Regulatory Mechanism Of The Extracytoplasmic ? Factor System BcrS And Its Role In Brucella Virulence

Brucella is an important zoonotic pathogen,which infects livestock and leads to miscarriage and sterility.It can also infects humans and causes diseases such as undulant fever,arthritis and endocarditis.Brucellosis is prevalent all over the world,causing serious harm to the development of animal husbandry and causing serious economic losses.Brucella is a typical intracellular parasite bacterium.In order to survive and replicate successfully in the cell,it has evolved many mechanisms to inhibit the host immune response to establish long-term persistent infection.One of which is expressed via Type IV secretion system(T4SS)to suppress the host immune response,which plays a key role in the pathogenesis of brucella.The other is the ability to resist intracellular cytotoxic oxidative killing.Antioxidant damage system is a defense system used by bacteria to protect themselves from the killing of endogenous and exogenous oxides.The Extracytoplasmic function Sigma factor(ECF)system is the third largest signal transduction system of bacteria,along with the single-component and two-component systems.It is commonly used to sense and respond to extracellular environmental signals and plays an important role in the process of environmental adaptation.The establishment of infection by pathogenic bacteria requires the bacteria to sense the surrounding environment and express virulence genes.However,the mechanism of how Brucella senses the environmental signals and coexpresses virulence factors is still unclear.In order to identify whether Brucella M28 B0022/B0023 genes constitute an ECF system,we found that B0022 contains two conserved domains,?2 and ?4,encoding extracytoplasmic ? factors by3 D structural and protein conserved domain prediction.Through transmembrane protein and homologous sequence alignment,it was found that B0023 contained six transmembrane helices,and contained conserved cysteine residues at positions 129 and 179 of amino acid sequence,encoding anti-?factor.Bacterial Adenylate cyclase-based two-hybrid(Batch)system was used to prove that B0022/B0023 can bind to each other at the protein level and identify them to form an ECF system.It was named bcrS/abcS((?)rucella R(?)S Resistance (?)igma Factor,bcrS;(?)nti-(?)rucella R(?)S Resistance Sigma Factor,abcS),B0019-B0021 was named bcrXQP.The gene bcr XQP encoding methionine-enriched polypeptide and methionine sulfoxide reductase system upstream of the ECF system is believed to be related to antioxidant function,and here it may act as a target gene regulated by the ECF system.In order to further study the regulation mechanism of ECF system,the reverse transcription polymerase chain reaction(RT-PCR)was used to prove that the gene bcrS/abcS formed a transcription unit,and the three upstream genes bcr XQP also formed a transcription unit for joint transcription.The transcription start sites of two operons,bcrS/abcS and bcr XQP,were identified by 5 'RACE method,and the regions of two operon promotors were deduced.Then,the total RNA of brucella was extracted with different oxides,and the gene expression was detected by fluorescence quantitative polymeric chain reaction(q RT-PCR).The results showed that the expression of ECF system gene bcrS/abcS was significantly induced by Reactive chlorine species(RCS)(p<0.01).The promoter of operator bcrS-abcS and lac Z reporter gene were constructed into plasmid vector and transferred into brucella.Brucella carrying reporter plasmid was treated under different RCS concentration conditions.The expression of bcrS gene was analyzed by fusion expression of lac Z reporter gene.The results showed that the expression of bcrS gene was affected by the concentration of RCS.In addition,brucella M28 mutants ?bcrS,?abcS and ?bcr XQP and complementary strains?bcrS+p BcrS,?abcS+p AbcS were successfully constructed by homologous recombination.The changes of differential expression of genes were detected by q RT-PCR.The results showed that the deletion of bcrS gene significantly down-regulated the expression of bcr XQP(P<0.01),and the absence of abcS significantly up-regulated the expression of bcr XQP(P<0.01).In addition,transcriptional fusion of lac Z reporter genes demonstrated significant autoregulation of bcrS(p< 0.01).To further identify other target genes regulated by the ECF system,transcriptome sequencing was performed on wild and mutant strains(?bcrS,?abcS)after HOCl treatment to obtain target genes regulated by the ECF system,including bcr X,bcr Q,bcr P,vir B1-vir B10,etc.In transcription,the primary function of ? is to recognize the promoter sequence and initiate the transcription of genes.Multiple potential bcrS identification sequences were obtained by genome-wide identification sequence search,suggesting that ECF system may directly regulate these genes.The results of transcriptome sequencing were verified by q RT-PCR.Moreover,it was further explained that AbcS not only regulates T4 SS through BcrS,but also independently regulates the expression of T4 SS.In order to study the function of ECF system,in vitro antioxidant survival experiments were carried out on wild strains and mutant strains,and the survival number of bacteria was counted.The results showed that the sensitivity of ?bcrS and ?bcr XQP to HOCl was significantly higher than that of wild strains(p<0.05),indicating that mutations in the ECF system gene affect the antioxidant killing ability of Brucella.In order to study the effect of ECF system on the intracellular and in vivo replication of Brucella M28,cell and animal models were used to infect Brucella,and then spleen weight and splenic bacterial load were counted.The results showed that the deletion of both bcrS and abcS did not affect the survival and replication of Brucella in murine passaged macrophages.In the mouse infection model,the spleen weight of the mutant ?abcS inoculated on the 3 week of infection was significantly lower than that of the wild strain M28(P< 0.01),and the bacterial load also decreased significantly(P<0.05).These results indicated that abcS were very important for maintaining long-term chronic infection of Brucella,while bcrS had no significant effect on the virulence of Brucella.This study lays a foundation for exploring the antioxidant damage system and pathogenic mechanism of Brucella,and provides insights into for the development of therapeutics methods for Brucella infection.