Study On FabT,A MarR Family Transcriptional Regulator,Regulating The Biosynthesis Of Capsular Polysaccharide And Teichoic Acids In Streptococcus Pneumoniae

Objective:Capsular polysaccharide and teichoic acid are important virulence factors and major components of the cell envelop on the surface of S.pn.They play a vital role in the survival,colonization and invasion when bacteria infect the host niche.However,the mechanism of transcriptional regulation on both two biosynthetic pathways is still obscure.We obtained a MarR family protein FabT?SPD₀379?that might bind to the promoter region of cps gene cluster in our previous study,screened out by DNA-pulldown using cps promoter as a probe.By further research we have found that it also interacts with the promoter region of the gene rafX?spd₁672?,which encodes a candidate ligase of teichoic acid,RafX.Thus,this study was aimed to determine the regulatory mechanism of FabT on capsular polysaccharide and teichoic acid biosynthesis,which may provide us a novel clue on the regulation of biosynthesis of the cell envelop,and to reveal the role of its regulation in the pathogenesis in S.pn.Methods:D39?fabT was constructed by LFH-PCR method.pJWV25,an ectopic integrative plasmid which contained a Zn2+-inducible promoter,and pEVP3,an insertion-duplication vector were employed to construct the complement strain of D39?fabT,Compll and Compl2,respectively.The alteration in content of capsular polysaccharide and teichoic acid in D39 WT,?fabT,Compll and Compl2 were confirmed by dot blot and ELISA.The phenotypic changes of D39 WT,?fabT,and Compll were observed by transmission electron microscopy.We used lysozyme to separate the cell wall fraction from the whole cell.The alteration in cell wall and protoplast content were compared by dot blot,ELISA,western blot and flow cytometry to further identify possible loci for the regulation of FabT to TAs and CPS.Electrophoretic Mobility Shift Assay?EMSA?was used to verify and confirm the interaction between FabT and the promoter of cps cluster and TAs synthesis-related gene promoter.A potential palindromic sequence which may bind to FabT specifically was predicted by MEME 4.12.0,and the binding was verified RT-PCR was used to detect changes in the transcriptional levels of downstream genes of the promoters that interact with FabT.Finally,the comprehensive effects of FabT on the virulence of S.pn were explored through anti-phagocytosis assay,adhesion invasion assay,andmouse pneumonia and bacteremia model in vivo.Results:Dot blot and ELISA showed that the TAs and CPS content of D39?fabT was significantly increased comparing to D39 WT in the whole bacteria lysates,and was decreased to a lower level in the Compll strain.Western blot analysis of separated subcellular components showed the pattern variation of both TAs and CPS.The small CPS molecular was significantly elevated with decreased macromolecule in ?AabT,and few distinct TA bands with a lower molecular mass appeared in cell wall fragment of ?fabT.In addition,FACS analyses demonstrated that the amounts of TAs and CPS are much higher in AfabT mutant strains,increased about 162.4%and 335.1%,respectively.Our results of TEM showed a loosen capsule in fabT mutant comparing to D39 WT and Compll.The direct binding of purified rFabT to the promoter of lic1/2,lic3,psr,lytR,rafX and cps were determined by EMSAs,however,it failed tobind with the predicted palindrome sequence?PPS?.qRT-PCR analysis with RNA isolated from D39,D39?fabT,and Compll was performed to examine the transcriptional levels of tarI,tacF,spr1226,spr1222,rafX,lytR and cps2A located in lic1,lic2,lic3,aroA,rafX,spr1761 and cps gene cluster,respectively.The results showed a remarkable increase of these genes in the absence of fabT.Since both the adherence and invasion might be reduced by the presence of polysaccharide capsule,we compared the adherence and the invasion to epithelial cell using nonencapsulated laboratory strain R6 and derived R6?fabT.The results showed the number of adherent and internalized R6?fabT mutants appears a significant increase.In addition,Anti-phagocytosis experiments showed that the anti-phagocytic ability of D39?fabT was significantly increased than that of D39 WT,which consistent with our observation of increased surface CPS content.In vivo,in the pneumonia model,we observed longer survival time after intraperitoneal injection with D39 than D39?fabT?p<0.05?,which may be related to the observation of loosen capsule phenotype,but the fabT mutant intranasal challenged mice were succumbed much earlier to the infections,whereas mice challenged with D3 9 and Compl2 strains survived much longer in the experiment?p<0.05?.Comparing to D39,the bacteria quantity of D39?fabT and Compl2 in blood,lung,liver,kidney and spleen of treated mice after 48 hours infection,an obvious increasing of bacteria loads in all tissues of D39 ?fabT was observed,respectively.Conclusion:Our results revealed that MarR-family regulator FabT act as a transcription repressor to the synthetic genes of teichoic acids and capsular polysaccharide in S.pneumoniae.Meanwhile,it may also positively affect the synthesis of macromolecular CPS.The variation of TAs and CPS caused by FabT deletion are found to contribute to virulence,which plays an important role in the invasive infection of S.pn,though the effects to different models are not exactly the same.