Our current Merit Award focuses on factors contributing to virulence in Clostridium difficile, and a number of surface-layer proteins (SLPs) have been identified, that mediate bacterial adhesion to host intestinal epithelial cells. We now propose to significantly expand on these studies with the ultimate goal of translating our findings to a safe, easily utilizable, probiotic-based intervention for prevention of C. difficile infection (CDI). Three Specific Aims are proposed. First, we will define mechanistic bases of CD adherence to host epithelia mediated by surface-layer proteins (SLPs). CD mutagenesis and animal models of CDI will be exploited for these studies. Second, we will characterize non-SLP factors involved in CD colonization. Both bacterial and host- response proteins involved in, and contributing to, CD colonization as well as colonization resistance will be studied. Third, we will translate our bench-research findings to a clinically relevant outcome by developing a new adherence-based CDI intervention. This will involve construction of a targeted CD colonization inhibitor, by engineering a probiotic bacterium (Lactobacillus acidophilus) to express the CD colonization protein SlpA. We will validate the probiotic developed above by testing it against frequently isolated single-episode and relapse strains of CD recovered from surveillance studies performed at our VA hospital. For all studies in this proposal, we will use both the hamster and mouse models of acute CDI and CD colonization. We will also incorporate state-of-the-art methodologies including automated mass spectrometry and live-animal bioluminescence imaging to track the fate of ingested CD spores.