Yersinia pestis, the Gram-negative bacterial agent of plague, remains an international public health concern, and recent outbreaks in human populations in India and Africa have led to its classification as a reemerging disease. Two new concerns add to the threat to human health posed by present and future plague outbreaks. The emergence of multiply-antibiotic resistant strains of Y. pestis makes possible pneumonic plague epidemics that could not be controlled by standard prophylactic treatment . The potential illegitimate release of Y. pestis by bioterrorists also increases the urgency for a more detailed understanding of the host-pathogen relationship at the molecular level that may lead to the design of improved medical countermeasures and diagnostics. Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission. The molecular and genetic mechanisms that enable Y. pestis, uniquely among the Enterobacteriaceae, to utilize a blood-sucking insect for transmission have not been investigated. We are studying the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild type or specific Y. pestis mutants. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce blockage of the flea foregut that is required for biological transmission. We have termed these genes transmission factors (analogous to virulence factors, the genes required for pathogenesis in the mammal). The strategy entails first identifying bacterial genes that are differentially expressed at a temperature typical of the cold- blooded flea host (20C) as opposed to the temperature of the warm-blooded mammalian host (37C). Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle, and may be applicable to other arthropod-borne agents. The work also provides a model that can be applied to other arthropod-borne bacterial pathogens.