HOST RESPONSE TO CYTOTOXIC PROTEINS

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High mortality rates are associated with nosocomial lung infections due to P.aeruginosa. As current antibiotic therapies are associated with a 50-80 percent mortality in this infection, improved methods for prevention and therapy clearly are needed. The airspace instillation of PA103, a cytotoxic strain of P.aeruginosa, recreates the lung injury and sepsis seen in many of the patients with nosocomial pneumonia; the instillation of the bacteria causes lung epithelial injury, bacteremia, organ failure and death of experimental animals. Over the last 4 years, our two laboratories have collaborated on bacterial genetic experiments and animal physiology experiments that have led to the discovery of novel P.aeruginosa extracellular products which are synthesized and secreted and coordinately controlled with exoenzyme S by a type III secretory system. Our previous investigations have documented that the lung injury and dissemination of the airspace PA103 to the circulation correlated with the production of exoenzyme S by the bacteria. Two of the novel extracellular products produced and secreted with exoenzyme S are ExoU, a novel cytotoxin, and PcrV, a homolog of the Yersinia pestis V antigen, which may affect host cytokine production. Our hypothesis is that these two bacterial products are the major virulence products of P.aeruginosa and therapies directed against these products would prevent the local and systemic injury due to the dissemination seen with this infection. To prove this hypothesis, we will compare the effects of these newly discovered bacterial products to P.aeruginosa endotoxin in terms of their individual and combined effects on lung injury, lung inflammation and the systemic inflammatory response. We will utilize isogenic transposon PA103 strains that are selectively missing the genes for ExoU, for PcrV or both of these genes. These 2 recombinant proteins are also available for experiments and we have obtained specific endotoxin antagonists and genetically deficient mice for lipopolysaccharide binding protein to determine the effects of these products in animals resistant to the effects of endotoxin. We will determine whether these two bacterial products are responsible for IL-10 production in vivo and if blockade of the IL-10 improves local host defense.