High-Throughput Identification of Regulators of the Locus of Enterocyte Effacement from Enterohemorrhagic E. coli




Pifer, Reed Allen

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Identification of pathways involved in the regulation of the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) of Enterohemorrhagic E. coli (EHEC) has been underway for more than twenty years, but significant knowledge gaps remain. We have created a generalizable, high-throughput method of identifying E. coli core genome components that facilitate T3S. Using this tool, we have generated a dataset of regulatory pathways that control the LEE under anaerobic conditions, a largely neglected but essential consideration for this enteric pathogen. As proof of principle as to the efficacy of this method, we have characterized two transcription factors, cutR and fadR, as previously unknown LEE regulators in EHEC and Citrobacter rodentium. We have determined that CutR functions as a transcriptional activator of the LEE in the presence of L-cysteine, in EHEC and C. rodentium. We have observed that CutR is capable of directly binding to the LEE1 regulatory region, suggesting a direct mechanism of action, while simultaneously controlling a network of LEE-governing genes. We have also determined that FadR functions as a transcriptional repressor of the LEE in EHEC and C. rodentium, directly binding to LEE1 of both organisms. Finally, we explore the broader dataset to begin assessing the potential of yet uncharacterized LEE regulators.

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