SUMMARY Klebsiella pneumoniae (Kpn) is a primary causative agent of healthcare-associated infections affecting hundreds of millions worldwide. Bloodstream Kpn infections are of particular concern as they contribute to high patient mortality and represent a major public health burden. Kpn colonizes the gastrointestinal (GI) tract of healthy and immunocompromised individuals. In healthy individuals, bacteria can breach the intestinal epithelial surface and enter the circulatory system, but are rapidly cleared. In patients lacking a fully competent immune system, Kpn can cause life-threatening systemic infections. There is an urgent need to understand the factors that control the spread of Kpn from the GI tract to sterile sites. We developed a mouse model of naturally acquired Kpn GI colonization to identify pathogen and host factors which regulate acquisition, carriage, and dissemination. Using this mouse model, we found a critical role for B cells in controlling GI bacterial burden, systemic dissemination, and mortality. In this proposal, we will investigate the mechanisms by which B cells regulate Kpn growth in the GI tract, translocation, and systemic spread. In Aim 1, we will examine: a) the roles of pre-existing and Kpn- induced (adaptive) mucosal and systemic antibody in regulating Kpn GI growth and dissemination and b) the impact of dysbiosis in driving unchecked GI growth and dissemination in B cell-deficient mice. This work will be complemented by an innovative technical approach in Aim 2 which will reveal bottlenecks and population dynamics experienced by Kpn along the path from the mouth to the gut to systemic sterile sites in both wild type and B cell-deficient mice. Here we will leverage a uniquely barcoded Kpn population in combination with high- resolution sequencing to identify frequencies of translocation events, expansion dynamics, and the origination of founding populations within distinct tissue sites and blood. Ultimately, this work is expected to reveal the importance of B cells in controlling Kpn growth and dissemination at various sites within the host and the mechanisms by which this occurs.