytogenes. By understanding host defense mechanisms against this organism, we will be better able to develop protective strategies.

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APPROACH: To determine if intestinal P-glycoprotein transports p60, we will purify L. monocytogenes-p60 and label with 125I. Subsequently, we will measure the apical to basolateral and basolateral to apical flux of 125I-p60 in Caco-2 cells. Caco-2 cells are a well validated model for studying the epithelial invasion of L. monocytogenes. To confirm P-glycoprotein involvement, we will utilize the P-glycoprotein monoclonal antibody and functional inhibitor MRK-16 to block transcellular transport of 125I-p60. Additional experiments will be conducted to determine the extent of intracellular penetration of p60 in Caco-2 cells. This will be accomplished by labeling newly synthesized L. monocytogenes proteins with 35S-methionine and measuring their intracellular concentrations in Caco-2 cells. Intracellular amounts of 35S-methionine-p60 will be determined via SDS-PAGE and autoradiography. MRK16 will be employed to confirm P-glycoprotein specificity. To elucidate the effect of in vivo modulation of P-glycoprotein on Listeria susceptibility, the FVB mouse will be used. Intestinal P-glycoprotein will be overexpressed by the chronic administration of a known P-glycoprotein substrate. Inhibition of intestinal P-glycoprotein will be accomplished using a pluronic block polymer. Following overexpression or inhibition, mice will be given an intragastric challenge of L. monocytogenes and dissemination from the GI tract measured over time.