Our work over the course of nearly 25 years of this grant have revealed insights onto the pore-forming mechanism of the cholesterol-dependent cytolysins (CDCs), which have laid the foundations to understand the basis by which this large class of toxins make the transition from soluble monomers to large oligomeric membrane-embedded pore complexes. In the next 5 years of the merit award we propose to address three specific aims, which will continue our work on the CDCs but will also open up new major fields of research in related areas. In the first aim we will address outstanding issues in the mechanism of the CDC pore-forming mechanism. The first of these is to understand how the B-barrel pore assembles and inserts into the membrane bilayer. This is a question that applies to all B-barrel pore-forming proteins but is fraught with complicating thermodynamic factors. Using our previous discoveries of the transition state barriers to pore formation and the ability to change the nature of the B-hairpins we will determine the mechanism of B-barrel assembly and membrane insertion. These studies will also address a contentious issue in the CDC-mechanism, which is whether incomplete oligomers (rather than complete rings) can insert a partial B-barrel into the membrane. In this aim we will also reassess the basis by which the listeriolysin 0 pH sensor functions, as we have recent published data and preliminary studies, which suggest that our original hypothesis made in 1995 is not the mechanism of pH sensing. In the second aim we plan to pursue the study of the CDC-like (CDCL) proteins that we have determined to be ancient relatives of the CDCs (perhaps the forerunners of the CDCs), the genes for which are widespread in numerous bacterial species (>300 so far) from at least 6 different phyla, as well as a few species of diatoms and fungi. Our primary goals are to (1) understand the pore-forming mechanism of these toxins, as some preliminary data suggest there are some fundamental differences with the CDCs, and (2) to identify the prokaryotic and/or eukaryotic targets of several of these proteins isolated from species from the oral and intestinal human microbiomes and from terrestrial environments, as none of the CDCL binding domains exhibit any similarity to the canonical binding domain of the CDCs. In the third aim we plan to study the mechanism of bacterial killing by a novel class of anti-bacterial toxins that are produced by a few species of the Provote/la and Bacteroides found in the oral and intestinal microbiome of humans. Our preliminary studies suggest that they are proteolytically-activated toxins that form higher order complexes (possibly pore-forming toxins), which target various species of bacteria. Our preliminary studies suggest that they are a unique class of antibacterial toxin, as there are no known class of prokaryotic or eukaryotic toxins that exhibit any similarity to the primary structure of these toxins. RELEVANCE (See instructions)...