MECHANISM OF BACTERIAL METASTASIS IN PLAGUE

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pla mutants of Yersinia pestis, the causative agent of plague, are specifically defective in ability to cause disseminated infections. Unlike fully virulent strains which rapidly spread from subcutaneous injection sites, the mutants produce only a local subcutaneous lesion. Their LD5O by subcutaneous injection is a million-fold higher than that of the isogenic parent. Nonetheless, they remain highly infectious and grow at the injection site, indicating substantial resistance to antibacterial defenses of the pre-immune host. Thee also remain highly virulent when injected intravenously and reach high titers in both liver and spleen. When subcutaneous injection-sites are examined histologically, the major difference observed between mutant and wild-type lesions is that the latter contain many fewer polymorph neutrophils (PMN). These results imply that (1) even bacteria with high levels of resistance to innate host defenses require some specialized function to produce disseminated infections in an otherwise healthy host, (2) this function is provided by pla, and (3) the crucial activity of this function may be reduction of PMN accumulation at infectious foci, blocking formation of a micro-abscess of sufficient integrity to prevent escape of the bacteria. The major goal of this proposal is to understand how pla acts to produce systemic disease. This gene encodes a 32 kD outer membrane protease which can cleave complement C3 and properdin at specific sites, block C3 consumption in human serum, and also activate plasminogen. Thus, disruption of the alternative complement pathway, a major source of chemoattractant early in infection, may account for limited PMN influx to lesions caused by Pla+ bacteria. Although it does not readily explain reduced PMN immigration, plasminogen activation could also play a role in dissemination of the bacteria by disrupting tissue integrity, a function it is known to perform in the metastasis of mammalian tumors. The complement hypothesis will be tested both in viva, with complement deficient mice, and in vitro, by analysis of the effect of Pla on complement components. and complement system function. The plasminogen activation hypothesis will be tested by substitution of an alternative plasminogen actIvator for Pla in Y. pestis, and by inhibition of plasmin activity in vivo. These approaches will be enhanced by a more thorough histo-pathological comparison Pla+ and Pla- mutants and further characterization of Pla structure and function, focusing on definition of the proteolytic active site. Because homologues of Pla occur in other Gram-negative bacteria, (including E. coli,) and may be widespread, their potential to enhance the development of disseminated infections will be assessed by determining if the E. coli Pla homologues correlate with bacteremia among clinical isolates, and determining if the E. coli enzyme can substitute for Pla in Y. pestis. Disseminated Gram-negative infections cause in excess of 100,000 deaths in the U. S. each year. This work should contribute significantly to dissecting the molecular mechanisms contributing to their genesis.