Our long-term goal is to develop human vaccines for the prevention of diarrhea and/or HUS caused by E. coli O157:H7 and diarrhea by other pathogenic E. coli. In the currently proposed studies, we will screen the genome of E. coli O157:H7 to identify protective antigens that would become components of a human vaccine, and such approach can be used to protect against pathogenic E. coli infections without affecting commensal flora. The central hypothesis is that identification of protective antigens unique to O157:H7 is a viable approach for development of a vaccine for prevention of diarrhea and Hemolytic Uremic Syndrome episodes. Progress in recent years identifying E. coli O157:H7 novel virulence factors suggest that they can become useful components of a vaccine candidate. Of particular note are those proteins expressed on the bacterial membranes or secreted to the extracellular milieu, which have been previously associated with virulence traits. Therefore, it is plausible to propose that E. coli O157:H7-specific genes can become excellent candidates as components of vaccines. Our proposed experimental approach will focus on two specific aims: Identify the O157-specific gene sequences of E. coli O157:H7 and use them to generate a library of expression constructs optimized for vaccine screening;and determine the protective capacity of O157-specific gene sequence using a murine model of E. coli O157:H7 infection, to identify a subset of subunit vaccine candidates. Our proposal is significant because it is expected to provide the basic knowledge needed to develop an optimal vaccine that will be used to prevent infections caused by E. coli O157:H7, a category B pathogen for its potential use as biothreat agents in food and agriculture, and eventually to treat infections caused by other related pathogenic E. coli strains.