PROJECT SUMMARY Within the colon, early life bacterial colonization events favorably influence host health, but simultaneously establish a vast reservoir for antimicrobial resistance genes. Bacteroides fragilis represents up to 2.5% of the human gut microbiota and is often found in neonates within the first month of life. B. fragilis is the leading cause of anaerobic sepsis and deep tissue infections. While long recognized as an antimicrobial resistance threat, an increasing number of B. fragilis clinical isolates now express high-level resistance to b-lactam antibiotics including the carbapenems which are considered agents of last resort. Genetic analysis has revealed two distinct phylogenetic clades of B. fragilis, termed clade I and clade II. Clade II strains exclusively harbor the cfiA locus which encodes for metallo-b-lactamase (MBL) activity - an antibiotic resistance enzyme for which no available inhibitors have yet been defined. We have discovered a novel B. fragilis toxin that is specifically expressed in clade II isolates, termed Bcf1. Bcf1 appears to play a critical role in interbacterial competition between B. fragilis isolates, positioning this toxin to enable dominance of clade II strains within the B. fragilis niche, expanding the reservoir for MBL-encoded resistance. The primary goal of this proposal is to examine the molecular mechanisms by which Bcf1 facilitates interbacterial competition. Our preliminary data suggest that competition may rely on the ability of Bcf1 to limit nutrient access within the niche. We will explore this through a series of in vitro and in vivo studies utilizing engineered bacterial genetic variants to discover the protein(s) targeted by Bcf1 and evaluate the necessity of Bcf1 in inter-clade competition within the B. fragilis niche in a mouse model of colonic colonization. These studies will benefit from the use of a novel model of B. fragilis vertical transmission in which the temporal and genetic determinants of niche colonization and interbacterial competition by B. fragilis can be evaluated in young mice. Through a comprehensive dissection of the mechanism by which Bcf1 ensures the success of clade II B. fragilis strains, this study has the potential to illuminate a novel mechanism by which antibiotic resistance to the potent metallo-b-lactam antibiotics is achieved. By focusing on the ecological niche, these studies may discern critical early events in niche occupancy that are subject to perturbation, or alternatively highlight novel probiotic-based strategies by which to re-shape interbacterial competition events mediated by Bcf1 to selectively reduce the prevalence of clade I MBL-expressing strains within the colonic microbiome.