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Role of DksA and Hfq in Shigella flexneri virulence
| dc.contributor.advisor | Payne, Shelley M. | en |
| dc.creator | Sharma, Ashima Krishankumar | en |
| dc.date.accessioned | 2008-08-28T23:23:05Z | en |
| dc.date.available | 2008-08-28T23:23:05Z | en |
| dc.date.issued | 2007 | en |
| dc.description | text | en |
| dc.description.abstract | Hfq is a post-transcriptional regulator playing an important role in virulence and cellular physiology by regulating the expression of several genes either directly or indirectly through interaction with small regulatory RNAs (sRNA). Hfq is highly abundant and its synthesis in E. coli is subject to auto-repression at the level of translation. My studies with Shigella flexneri showed that hfq gene expression is regulated at the transcriptional level by a pleiotropic regulatory protein, DksA. I compared the gene expression profiles of wild type and dksA mutant S. flexneri by microarray and real time PCR analyses and determined that hfq expression was reduced in the dksA mutant. Significantly reduced Hfq levels in the dksA mutant were restored to wild type levels in the dksA mutant complemented with wild type dksA. Characterization of an hfq mutant in S. flexneri showed several phenotypes in common with the dksA mutant including reduced ability to survive in stress conditions and formation of elongated cells within cultured epithelial cells. Because DksA is required by S. flexneri to form plaques in cultured epithelial cell monolayers, a measure of virulence, the role of Hfq in the dksA virulence phenotype was assessed. Inducing expression of hfq in the dksA mutant restored plaque formation, and an S. flexneri hfq mutant failed to form wild type plaques. These data suggest that DksA plays a role in regulating hfq gene expression and that this regulation is important for S. flexneri virulence. In an in vitro transcription assay, addition of purified DksA increased transcription of hfq and this effect was greater with one of the two known hfq promoters. Addition of ppGpp, a stringent response molecule, along with DksA in the in vitro transcription assay resulted in a further increase in transcription of hfq, indicating that DksA is required for maximal transcription of hfq during both exponential and stringent response growth conditions. Real time PCR analysis showed reduced mRNA levels of the three major transcriptional activators of S. flexneri virulence genes, VirF, VirB and MxiE in the hfq mutant providing an explanation for its reduced ability to invade and form plaques in cultured monolayers. | |
| dc.description.department | Microbiology | en |
| dc.format.medium | electronic | en |
| dc.identifier | b68664333 | en |
| dc.identifier.oclc | 166353823 | en |
| dc.identifier.uri | http://hdl.handle.net/2152/3029 | en |
| dc.language.iso | eng | en |
| dc.rights | Copyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works. | en |
| dc.subject.lcsh | RNA-protein interactions | en |
| dc.subject.lcsh | Genetic transcription--Regulation | en |
| dc.subject.lcsh | Shigella flexneri | en |
| dc.subject.lcsh | Virulence (Microbiology) | en |
| dc.subject.lcsh | Proteins | en |
| dc.subject.lcsh | Non-coding RNA | en |
| dc.subject.lcsh | Messenger RNA | en |
| dc.title | Role of DksA and Hfq in Shigella flexneri virulence | en |
| dc.type.genre | Thesis | en |
| thesis.degree.department | Microbiology | en |
| thesis.degree.discipline | Microbiology | en |
| thesis.degree.grantor | The University of Texas at Austin | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |