ABSTRACT Extraintestinal Pathogenic Escherichia coli (ExPEC) comprise a broad group of E. coli isolates that cause disease outside of the intestine and are among the most common causes of clinical bacteremia in humans. Most bacteria that gain access to the circulatory system are captured and killed by tissue-resident macrophages in the liver. In contrast to related commensal and intestine-restricted pathogenic E. coli, ExPEC isolates possess mechanisms to survive longer within these hepatic defenses. To uncover genes underlying such mechanisms, we performed a genome-scale transposon insertion sequencing screen on ExPEC isolated from murine livers following intravenous injection of mice with an ExPEC transposon insertion library. This screen uncovered a core set of genes required for optimal ExPEC fitness in the liver, ~90% of which encode components or regulators of the cell envelope. Unexpectedly, analysis of the population-level distribution of transposon-insertion mutants revealed that a random subset of the bacteria within the liver expands clonally. Together, these observations underlie the two central hypotheses that will be explored in this proposal: 1) ExPEC pathways that control distinct cell envelope homeostasis axes protect bacteria against liver macrophages to maintain bacterial survival, and 2) interactions between liver macrophages and specific bacterial genes that support intra-hepatic survival govern how the pathogen is distributed within the host during clearance. Findings from the proposed studies, which rely on both pathogen- and host-centric approaches, will provide knowledge of how ExPEC survive within and are cleared from extraintestinal organs. Ultimately, these fundamental insights may assist in the development of therapeutic strategies to accelerate clearance of systemic infections.