Extraintestinal pathogenic Escherichia coli (ExPEC) cause a diverse array of diseases including urinary tract infections, sepsis, and neonatal meningitis. These infections affect 6.7 to 8.6 million individuals each year in the United States, at a cost of $1.5 to $2.3 billion. Current treatment protocols for ExPEC-induced infections are often ineffective or rendered useless by the acquisition of antibiotic resistance. Genomic sequencing of several ExPEC isolates, in addition to epidemiological studies and genetic screens have identified numerous putative virulence factors. However, in most cases the functional roles of these bacterial factors during infection remain enigmatic. Recently, we have developed a novel infection model employing zebrafish embryos that allows for large-scale screening and functional analysis of ExPEC virulence mechanisms following bacterial inoculation into either the blood or pericardial chamber. An initial screen of several sequenced ExPEC isolates in this model system demonstrated that the virulence potential of these microbes can vary significantly in a niche-dependent fashion. These data suggest that ExPEC isolates have evolved multiple, probably overlapping strategies for enhanced survival and growth within specific host environments. Here we propose to utilize the zebrafish model system, in association with genetic screens and gene expression profiling, to delineate ExPEC virulence factors and host response pathways that come into play during both localized and systemic infections. The ultimate goal of the proposed research is to identify microbial targets that will aid the development of improved tools for the prevention and treatment of ExPEC-induced infections. If successful, this work has the potential to impact the quality of life, health, and longevity of millions. The emerging prevalence of antibiotic resistance among ExPEC isolates, as well as growing numbers of highly susceptible elderly and immunocompromised individuals, provide additional incentive for advancing our understanding of the myriad strengths and weaknesses of ExPEC. PUBLIC HEALTH RELEVANCE: Strains of Extraintestinal pathogenic Escherichia coli (ExPEC) cause a diverse array of serious illnesses that affect several million individuals each year, costing billions in health care and time loss at work. Using a novel zebrafish infection model, we propose to identify bacterial factors and host responses that modulate the ability of different ExPEC strains to cause disease during both systemic and localized infections.