The Molecular Regulatory Mechanism Of Plasmid PPCP1-encoded SRNA HmsA On Virulence In Yersinia Pestis

Small regulatory RNAs(s RNAs)are non-coding RNAs with the size of 50 to 500 nts.s RNAs commonly exist in prokaryotes and eukaryotes and in the post-transcriptional regulation they play key roles.s RNAs affect the stability of m RNAs or the activity of proteins in microorganisms by up-regulating or down-regulating target genes.According to the location of the s RNAs at the genome,small RNAs are subdivided into two categories,trans-encoded s RNAs and cis-encoded s RNAs.In bacteria the most common s RNAs are the trans-encoded s RNAs which requiring the help of the chaperone protein Hfq to promote or inhibit genes expression by incompletely base-pairing with target RNAs.Another one synthesized from the strand complementary to the m RNA they regulate.Hfq is a homo-hexameric protein in bacteria that is common in prokaryotes and occupies an important position in the complex network of gene transcription regulation.Hfq is generally binding to “AU”-rich regions in bacterial s RNAs.Hfq binding to s RNAs in the vicinity of the 5’ ribosome binding site and the translation initiation site of the target m RNA,inhibits translation of m RNA or directly leads to degradation of m RNA.s RNAs play an important regulatory role in the pathogenic process of bacteria,and its regulation of bacterial virulence is manifold.s RNAs can affect bacterial invasiveness,virulence and the ability to against host immunity by regulating the expression of bacterial virulence genes.Bacterial outer membrane proteins and LPS are the main component of the bacterial cytoderm.Out membrane proteins carry antigens which are the mediate interactions between bacteria and host.s RNAs affect bacterial adhension ability and invasiveness by regulating the synthesis of bacterial outer membrane proteins and modifying LPS.Otherwise,they also can affect the formation of bacterial biofilms and regulate the quorum sensing capacity of bacteria by secreting self-inducing molecules(AI).When bacteria adapt to the changing external environment,they form a complex and effective regulatory network,and s RNAs constitutes an important part in the pathogenic bacteria regulatory network.Yersinia pestis is the cautive pathogen of plague.It is a foci of bacteria that usuallyonly spread and cause plague among animals.Human beings infect plague through infected flea biting.The clinical symptoms of the plague is more obvious,swelling of the lymph nodes,sepsis and septicemia.The epidemic has occurred three pandemic in history,is the legal A-class infection disease in our country.Yersinia pestis,a more "young" pathogen,evolved from Yersinia pseudotuberculosis about 5021-7022,is an important biological warfare agent.Yersinia pestis contains many plasmids,among these the plasmids p CD1,p MT1 and p PCP1 are closely related to the virulence of Y.pestis.The p MT1 and p PCP1 which were the plasmids of Y.pestis obtained in the course of evolution and have an important role in virulence and respond to changes in the environment of Y.pestis.Here we focused on an s RNA Hms A,located on the plague p PCP1 plasmid.Therefore it is necessary to investigate the role of Hms A in virulence of Y.pestis.According to laboratory results,it was found that the small RNA Hms A is located on the base of chromosome 9435-9500 of p PCP1 plasmid of Y.pestis.In this study we found that s RNA Hms A has an inhibitory effect on the virulence of Y.pestis by competition index measured in vivo.In total,413 genes were showed differently expressed between hms A mutant strain and WT.among these,105 were up-regulated and 308 were down-regulated.According to the different functions of gene expression classified genes and screened for virulence-accociated genes(psa EF hms T and hms S).It has been confirmed that Hms A has some regulatory effect on these genes,but the direct target gene of small RNA Hms A which related to virulence has not been determined yet.In order to search for its direct target genes,we constructed s RNA Hms A overexpression strain and compared genome-wide differential genes expression by RNA-seq.We screened out 25 candidate target genes,to determine whether Hms A regulate the candidate target genes we used q RT-PCR and β-galactosidase(Lac Z)reporter gene fusion assays to screened direct target genes.The results displayed that Hms A positively regulated YPO3620,YPO362 and YPO3905 at transcriptional level but no significant changes at the post-transcriptional level.Therefore,the regulatory mechanism of Hms A on Y.pestis virulence needs further investigated.