Characterization Of Virulence-required Regulator Cyclic AMP Receptor Protein (CRP) In Yersinia Pestis

Plague is a deadly natural focus-based disease.There were three human plague pandemics in history.Moreover,the morbility of plague was exhibiting an increasing trend since 1990s,and it was therefore classified as re-emergence infectious diseases by WHO in 2000.In addition,Yersinia pestis is one of traditional biological warfare agents,so it is used as one of the preferred microorganisms by terrorists.The pandemic of plague in China is rigorous,and the foci of plague is about 15%of the total mainland area,mainly distributing in the Northwestern China.The causative agent of plague is Y.pestis,and the main host is rodent.Plague is transmited among different hosts and ultimately transmited to human by flea biting or other routes,leading bubonic plague,pneumonic plague and septicaemia plague.Y.pestis experiences various environmental changes such as temperature, osmolarity,ion concentration,PH,oxygen tension when it transmits from flea to host,in order to survive and maintain its virulence and pathogenicity,bacterial must adapt to these stresses,accompanying with transcriptional changes of genes expression,that is some certain regulators could respond to their certain environmental signals,mediating the expression of certain genes(including virulence-related genes) up-regulated and down-regulated.The regulators and the targeted virulence-related genes ultimately construct a complicated virulence regulatory networks,controlling the survival and pathogenicity of Y.pestis intermediary and reservoir.CRP is a bacterial regulator that controls more than 100 promoters,including those involved in catabolite repression.In the present study,a null deletion of the crp gene was constructed and its complementary stain(C-crp) was contructed accordingly for Y.pestis bv.microtus strain 201.The virulence-related phenotype assays carried both in vivo and in vitro demonstrated CRP could regulate the virulence-related genes of Y.pestis.Microarray expression analysis disclosed that at least 6%of Y.pestis genes were affected by this mutation.Bioinformatics methods based on the E.coli CRP consensus was used to predicted the possible targeted genes regulated directly by CRP,and then 38 genes was found.The precise mechanism of transcriptional regulation by CRP was validated furtherly.Firstly,LacZ reporter fusion analysis validated CRP could positively regulated pla and pst,but negatively regulated ypkA. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 36 genes or putative operons to be the direct targets of CRP,and thus they constitute the minimal CRP regulon in Y.pestis.Subsequent Primer extension and DNase I footprinting assays mapped transcriptional start sites,core promoter elements,and CRP binding sites within the DNA regions upstream of pla and pst,revealing positive and direct control of these two laterally acquired plasmid genes by CRP.The crp disruption affected both in vitro and in vivo growth of the mutant and led to a＞15,000-fold loss of virulence after subcutaneous infection but a＜40-fold increase in the 50%lethal dose by intravenous inoculation.Therefore,CRP is required for the virulence of Y.pestis and,particularly,is more important for infection by subcutaneous inoculation.It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute,at least partially,to its attenuation of virulence by both routes of infection.The C-crp strain could complement the defect of virulence phenotype compared with WT in vivo and in vitro due to crp mutant. Consistent with a previous study of Y.pestis bv.medievalis,LacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity.The plasminogen activator encoded by pla was previously shown to specifically promote Y.pestis dissemination from peripheral infection routes (subcutaneous infection[flea bite]or inhalation).The above evidence supports the notion that in addition to the reduced in vivo growth phenotype,the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.In our study,gene expression profile differences between the WT and△crp strains grown in TMH-1mM were compared by using microarray expression analysis based on Y.pestis whole-genome cDNA,278 genes was screened as differentially expressed genes,among them,104 genes up-regulated and 174 genes down-regulated by comparing the expression of genes in△crp with that of WT.Presenting frequency of four bases at each site of CRP binding sequence in E.coli was obtained by consensus-matrix and convert-matrix program,then the sequence logo were shown by WebLogo software.Finally,CRP binding box in Yersinia pestis was confirmed to be TGTGA-N6-TCTCA.The differentially expressed 278 genes was scanned for the conserved motif with Matrix scan software,and 38 genes whose matching score was high(cutoff value is 8) was found as candidate genes regulated by CRP directly.Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 36 genes or putative operons among 38 genes/operons to be the direct target genes of CRP,and thus they constitute the minimal CRP regulon in Y.pestis(except the genes YPO2468 and yopD).LacZ reporter fusion analysis also validated that CRP could positively regulated two laterally acquired genes pla and pst,while regulated ypkA negatively.Subsequent Primer extension and DNase I footprinting assays mapped transcriptional start sites,core promoter elements,and CRP binding sites within the DNA regions upstream of pla and pst,the CRP sites on pla and pst both located on the upstream of-35 region,revealing positive and direct control of these two laterally acquired plasmid genes by CRP.The sycO,ypkA and yopJ genes constituted a single operon in Yersinia pestis. CRP specifically bound to the promoter region upstream of sycO and thus directly repressed the expression of the sycO-ypkA-yopJ operon.A single CRP-dependent promoter was employed for sycO-ypkA-yopJ,but two CRP binding sites(site 1 and site 2) were detected within the promoter region.A CRP box homologue was found in site 1 other than site 2,therefore only the site 1 was the CRP binding site and the site 2 was just a nonfunctioning one.The determination of CRP-binding sites,transcription start site,and core promoter element(-10 and -35 regions) promoted us to depict the structural organization of CRP-dependent promoter,giving a map of CRP-promoter DNA interaction for sycO-ypkA-yopJ.The CRP site was located on the downstream of -10 region and exerting repressive transcriptional regulation on sycO-ypkA-yopJ. Either the CRP protein or pla,pst and the sycO-ypkA-yopJ promoter region was extremely conserved in all the four Y.pestis biovars,namely Antiqua,Mediaevalis, Orientalis and Microtus.Therefore,data presented in biovar Microtus here can be generally applied to other Y.pestis biovars.Our data was not only helping us to define the minimal CRP regulon in Yersinia pestis bv.Microtus and predict the possible CRP regulon,but also demonstrate the subtile regulation mechanism of CRP on the virulence-related genes such as pla,pst and operon sycO-ypkA-yopJ,combined with the in vivo and in vitro virulence-related phenotype for the first time.The regulation of CRP on virulence might provide us new target for developing vaccine and medicine for Y.pestis,and it is necessarily helpful for the prevention and cure of plague.