To understand the molecular mechanism of the foodborne pathogen, Listeria monocytogenes pathogenesis during intestinal phase of infection, and to study the effect of a bioengineered Lactobacillus probiotic strain for immunomodulatory benefits in the intestinal tract to improve gut health and reduce Listeria colonization, shedding, and infection.Specific Objective 1. To understand the molecular mechanism of the foodborne pathogen, Listeria monocytogenes pathogenesis during intestinal phase of infection in mouse, gerbil and swine models.Listeria monocytogenes is a foodborne pathogen. During the gastrointestinal phase of infection, Internalin A (InlA) has been implicated to aid in L. monocytogenes translocation across the gut epithelial barrier (37). However, L. monocytogenes expressing truncated InlA (defective) can infect laboratory animals (17) and the humans (16). The host cell receptor for InlA is E-cadherin (E-cad); however, InlA does not interact with the E-cad of the non-permissive hosts such as mouse or rat, but it interacts with the E-cad of permissive hosts such as humans, guinea pigs, rabbits, and gerbils (13, 26), thus raising a serious question about the involvement of InlA during gastrointestinal phase of infection.Previously, we have shown that Listeria adhesion protein (LAP), a 104-kDa alcohol acetaldehyde dehydrogenase (lmo1634), present on the bacterial cell surface and exhibits adhesion properties in pathogenic Listeria species and promotes adhesion to cell lines of intestinal origin (19, 21, 34). We also showed that human heat shock protein 60 (Hsp60) is the epithelial receptor for LAP, and an anti-Hsp60 antibody blocked LAP-mediated L. monocytogenes interaction with host cells (18, 40). We have also shown that LAP promotes L. monocytogenes translocation across the epithelial cell barrier in vitro (6). An InlA-mutant strain also successfully crossed the epithelial barrier in cell culture and in a mouse model, and L. monocytogenes translocation was facilitated by activation of NF-κB, inflammatory cytokine release (TNF-α, IL-6) and stimulation of myosin light chain kinase (MLCK) for epithelial tight junction protein dysregulation (14). These findings demonstrate that the translocation of L. monocytogenes across the epithelial barrier is dependent on an interaction of LAP with the host cell receptor Hsp60 and is independent of InlA. However, the involvement of LAP in InlA/E-cad permissive animal models, such as in gerbil and possibly in swine is not well understood. In this project, we plan to investigate whether LAP contributes to translocation of L. monocytogenes across the intestinal barrier in vivo in mice, Mongolian gerbil, and swine models and elucidate the molecular mechanism by which LAP facilitates L. monocytogenes translocation.Specific Objective 2. To demonstrate the effect of a bioengineered Lactobacillus probiotic strain for immunomodulatory benefits to improve gut health and reduce Listeria colonization, shedding, and infection.The gut mucosa represents the first site for the dynamic interaction of the enteric pathogens with the host. Therefore, averting this critical pathogen interaction step should help prevent extra-intestinal dissemination of pathogens and the consequent pathology. Live probiotic bacteria such as lactobacilli and bifidobacteria are known to colonize and proliferate in the intestine to improve intestinal microbial balance and protect the host from pathogens (5, 38). Among the different probiotic bacteria used, Lactobacillus species are common because they are a natural inhabitant of the gut, modulate immune system (1, 39), and enhance epithelial innate defense and restore epithelial barrier function (5, 33). One of the major drawbacks of probiotics for prophylactic or therapeutic use is that the antimicrobial effect is inconsistent and may be strain specific thus may have limited efficacy against a target pathogen. Therefore, pathogen-specific proteins are expressed on probiotic bacteria to prevent pathogen interaction with the host (2, 29, 31). Here, we want to investigate whether a probiotic bacterium expressing LAP on their surface can competitively exclude pathogen interaction on the host epithelial cell, thereby preventing listeriosis in a high-risk population in the background of the probiotic's natural beneficial attributes. In a previous study, as a proof of concept, we showed that LAP of L. monocytogenes expressed on Lactobacillus paracasei was able to reduce L. monocytogenes interaction with the enterocyte-like Caco-2 cell model (24), however, its effectiveness in an animal model and the host response were unknown. Here, we want to investigate if the LAP, especially from a non-pathogenic Listeria (L. innocua),expressed on a more commonly used probiotic strain, Lactobacillus casei to competitively exclude pathogen interaction in mouse and swine models.