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Show SCHOOL OF MEDICINE AND HEALTH SCIENCES UNDERGRADUATE RESEARCH ABSTRACTS Emily Callaway Mark Fisher 114 IDENTIFICATION AND CHARACTERIZATION OF Y. PESTIS GENES REQUIRED FOR SURVIVAL IN THE FLEA VECTOR Emily Callaway (Mark Fisher) Department of Pathology University of Utah Yersinia pestis is the bacterial agent of the plague, which is a rapidly progressive and often fatal disease. This organism has the ability to survive in both mammalian hosts and insects, namely the flea, which it exploits for transmission between mammalian hosts. However, little is known about what genetic traits allow Y. pestis to infect and use fleas as a vector for transmission. Understanding more about the transmission factors associated with Y. pestis m a y lead to the development of a broadly effective vaccine as well as more therapeutic options to combat plague. To date, only three Y. pestis gene loci have been implicated in survival in the flea: ymt (Yersinia murine toxin), h m s (hemin storage system), and g m h A which encodes an enzyme responsible for full-length lipo-polysaccharide production. Mutations in these loci have not demonstrated an absolute survival defect, indicating there are other genes involved in allowing Y. pestis to survive in the flea. As a first step toward understanding and identifying additional genes necessary for survival of Y. pestis in fleas, signature-tagged mutagenesis (STM) was utilized to randomly generate Y. pestis mutants, which were introduced into flea hosts. Seven mutants were identified with decreased survival in the flea vector, and transposon insertion sites were mapped by D N A sequencing. Initial data suggests that some of these mutants might be defective in cell wall or membrane integrity. Consistent with this hypothesis, several mutants displayed increased sensitivity to a cationic antimicrobial peptide (CAMP). In the current project, we engineered mutagenesis constructs (utilizing a X-red recombineering strategy) that were designed to yield in-frame deletions in selected genes identified by STM. In addition, constructs were engineered to complement the deleted genes in trans. Using these strains, w e are exploring alternative mechanisms of sensitivity to C A M P s and other stresses that Y. pestis could encounter in the flea. Through this work, w e expect to uncover novel Y. pestis transmission factors as well as mechanisms the bacteria use to respond to the environmental stresses encountered inside the flea vector. |