Regulation and function of the global virulence regulator RovA of Yersinia pseudo-tuberculosis, a transcriptional activator of the SlyA/Hor family
Final Report Abstract
Initiation of a Yersinia pseudotuberculosis infection by colonization and subsequent penetration of the intestinal epithelial layer requires the expression of a special set of early-stage virulence genes, such as the internalization factor invasin and the PsaA pili. These virulence genes are activated by the regulator RovA, which itself is under the control of several environmental parameters, such as temperature, growth phase, ions and nutrient content of the growth medium. In our attempt to analyze the temperature dependent control mechanism of RovA, we found that RovA is a protein thermometer. We could show that thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator protein RovA which reduces its DNA-binding functions. In particular cooperative binding of RovA to its own promoters is significantly reduced at 37°C showing that temperature control of rovA transcription is primarily based on the positive autoregulatory loop. Thermally induced reduction of DNA-binding is further accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control, and studies with modified or chimeric RovA proteins indicated that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Taken together our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation. Using a genetic approach to identify additional regulatory components influencing rovA expression, we identified a new LysR-type regulatory protein, designated RovM, components of the carbon starvation regulatory (Car) system including the RNA binding protein CsrA plus two regulatory RNAs CsrB and CsrC, as well as the histone-like nucleoid-associated protein YmoA. RovM exhibits homology to the virulence regulator PecT/HexA of plant pathogenic Erwinia species and interacts specifically with a short binding site between promoters PI and P2 within the rovA regulatory region. The rovMqene itself is under positive autoregulatory control and is significantly induced during growth in minimal media. Disruption of the rovM gene leads to a significant increase of RovA and Invasin synthesis, enhances internalization of Y. pseudotuberculosis into host cells and increases virulence with significantly higher numbers of bacteria in the gut-associated lymphatic tissues and organs in the mouse infection model. In contrast, elevated levels of the RovM protein, which exert a positive effect on flagellar motility, severely attenuate the ability of Y. pseudotuberculosis to disseminate to deeper tissues. We further demonstrate that the Csr system affects rovA through control of RovM. CsrA activates RovM synthesis but is sequestered and blocked by the CsrC RNA under rovA inducing conditions. The CsrC RNA is highly induced in complex but not in minimal media, indicating that medium-dependent rovM expression is mediated through CsrC. Furthermore, we could demonstrate that YmoA in association with another histone-like protein H-NS and the RNA chaperone Hfq activate CsrC synthesis. In contrast to synthesis of the CsrB RNA is generally very low under all tested conditions, but can be induced upon overexpression ofthe response regulator UvrY. A genetic screen and microarray analysis to identify RovA-dependent genes have shown that several early stage virulence genes of Yersinia, including invasin, the pili PsaA, and additional adhesins are coregulated with numerous stress adaptation proteins, metabolic enzymes and transport proteins. This demonstrated that RovA has a major impact on Yersinia fitness and virulence and is important for successful adaptation to environmental changes during an infection.
Publications
- (2006) RovM, a novel LysR-type regulator of the virulence activator gene rovA, controls cell Invasion, virulence and motility of Yersinia pseudotuberculosis. Mol. Microbiol. 62:1469-83
Heroven A.K. and P. Dersch
- (2007). Regulatory Elements Implicated In the Environmental Control of Invasin Expression in Enteropathogenic Yersinia. In: The Genus Yersinia. Edt: Springer Adv. Exp. Med. Blol. 603:156-166
Heroven AK., Böhme K., Tran-Wlnkler H. and Dersch P.
- (2008) A Csr-type regulatory system, including small non-coding RNAs, regulates the global virulence regulator RovA of Yersinia pseudotuberculosis through RovM. Mol. Microbiol. 68:1179-1195
Heroven A.K., Böhme K., Rohde M. and Dersch P.
- (2009) Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA. PLoS Pathogens 5(5):e1000435
Herbst K., Bujara M., Heroven AK., Opitz W., Weichert M., Zimmermann A. and Dersch P.
- (2010) Global virulence gene regulation networks in enteropathogenic Yersinlae. In: Mohan RM (Ed.): Research Advances in Molecular Microbiology. Kerala, Indien: Global Research Network, p.1-25
Heroven A.K. and Dersch P.
