Pseudomonas aeruginosa is an opportunistic pathogen that is capable of causing a variety of infections, including a significant number of nosocomially-acquired urinary tract and lethal septicemic infections. It is also a chronic colonizer of the lungs of individuals with cystic fibrosis and leads to much of the lung pathology seen in these patients. The virulence of this organism is, in large part, due to its ability to export a number of hydrolytic enzymes and toxins. The extracellular secretion of these proteins requires an export pathway composed of at least twelve different gene products. Protein export mechanisms requiring nearly identical gene products, and now referred to as the Type II pathway, have been found in a number of gram-negative pathogens. Homologues of Type II export proteins are also known to be required for the biogenesis of the Type IV pilus of P. aeruginosa that is required for specific attachment to host tissues during infection. The similarities between these two systems suggests that the assembly and structure of the Type II protein export apparatus is reflected in the assembly and macromolecular structure of the Type IV pilus. The formation and operation of the export apparatus is likely to require complex interactions between its component parts. We propose to study the Type II Xcp export apparatus of P. aeruginosa by using a genetic approach to determine the structure and function of the individual components and the interactions between proteins required for the assembly and function of the export apparatus. Conditional mutations in two of the xcp genes, encoding XcpR and XcpT, will be generated using a strategy that allows for simple allelic exchanges and maintenance of gene organization and stoichiometric expression of the gene products. These mutants will be used to generate suppressors that restore protein export using a selection strategy we have developed. We propose to complement these studies using a biochemical approach to confirm and extend our observations of any protein-protein interactions determined by the suppressor analyses. Antibodies specific to each of the Xcp proteins will be used identify protein complexes generated by crosslinking experiments. Comparisons of complexes isolated from wild-type and xcp mutant strains should also allow us to define interactions between Xcp proteins and speculate on their roles in the assembly process.
