Burkholderia pseudomallei (Bp), and its diminutive deletion clone Burkholderia mallei (Bm) cause fatal human diseases. Their inherent antibiotic resistance, ability to infect via aerosol, and lack of protective vaccines has led to their classification as major biothreats and select agents. Although reports of natural infections by these bacteria date back over 100 years, and dozens of new cases still arise each year, relatively little is known about their virulence and pathogenicity mechanisms. However, the recent availability of complete genome sequences for multiple strains of Bp, Bm, and their nonpathogenic relatives, as well as the development of new shotgun proteomic technologies, has opened up new avenues to quickly identify dozens of novel extracellular virulence factors of these and other pathogens. We propose to use in-place ion-trap tandem mass spectrometry methods to rapidly identify the genes encoding >100 secreted and cell surface-exposed proteins of Bm and Bp. Next, to find the 15-20 of these genes with the highest probability of involvement in disease, we will search these genes against: 1) our microarray (expression profiling) databases containing Bm genes showing altered expression in mouse spleen and liver; and 2) against an in silico-generated database of 390 genes shared by Bp and Bm, but missing from 5 closely-related, but nonpathogenic Burkholderia. Genes thus selected will be site-specifically inactivated and the resultant mutants assessed for virulence in our Galleria mellonella (wax moth larvae) surrogate disease model, in which behavior of Bm and some of its virulence mutants correlates well with what is observed in infected hamsters. We will also assess the ability of these mutants to invade and survive in human and mouse macrophages and lung epithelial cells. Mutants showing significantly reduced virulence in more than one of these assays will then be tested in a Syrian hamster animal model. These experiments will discover and partially define new secreted and cell surface proteins of Bm and Bp that interact in diverse ways with animal hosts during disease. The results obtained may not only lead to new paradigms and insights into microbial pathogenesis, but also to new diagnostics, vaccines and therapeutics for Bm and Bp and other important pathogens and biothreat agents. [unreadable] [unreadable] [unreadable] [unreadable]