| Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause lethal infections in immunocompromised individuals. The ability to cause sepsis and death is highly correlated with the expression of a type III secretion/translocation system whereby P. aeruginosa injects proteins with toxic enzymatic activities directly into target cells. Of the four enzymes injected into cells, ExoS, ExoT, ExoY and ExoU, intoxication with ExoU is associated with more severe outcomes including lung injury, bacterial dissemination, and sepsis in animal model and human infections. During infection of tissue culture cells, ExoU expression correlates to an acutely toxic phenotype with a rapid increase in cell permeability and necrotic death. Although type III-mediated injection is the natural route of intoxication, in order to study the activities of ExoU in the absence of other bacterial factors, a cellular transfection system was developed. Similar to the results of infection, expression of recombinant ExoU after transfection correlates with the release of an intracellular marker enzyme such as beta-galactosidase or lactate dehydrogenase. Although transfection analyses were useful for domain mapping studies, the acute activity of ExoU in mammalian cells prevented detection of the toxin and localization of ExoU within cellular compartments. To determine if ExoU interacts with cellular proteins, we performed a yeast two-hybrid analysis, however, we were unable to obtain transformants expressing full-length forms of ExoU. To assess the cytotoxic activity and to search for ExoU targets in a genetically tractable system, Saccharomyces cerevisiae was used as a host to express the toxin.; ExoU was cloned in both high and low copy number vectors and the biological activity was measured relative to derepression or induction of a GAL1 promoter. ExoU was acutely toxic to yeast and this biological activity appeared to be mediated by minute amounts of the expressed protein. Genetic screens were designed to identify yeast suppressors of ExoU toxic activity. These experiments resulted in the isolation of only intragenic deletions or truncations of ExoU, confirming previous mapping data from transfection analyses. GFP-tagged ExoU expression was detectable in yeast by fluorescent microscopy and by immunomicroscopy with a monoclonal antibody specific to ExoU. The full-length molecule and non-toxic forms of ExoU were localized to the cytoplasmic compartment of yeast cells. Using a combination of Nomarski and fluorescent microscopy, live staining, and immunostaining, ExoU expression correlated to vacuolar fragmentation and disruption of the yeast actin cytoskeletal structure. (Abstract shortened by UMI.)... |
