Small non-coding RNAs (sRNAs) post-transcriptionally regulate a myriad of cellular processes in prokaryotes. However, there is relatively scant knowledge of sRNAs that regulate bacterial virulence, despite indications that this occurs in several pathogens. The principal goal of this application is to identify and characterize sRNAs that control the virulence of Enterohemorrhagic Escherichia coli (EHEC), an emerging group of food-borne non-invasive intestinal pathogens that cause illnesses ranging from non-bloody diarrhea to the potentially fatal hemolytic uremic syndrome (HUS). In order to address this goal, I propose the following three aims: 1) Examine the role of sRNAs in the regulation of known EHEC virulence factors. Strong preliminary evidence indicates that a sRNA may regulate the mRNA coding for Tir, one of the crucial proteins in the colonization abilities of EHEC. After confirming the preliminary data I will carry out a genetic screen to identify the sRNA of interest. 2) Identify EHEC sRNAs likely to play a role in virulence regulation. Both bioinformatic and 'deep-sequencing'approaches will be used to conduct a comprehensive screen for EHEC sRNAs. 3) Characterize the role in virulence, the target(s), and the mechanisms of action of selected EHEC sRNAs identified in Aims 1 and 2. I will create in-frame deletions in the genes encoding the sRNAs of interest and examine those mutants for virulence-related phenotypes both in vitro and in vivo. These mutants will also be used in microarray analyses to identify the targets regulated by these sRNAs. I will then determine the mechanisms by which sRNAs govern virulence gene expression. This approach appears to be sound as I have already confirmed several predicted sRNAs which may be involved in virulence.
This work will yield fundamental new knowledge of the small transcriptome in an important pathogen as well as provide new insights into the processes that govern bacterial virulence. In addition, these studies will contribute to the growing body of knowledge of the mechanisms of small RNA regulation, an area of great interest in both prokaryotic and eukaryotic fields. Finally, a greater understanding of the regulation of EHEC virulence may lead to better understanding of treatment options and to the development of novel therapeutics for this important human pathogen.
| Shakhnovich, Elizabeth A; Davis, Brigid M; Waldor, Matthew K (2009) Hfq negatively regulates type III secretion in EHEC and several other pathogens. Mol Microbiol 74:347-63 |
