2005 Annual Report 4d.Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Wisconsin. Additional details can be found in the parent CRIS 1935-42000-052-00D Validation of the Effect of Interventions and Processes on Persistence of Pathogens on Foods. The purpose of this project was to investigate the role of serotype 4b surface associated antigens of L. monocytogenes in its virulence in vivo and intestinal epithelial cell invasion in vitro. There are three specific aims that will be addressed during this collaborative project: i) investigate the role of p60 and serotype 4b specific surface antigens in the ability of L. monocytogenes to colonize and grow on ready-to-eat meat products; ii) evaluate the interaction between P-glycoprotein and these surface antigenic determinants in the ability of L. monocytogenes to attach to, invade, and multiply in human intestinal epithelial cells using differentiated Caco-2 cells; and iii) investigate whether these determinants are required for virulence in the gastrointestinal tract using a mouse model, and whether the administration of specific antibodies, or the generation of such antibodies during immunization or infection, might protect against gastrointestinal infection with L. monocytogenes. Thus far, one of the serotype 4b antigen mutants was less virulent in a mouse model of gastrointestinal listeriosis. This mutant was also less able to adhere to and invade Caco-2 human intestinal epithelial cells in vitro. This finding must be confirmed, and examination of the other serotype 4b mutants completed, during the coming year. We will also assess the role of P-glycoprotein in intestinal epithelial cells on their susceptibility to infection, and in particular the ability of p60 to modulate this process, using antibodies against the serotype 4b antigens to provide a second means of assessing the role of these molecules in cell invasion in vitro and virulence in vivo. We encountered a technical difficulty in our effort to assess the virulence of the p60 mutant. Because this mutant lacks an enzyme (murein hydrolase) that cleaves the peptidoglycan cell wall during normal growth, it tends to grow as long filaments or clumps of rods. This confounds our efforts to quantify numbers of listeriae by plate counts. We have evaluated various growth conditions and found that incubation for an extended period (30 hr) diminishes filament formation to an extent that will allow us to perform in vivo (mouse infection) and in vitro (Caco-2 cell invasion) experiments in the coming year. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Neudeck, B.L., Loeb, J.M., Faith, N.G., and Czuprynski, C. J. 2004. P-glycoprotein involvement in defense against Listeria monocytogenes. Infect. Immun. 72:3849-3854.