Biological Functional Of Transcription Regulator Rex In Streptococcus Suis Serotype 2

Streptococcus suis(S.suis)is considered an important zoonotic agent responsible for a wide variety of diseases in pigs and human,including meningitis,septicemia,arthritis and endocarditis.S.suis serotype 2(SS2)is one of the most virulent,hazardous and frequently isolated serotype among 33 serotypes,causing important economic losses to porcine industry and endangering public health security.Adaptation to any unfavorable changes of environment and survival are the primary steps for the pathogenesis of pathogen.Regulatory mechanisms to survive various kinds of environmental insults is very important.Rex is an important global regulator in most gram-positive bacteria,and contributesto survival of bacteria.In this study,biological function of gene Rex was analyzed by multiple experiments.1.Prokaryotic expression of Rex transcription regulator in streptococcus suis and in vitro binding assayTo clone and prokaryoticly express the gene encoding Redox regulator(Rex)of Streptococcus suis serotype 2 and to analyze biological information and in vitro binding activity.The encoding Rex gene of SS2-1 strain was amplified by PCR with the designed primers,and then cloned into prokaryotic expression plasmid pET28a.The recombinant plasmid pET28a-Rex was transformed into E.coli BL21.After induced expression by IPTG,the Rex protein was obtained.The binding activity of Rex protein and DNA was analyzed by gel mobility shift assay(EMSA)in vitro.Purification of recombinant protein Rex was successfully expressed.The presence of NAD+ did not have major effect on mobility shift,but addition of NADH almost abolished such a binding activity.By in vitro binding assay,Rex was found to regulate the expression of Prex in response to NADH/NAD+ equilibrium.2.Construction of the Rex deleted mutant in streptococcus suisTo investigate the role of the Redox regulator(Rex)in the pathogenicity of Streptococcus suis type 2(SS2)virulent strain SS2-1,an isogenic Rex mutant strain(?Rex)and a complemented strain(C?Rex)were constructed.The recombinant gene knock-out vector was constructed consisting chloramphenicol resistant(CatR)cassette with flanking homology regions of Rex by using Streptococcus suis-Escherichia coli shuttle vector pSET4s.The plasmid pSET4s::Rex was transformed into wild-type strain SS2-1.The resulting mutant strains were screened by chloramphenicol resistant plates and further confirmed by PCR analysis and DNA sequencing.3.Characterization of the Rex mutant in Streptococcus suis serotype 2To futher determine the function of Rexgene in S.suisSS2-1,the charateristics of wild type strain,Rex gene deletion strain were in vivo and in vitro.The ?Rex mutant and CARex showed no significant differences in growth characteristics and adherence to HEp-2 cells compared with the wild-type strain.However,the Rex mutant showed increased susceptibility to H2O2 and inextreme acidity environment,suggesting that Rex is involved in counteracting environmental stress.Using a murine infection model,we demonstrated that the abilities of the mutant strains to survive the tissues were significantly reduced compared to that of the wild-type strain.The mutant strains also showed a decreased level of survival in swine blood.To further evaluate the in vivo relevance of Rex,we studied the intracellular bacterial survival of the Rex mutant strain in macrophages of culture infection model.The isogenic Rex mutant displayed reduced survival in macrophages compared to the wild type.The iTRAQ analysis identified 96 differentially expressed(39 up-and 57 down-regulated)proteins in ARex and SS2-1.Dpr,was down expressed in Rex mutant,which accorded with the phenomenon that mutant strain was more susceptible to oxidative stress.In conclusion,Rex contributes to the intracellular and environmental stress survival of S.suis.