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The role of the RNA-binding protein Hfq in the model pathogen Salmonella Typhimurium
Hfq is a RNA-binding protein which exists in homohexamers in vivo. Based on its folding, containing the highly conserved Sm1 and Sm2 motifs, it belongs to the growing family of Sm and Sm-like (Lsm) proteins. It has been shown, that Hfq is a pleiotropic regulator in bacteria which is involved in a br...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2008
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| Zusammenfassung: | Hfq is a RNA-binding protein which exists in homohexamers in vivo. Based on its folding, containing the highly conserved Sm1 and Sm2 motifs, it belongs to the growing family of Sm and Sm-like (Lsm) proteins. It has been shown, that Hfq is a pleiotropic regulator in bacteria which is involved in a broad variety of functions.
The RNA chaperone Hfq is essential for the virulence of
Salmonella typhimurium
Even though hfq has turned out to have no severe influence on the growth or the viability of the pathogenic bacterium Salmonella Typhimurium under laboratory conditions, we could show that it is strongly involved in the regulation of pathogenicity. A Δhfq mutant leads to loss of effector protein expression and secretion and thereby to reduced invasion of non-phagocytic cells and to reduced ability of intracellular replication in macrophages. Based on these observations, loss of infectivity in a mouse-model of infection could be proven. Further studies revealed not only lack of secreted proteins in the Δhfq mutant, but showed severe changes in the overall protein pattern when compared to its isogenic wild type strain, with an overrepresentation of membrane and membrane-associated proteins. Concerning the virulence phenotype, we have been able to restore effector protein expression (even if not their secretion) by overexpression of one of the major transcription factors involved in expression of virulence genes encoded in Salmonella pathogenicity island 1 (SPI1), namely HilA. Additionally, we could show that alteration in mRNA stability is causing for example the increase of the major outer membrane protein, OmpD or the decrease in the flagellar protein, FliC.
Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq
Our analysis represents a demonstration for usage of high throughput pyrosequencing (HTPS) in bacteria to determine the large regulon of the pleiotropic regulator, Hfq. The combination of transcriptomics with co-immunoprecipitation (coIP) of direct binding partners of Hfq and subsequent cDNA library synthesis and its sequencing allowed the dissection of genes directly influenced by Hfq and downstream effects based on deregulation of transcription factors. By analysis of RNA co-immunoprecipitated with Hfq compared to control coIPs in Salmonella Typhimurium lysates we were able to determine specific enrichment factors for a large set of mRNAs as well as sRNAs. Comparison with the transcriptomic data showed that Hfq regulates multiple major transcription factors, like a transcription factor of SPI1, HilD, and the major transcription factor, FlhD2C2, regulating the large class of flagellar genes in Salmonella and other bacterial species. By overexpression of these transcription factors we could restore phenotypes of a Δhfq mutant, e.g. loss of effector protein expression and secretion and reduced expression of the class III flagellar gene, FliC. Concerning sRNA expression in Salmonella, we found 10 new sRNAs in this pathogen and were able to verify the expression of a large set of sRNAs that have been known to be conserved in the model organism, Escherichia coli. Aside noncoding RNAs also two mRNAs encoding for small open reading frames (ORFs) in E. coli could be detected in the coIP RNA sample from Salmonella Typhimurium. |
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| Umfang: | 217 Seiten |
| DOI: | 10.17192/z2008.0905 |