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Shigellosis, a several diarrheal disease caused by infection with bacteria of the genus Shigella, remains endemic throughout the world. The global burden of shigellosis is due in part to the lack of a vaccine to prevent the infection and the lackof a universally safe and available antibiotic regimen to treat the infection. While the potential f using small RNA molecules (sRNAs) as models for, or targets of, RNA-based anti-Shigella therapeutics is promising, what is lacking is a comprehensive understanding of the role that sRNAs play in controlling Shigella physiology and pathogenesis. A necessary first step towards achieving the long-term goal of developing RNA-based antibiotics to treat shigellosis is to reveal the full extent to which sRNAs control the physiology and virulence of Shigella species. To this end, the overall objective of this study is to elucidate the role of newly identified duplicate sRNAs RyfA1 and RyfA2 in controlling the physiology and pathogenesis of S. dysenteriae. <p/>The central hypothesis being tested is that RyfA1 and RyfA2 are differentially produced under unique environmental conditions and modulate S. dysenteriae virulence by regulating the expression of distinct over-lapping sets of genes. This hypothesis will be tested by achieving the following specific aims: 1) identify the iron- and temperature-responsive factors regulating the production of RyfA1 and RyfA2; 2) elucidate the function of RyfA1 and RyfA2 by identifying the regulatory targets of each; and 3) determine the effect of RyfA1 and RyfA2, individually and in combination, on S. dysenteriae virulence.<p/> The proposed systematic characterization of duplicate sRNAs, RyfA1 and RyfA2, will contribute to answering fundamental questions in the fields of Shigella pathogenesis and bacterial sRNAs, contributions that will facilitate achievement of the long-term goal of developing therapeutics to treat shigellosis. The proposed study puts forth the innovative hypothesis that two sRNA molecules that share 95% sequence identity and a identical predicted structure have non-redundant functions. This hypothesis will be tested using a balanced approach of standard, student friendly, bacterial genetics and innovative high-tech assays. Student involvement in every step of the proposed study form the foundation of an innovative training program that will provide students with practical experience in the fields of bacterial pathogenesis and RNA-based regulation. <p/>The resulting combination of skills and experience will position students well to contribute to the rapidly advancing fields of ribo-regulation in pathogenic bacteria, commensal bacteria and eukaryotic systems.
PUBLIC HEALTH RELEVANCE: The proposed research will investigate how two newly discovered regulatory molecules influence the survival and physiology of Shigella dysenteriae, a bacterium that causes severe diarrheal disease in humans. Targeted disruption or inactivation of regulatory molecules that control the ability of the bacterium to survive within the human body would limit the ability of the bacteria to cause human disease.