is the causative agent of cholera, an acute dehydrating diarrhea that is epidemic in many developing countries. Extensive in vitro studies have demonstrated that the ability of V. cholerae to colonize and cause disease requires expression of virulence factors during its infection of hosts. However, how V. cholerae controls its gene expression in response to the changes in environmental signals is largely unknown. Therefore we intend to address these questions: how does V. cholerae alter its gene expression to colonize the human intestine from its inter-epidemic reservoir? What genes must be silenced to facilitate bacterial survival in the gut? In what sequence does V. cholerae initiate these genetic controls? Three specific aims are outlined in this proposal to investigate these situations using an animal model. First, we have developed a novel screening method and have used it to identify a set of genes that are expressed m vitro but repressed during V. cholerae early colonization of infant mice. Among them, repression of a type IV pilus synthesis genes is crucial for colonization. We will study why and how this type of genetic regulation is important for V. cholerae pathogenesis. This will allow for a broader sense of how pathogens overcome host defenses to initiate infection. Second, we have developed a genetic tool allowing us to monitor both gene activation and repression at the single-cell level. Using this novel approach, we will shed light on currently little known temporal changes in gene transcription during the V. cholerae infectious process. The regulation of these changes over time may be important for V. cholerae pathogenesis. Thirdly, we will investigate the role quorum sensing plays in the repression of genes late in V. cholerae infection. This regulation might be of key importance because of its ability to coordinate genetic expression in a broad population of cells, thus orchestrating a shift in the infectious process as a whole. This proposal represents a new focus away from research based solely on genetic activation events, and so will provide a more complete picture of V. cholerae pathogenesis. By understanding the complete genetic properties of the infectious process, we can illuminate the underlying factors that enable V. cholerae to survive and cause disease within the human intestines. The ultimate goal of this research is to gain knowledge that will potentially lead to novel treatments of such diseases. ? ? ?
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