Protein export or secretion is one means by which pathogenic microbes injure the host cells of infected animals and humans. For example, bacteria export proteins that can kill host cells or promote adherence to tissues and block host defense mechanisms. Gram- negative bacterial pathogens utilize at least six distinct extracellular protein secretion systems to export proteins through their multi-layered cell envelope and in some cases into host cells. We propose to study the function of one such system, the Type VI secretion system (T6SS) that is expressed by numerous bacterial pathogens including Pseudomonas aeruginosa. P. aeruginosa is an opportunistic pathogen that frequently causes chronic lung infections in cystic fibrosis patients. The T6SS of this organism is an important virulence factor within the lung and thus is an exciting new target for vaccine and drug discovery. T6S apparatus is thought to be composed of 15-20 proteins whose biochemical function is not well understood. Our preliminary data suggest that proteins involved in T6SS function are structural analogs of a protein complex called the `phage tail' of the bacteriophage T4. This complex is a special machine that attacks the outer surface of host cells during the phage infection process. We propose to study whether the T6S apparatus of P. aeruginosa is a nano-machine similar to the phage tail complex, and introduces exported proteins into target cells via a similar membrane puncturing device. Thus, this proposal seeks funding for a research program to address the following general questions about T6SS: 1) Can we visualize components of the T6S apparatus and confirm its structural relationship to phage tail-like structures? 2) Does the T6S apparatus form channels in membranes that could be involved in protein deliver into target cells? 3) What controls the expression of the two other highly conserved T6SS gene clusters in P. aeruginosa? 4) Do components of the T6S apparatus appear on the surface of cells and do these actually spin? 5) Is it possible to control bacterial infection by developing drugs or vaccines targeting components of the T6SS?