Function Study Of Salmonella Paratyphi A Operon:norRVW

Salmonella Paratyphi A(S.Paratyphi A),a gram-negative bacterium,distributes in nature widely which is a great dangerous pathogenic bacterium in intestinal tract..Paratyphi A can cause symptoms of paratyphoid such as bellyache,fever,splenomegaly and so on.Severe cases can lead to death.Innate immunity is the first defense when pathogenic bacteria enter human body.Phagocyte plays an important role in innate immunity that can kill a vast majority of invasive pathogens.But S.Paratyphi A is an intracellular pathogen which can survive in the phagocyte that will carry S.Paratyphi A to spread to other place in human body.Researching the mechanism is beneficial to targeted prevention and control for dissemination of S.Paratyphi A.NO is an important weapon in the phagocyte to kill swallowed bacteria.It has been reported that norRVW operon of Salmonella typhimurium or E.coli can produce functional molecules which can decompose NO inside the phagocyte and the relevant research about S.Paratyphi A has not been reported.Therefore,this study is a tentative exploration to norRVW operon and pathogenicity of S.Paratyphi A.In this paper,there are some homologous arms(R12,R34,V12,V34,W12,W34)amplified respectively with primers according to the sequences of norR?norV?norW inside norRVW operon of.Paratyphi A genome(GI:CP009049).Upstream and downstream homologous arms are fused by fusion PCR.After TA cloning and being identified correctly,the fused homologous arms digested by Bgl ? are recycled to connect with suicide plasmid vector pYG4 digested by the same restriction enzyme and then being transfered to S17-1?kpir by chemical conversion.Positive transformant can transfer recombinant plasmid into Rifampin resistance S.Paratyphi A named 50973(Rif+)by conjugative transfer and then taking a deletion mutation of corresponding gene so that constructing gene deletion mutant strain:50973(?norR),50973(?norV)and 50973(?norW).Meanwhile,norR gene including promoter and terminator are amplified to connect with pMD19T vector(simple)and then being transfered to 50973(Rif+)for constructing norR overexpression strain 50973(NorR).In order to research the influence of norR,norV and norW gene on pathogenicity of S.Paratyphi A.There are 75 female BALB/c mice randomly divided into a control group named 50973(Rif+)and four experimental groups named 50973(?norR),50973(?norV),50973(?norW)and 50973(NorR).Each group is randomly divided into three groups which include five mice respectively.There are three amounts of bacteria about 105,106 and 107 injected into corresponding mouse peritoneal respectively and survival condition of mice injected in 72 hours is recorded.Thereby calculating the LD50 of each strain:LD50/50973(Rif)+ =1.25 × 106,LD50/50973(?norV)11.99×106,LD50/50973(?norW)=1.99×106,LD50/50973(?norR)>3.16× 107,LD50/50973(NorR)=1.77 × 105.The results show that:Comparing with 50973(Rif+)group,the lethal force of 50973(?norV)and 50973(?norW)are slightly reduced,the lethal force of 50973(?norR)are significantly reduced,the lethal force of 50973(NorR)are significantly increased.The viability of different strains is comparing by testing the unit amount of bacteria in three spleens of mouse which are derived from the highest survival rate group respectively.Thereby calculating the bacteria survival amounts in the spleen after injecting 72 hours:50973(Rif+)=1.89×104±1.96×104,50973(?norV)-1.08×104 ±536.69,50973(?norW)=5.89×104±5.27×104,50973(?norR)=0×0,50973(NorR)=5.03×103±2.31×103?The results showed that:Comparing with 50973(Rif+)group,the viability of 50973(?norV)and 50973(NorR)are reduced,the viability of 50973(?norW)are increased,the viability of 50973(?norR)are zero.In order to research the relationship between norR,norV and norW gene,the total RNA of the five aforementioned strains in logarithmic phase are extracted to be tested by qPCR with internal reference 5SrRNA:testing the transcriptional level of norR in 50973(?norV)and 50973(?norW);testing the transcriptional level of norV in 50973(?norR)and 50973(NorR);testing the transcriptional level of norW in 50973(?norR)and 50973(NorR).The data from qPCR are analyzed by the method of 2-??Ct in relative quantification.The results show that:1)In S.Paratyphi A,the deletion of norV or norW will increase the transcriptional level of norR that suggest the reduction of norV or norW will feedback to bacterial regulating system thereby enhancing the transcription of norR.2)In S.Paratyphi A,the deletion of norR will reduce the transcriptional level of norV and norW,the increase of norR will also increase the transcriptional level of norV and norW.It suggests norR regulates the transcription of norV and norW positively.In summary,norR,norV and norW gene deletion mutant strains and norR gene overexpression strain are constructed in the study.The research of bacterial lethal force and viability show that norRVW operon is able to influence bacterial pathogenicity and norR play a critical role.The results of qRT-PCR suggest that norR regulates the transcription of norV and norW gene positively and the latter have negative feedback to the former.This preliminary research results will lay a foundation for further researching norRVW regulation and its resistance to host innate immunity.It also provides a new mentality to treat,prevente and control S.Paratyphi A.