Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.

SummaryThe MarR-type regulator RovA controls expression of virulence genes of Yersinia pseudotuberculosis in response to environmental signals. Using a genetic strategy to discover components that influence rovA expression, we identified new regulatory factors with homology to components of the carbon storage regulator system (Csr). We showed that overexpression of a CsrB-or a CsrC-type RNA activates rovA, whereas a CsrA-like protein represses RovA synthesis. We further demonstrate that influence of the Csr system on rovA is indirect and occurs through control of the LysR regulator RovM, which inhibits rovA transcription. The CsrA protein had also a major influence on the motility of Yersinia, which was independent of RovM. The CsrB and CsrC RNAs are differentially expressed in Yersinia. CsrC is highly induced in complex but not in minimal media, indicating that medium-dependent rovM expression is mediated through CsrC. CsrB synthesis is generally very low. However, overexpression of the response regulator UvrY was found to activate CsrB production, which in turn represses CsrC synthesis independent of the growth medium. In summary, the post-transcriptional Csr-type components were shown to be key regulators in the co-ordinated environmental control of physiological processes and virulence factors, which are crucial for the initiation of Yersinia infections.

Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathogen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the virulence regulator RovA. Switching of the Csr RNA involves Crp-mediated repression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transcriptome, and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favored when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successful Y. pseudotuberculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize biological fitness and infection efficiency through the infectious life cycle.

This review emphasizes the function and regulation of the Csr regulatory system in the human enteropathogen Yersinia pseudotuberculosis and compares its features with the homologous Csr/Rsm systems of related pathogens. The Csr/Rsm systems of eubacteria form a complex regulatory network in which redundant non-translated Csr/Rsm-RNAs bind the RNA-binding protein CsrA/RsmA, thereby preventing its interaction with mRNA targets. The Csr system is controlled by the BarA/UvrY-type of two-component sensor-regulator systems. Apart from that, common or pathogen-specific regulators control the abundance of the Csr components. The coordinate control of virulence factors and infection-linked physiological traits by the Csr/Rsm systems helps the pathogens to adapt individually to rapidly changing conditions to which they are exposed during the different stages of an infection. As Csr/Rsm function is relevant for full virulence, it represents a target suitable for antimicrobial drug development.