Molecular Characterization Of The Transcriptional Regulation Of Psa Locus By PhoP And RovA In Yersinia Pestis

Yersinia pestis is the causative agent of plague, an acute zoonotic infection fatal to humans. During the transmission cycles and pathogenesis processes, Y. pestis will encounter multiple environmental adverse simulation, such as antibiotic peptides, temperature changing, pH and osmotic pressures. Y. pestis can sense and response to these diverse environmental signals, then initialize self-regulation mechanisms secrete virulence factors and archive successful survive. One of the most important self-regulation mechanisms is that regulators response the environmental signals and regulate the transcription of their target genes. In Y. pestis, both PhoP and RovA are the virulence regulators, and pH6 antigen is an important virulence related factor, but the relationship between them is still unknown.PhoP is the regulator protein of two-component system PhoP-PhoQ. The PhoP-PhoQ system can response to the signals of low-magnesium, mild acid pH and antibacterial peptides, and then activate PhoP protein, the activated-PhoP can activate or repress the target genes'transcription. Our previous results in Y. pestis revealed that, PhoP is a globe regulator. For bacteria growing in low-magnesium culture media, PhoP regulate at least 706 targets at transcriptional level, including psa locus, phoP-phoQ system and rovA.It has been established that RovA is a virulent regulator, as the virulence of Y. enterocolitica rovA null mutant strain to mice declined significantly compared to wild strain. RovA can regulate the expression of target genes through binding to their promoter regions in dimeride form. In Y. enterocolitica and Y. pseudotuberculosis, RovA not only stimulate the transcription of inv, but also other virulence related genes and itself as well. Our study based on microarray transcriptional analysis indicated that, dozens of genes(including the gene cluster of pH6 antigen, psa locus) were regulated by RovA at transcriptional level. Although RovA is proven a global regulator, there is no data about its regulate mechanism in Y. pestis by now.The pH6 antigen is an important virulence related factor in Y. pestis, and it is closely related to bubonic plauge. pH6 antigen in Y. pestis is encoded by psaEFABC operon. Cluster psaABC encode subunit protein PsaA, putative chaperone PsaB and membrane protein PsaC respectively. Meanwhile PsaEF may get involved in the transcriptional regulation of psaA. Expresson of pH6 antigen is affected by both temprature and pH: it won't get expression at the temperature lower than 34℃and will have the highest expression at pH6.0.According to microarray and bioinformatics analysis, the transcription of psaA, psaE, phoPQ operon and rovA can be regulated by PhoP, meanwhile rovA and psaA, psaE may be regulated by RovA. The aim of this research is to clarify the regulation mechanism of PhoP and RovA to their possible target genes.We firstly obtained Y. pestis phoP and rovA null mutant, by using Red-based one-step gene inactivite system. Then we employed the E. coli BL21λDE3 to express recombinant PhoP and RovA proteins. Then we applied the classical melocular experiments, such as Electrophoretic mobility shift assays (EMSA), DNaseⅠfootprinting, lacZ fusion assay and primer extension, to decipher the regulate machanism of PhoP or RovA to it's own targets.We found out that, the transcription of psaA, psaE and rovA were all inhibited by PhoP under low-magnesium and medial log-phage conditions; meanwhile it's own operon (YPO1635 and phoP) were both activated. For pH5.8 and mid-log phase conditions, the transcription of psaA was repressed by PhoP, but other targets were no affected. For mid-log phase conditions, RovA activates the transcription of psaA, psaE and rovA. We also found out that the transcription of psaA was regulated by acid as the expression of pH6 antigen genes was higher in pH5.8 than in pH 7.2; while temperature had no influence on the expression of psaA.For rovA, we identified two transcription starts in it's promoter region, which were named P1 and P2 respectively. P1 was much more closer to the translation start"ATG"than P2. P1 was PhoP-repressed and RovA-activited, while P2 was only RovA-dependent. DNaseⅠfootprinting indicated that there are two sites (named site1 and site2) for RovA in rovA promoter region, and RovA had a much higher affinity to site1 than site 2, while site1 located upstream close to P2. We postulated that rovA would be activated from P2 when RovA binding to site 1, but inhibited when RovA binding to site 2, as site 2 was downstream of P1. This hyphothsis proposed RovA had double effects on the transcription of rovA.For psaE, the binding sites of PhoP and RovA were identied, and three transcriptional starts were also found. PhoP binding site in psaE almost covered all the three transcriptional starts, while RovA binding site located upstream of the PhoP's, farther to the transcription starts. According to in vivo experimentsresults, the transcription of psaE was inhibitated by PhoP, but activiated by RovA under special conditions.Binding sites of PhoP and RovA in the promoter region of psaA overlaps, and both sites were downstream of transcriptional start. Results from in vivo experiments showed that, in low-magnesium or mild acid pH culture media, RovA activiated psaA transcription, while PhoP inhibitated it, So we can induced that PhoP and RovA co-regulated the transcription of psaA through competing for the same binding site of psaA promoter.Under low-magnesium condition, the activiation of the expression of YPO1635 by PhoP has been demonstarted in other papers.In this study, we confirmed this phenomenon. We also found out that PhoP activated self-transcription. For the two transcriptional starts identified in phoP promoter, namely P1 and P2, but only P2 was PhoP-dependent in mid-log phase.In shortly, this paper applied molecular and biochemical test methods, firstly discovered the transcriptional regulation cycle of PhoP-RovA-psa, that RovA activated the transcription of psa locus and rovA, and under low-magnesium condition, PhoP inhibited the transcription of psa locus and rovA, but activated its'own operon, PhoP can sense and response to acid signal to inhibited the transcription of psaA; The transcription of psaA was also regulated by acid , one may reap more as the expression of pH6 antigen genes were higher in pH5.8 than in pH 7.2, however, the temperature had no influence on the expression of psaA.