Action Mechanism Of Virulence Regulation Factors Hfq And CRP In Yersinia Pestis

Background: Plague, a lethal disease of natural focus, is caused by Yersiniapestis. In the infection courses, bacteria had to adjusted themselves to various signalsfrom the environment and hosts, while maintaining their virulence and pathogenicity.The virulence regulation factors control up and down regulation of various genesthrough the network of virulence regulation, of which regulators Hfq and CRP are ofimportance. Hfq, a molecular chaperone of sRNAs, is a global post-transcriptionalregulator in bacteria which mediates interactions between many regulatory sRNAsand their mRNA targets. Loss of hfq attenuated the virulence of Yersinia pestis. ThecAMP receptor protein (CRP) plays a crucial role in the carbon catabolite repressioncourse. Also the CRP is required for full virulence of Yersinia pestis, and controls alarge regulon, including several virulence-associated genes.Methods: The-Red recombination system was adopted to construct the hfq andcrp mutants. As for the study of Hfq, colony morphology, crystal violet andnematodes assays were conducted to evaluate the effect of hfq on biofilm formation.The-galactosidase assay were adopted to validate the effect of Hfq on hms-genes.The relative mRNA levels of different strains were determined with the intensities ofprimer extension product. The hms-genes were expressed using the pet28a vector.Antibodies against recombinant His-tagged proteins were obtained from rabbits,which were used in subsequent western blot analysis. The intracellular levels ofc-di-GMP were determined by the HPLC-MS/MS method. As for the study of CRP,the LacZ fusion experiments and primer extension assay were adopted to verify theeffect of CRP on its own encoding gene and cyaA gene. EMSA and DNase Ifootprinting assay were used to obtain the binding sequence of CRP on the promoterregion of cyaA gene. The intracellular levels of cAMP were compared between WTand crp in different mediums. Results: As for Hfq regulation, when the plasmid pCD1was present, hfqpositively regulated biofilm formation; while with the plasmid missing, hfq had anegatively effect on biofilm formation. The-galactosidase assay indicated that hfqnegatively regulated the hmsP transcription, but did not change the transcriptionalactivity of other hms genes. The primer extension experiments showed that hfq had apositive effect on hmsHFRS，hmsT, while negatively regulated hmsP. Hfq did notregulate hmsD. Western blot assays confirmed that the regulation effects of hfq onhms genes were consistent with that of primer extension. As for CRP regulation, theLacZ fusion experiments revealed that the cyaA promoter activity was significantlyenhanced in crp relative to WT, but the crp promoter activity gave no changebetween WT and crp. The results of primer extension assay were consistent with thatof LacZ fusion. His-tagged CRP protein was able to bind to the cyaA fragment in adose-dependent manner in vitro, but the negative EMSA results were observed for thecrp fragment. CRP protected a single region from155bp to124bp upstream of cyaAagainst DNase I digestion in a dose-dependent manner.Conclusions: In our work, the mechanism of two virulence regulation factorsHfq and CRP were studied in their own regulatory pathways. As for Hfq regulation,the plasmid pCD1had an important effect on the regulation of biofilm by Hfq.Moreover, Hfq positively regulated hmsHFRS and hmsT, while negatively regulatedhmsP. HmsD did not involve in the pathway of Hfq regulation. As for CRP regulation,it bound to the promoter-proximal region of cyaA to repress the cyaA transcription,but it had no regulatory effect on its own gene. Repression of cAMP production byCRP through acting on the cAMP synthesase gene cyaA would represent a mechanismof negative auto-modulation of cellular CRP function in Y. pestis.