This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Site-directed mutagenesis is typically used to inactivate wild-type (wt) Gram-negative bacteria. However, virulence genes have often not been identified, thus making attenuated strains difficult to construct. Attenuating virulent bacteria by forcing the expression of various appendages, e.g., fimbriae, needles, or capsules, represents an alternative approach to inactivating wt bacteria, thereby allowing these mutants to be used as live vaccines while still stimulating a robust immune response. To test this hypothesis, the Yersinia pestis (Y. pestis) capsule antigen F1 (F1-Ag) was selected. Secretion of F1-Ag is dependent upon the formation of a secretion apparatus encoded by the caf operon, which includes an usher protein that forms into channels in the bacterial outer membrane (OM), allowing secretion of F1-Ag. We questioned whether over expression of this protein secretion apparatus in the OM would adversely affect the bacterium, thereby, influencing channel-mediated attenuation. We hypothesize that over expressed usher Caf1A protein, when assembled into channels, will attenuate Salmonella, and this avirulent mutant will render protection against wt Salmonella challenge. To study this possibility, we investigated whether over expression of the entire caf operon in wt Salmonella enterica serovar Typhimurium (S. typhimurium) would attenuate Salmonella and found that over expression of the Caf1 capsule can significantly attenuate S. typhimurium both in vitro and in vivo. Through a series of gene deletions in the caf operon, we demonstrated that the observed capsule-mediated Salmonella attenuation resulted from over expression of Caf1A. In the future, we will investigate whether the Caf1A-attenuated Salmonella are able to serve as a live vaccine for salmonellosis. We will further investigate the underlying mechanism involved in this novel attenuation strategy via microarray technology.