B-217. Development of In Vitro Assays to Identify Common Modes of Interaction of the Pathogens Yersinia pestis and Bacillus anthracis with Macrophages as a Model for In Vivo Pathogenesis

A. Jenkins, C. Cote, S. Welkos;
USAMRIID, Frederick, MD.

Bacillus anthracis (BA), the causative agent of anthrax, and Yersinia pestis (YP), the causative agent of plague are known bioterrorism agents. An understanding of the early events involved in BA and YP infection is critical in the development of novel therapeutics and vaccines. It is clear that macrophages play a key role in the early stages of BA and YP infections, but little is known about these early stages, especially at a molecular level. Candidate genes involved in early infection and host response to YP and BA have been identified using mutagenesis libraries and sequence homology searches. This approach allows for the identification of genes of interest that are involved both in BA and YP infections and host responses. Currently, two such proteins were identified by various methods. Sequence homology searches identified a putative adenylate cyclase in YP that shares sequence homology with BA edema factor. The putative adenylate cyclase from YP was cloned and overexpressed in E. coli and characterized in vitro. Exposure of Chinese hamster ovary (CHO) cells to crude cell lysate containing the putative adenylate cyclase resulted in a 10-fold increase in cAMP production in the cells as compared to control samples. A BA delta-Ames mutagenesis library prepared using the mini-Tn10 transposon was screened with an assay that identifies clones that were resistant to the germination inhibitory activity of anti-spore antibodies. This assay also has the potential to identify candidate genes that may be involved in spore opsonization and macrophage uptake. The screen identified a mutant, which was determined, by rescue cloning, to be a MarR-like transcriptional regulator; a similar regulator, RovA, was found in YP, where it is an important regulator of virulence genes. Studies to determine the roles of these two proteins in virulence and early pathogenesis are ongoing using deletion mutants in virulent strains of YP and BA