Study On FabT,a MarR Family Transcriptional Regulator,Influencing Colony Phase Variation Of Streptococcus Pneumoniae

Objective: Streptococcus pneumoniae,an important opportunistic pathogen can cause many invasive diseases.Streptococcus pneumoniae undergoes phase variation or spontaneous,reversible phenotypic variation.The phenotypic diversity in bacterial populations is complicated,but the variation in colony morphology represents the typical characteristics.The different colony phases are fundamentally distinct phenotypes in their metabolism and multiple characteristics,and finally reflect in phenotypes in colonization and virulence.The factors that affect the cause of phase variations are not yet fully clear.Recent studies show that in S.pneumoniae,the phase variation is manipulated by the recombination of allelic hsdS genes in the type I RM system SpnD39? locus,and the recombination of hsdS alleles are catalyzed by invertase PsrA.Besides,unknown endonuclease or recombinase existed in the S.pneumoniae might be involved in the recombination of this locus.Here,we demonstrated a Mar R family transcriptional regulatory protein FabT(SPD?0379),which can cause significant phenotypic variation after knockout mutation.Further study showed that it may be related to the phase variation in colony opacity.Thus,this study was aimed to determine the regulatory mechanism of FabT on the known mechanism in phase variation and provide new evidence for revealing the mechanism of phase variation.Methods: We first construct mutant strains by using Janus cassette1,frameshift mutant or plasmid double crossover as engineering approaches.Observation of pneumococcal colony opacity was carried out with TSA plates supplemented with catalase.The degree of encapsulation was evaluated by measuring the zone of exclusion of FITC-dextran,immunofluorescence method,transmission electron microscope,uronic acid quantifucations.Using qRT-PCR to exclude the influence of lyt A or cps2 A on the phase variation in fabT mutants.Finally,the comprehensive effects of FabT on the virulence of S.pn were explored through anti-phagocytosis assay,adhesion invasion assay,and model of mouse pneumonia and bacteremia in vivo.Results: We construct the unmarked fabT and cps mutants in an streptomycin resistant strain JH1900.We replaced the pregenetic part of the fabT coding region(with C-terminal 54 bp retained)with the Janus cassette1 by allelic exchange JH1900,resulting in fabT replaced strain JH0004.To construct the cps mutant JH0002,we remove the entire cps2 A promoter region dex B-cps2 A.Encapsulated fabT mutant displayed significant growth defects when grown in THY medium,and was with premature autolysis.We observed rough and small colony morphology in Blood agar plate(BAP)and a more intriguing finding is the opacity variation displayed in TSA plate in fabT mutants,where ?fabT mutant correlates with an increased ratio of transparent(T)variants.The plate was put on the stage with the open side up,and a LED spotlight diagonally above was used to provide an oblique and transmitted light to observe the colony.Consistently,the unencapsulated fabT mutant displayed much more transparent colony compared to the unencapsulated wild type JH0002.We measured the capsular thickness by exposing the bacteria with FITC-dextran,immunofluorescence microscopy and TEM.These experiments consistently displayed a considerably thinner capsule layer in?fabT mutant compared to JH1900.We hypothesized that the reduction of surface capsule is due to the variation of fatty acid composition ratio of the membrane affected by FabT,thus affects the polymerization and maturation of the capsular precursor on the membrane.The reduction of the capsular level at the whole bacterial level may be due to an FabT regulated unknown mechanism regulates the synthesis of capsular polysaccharides.FabT may attenuate the virulence of S.pneumoniae by changing the phenotypic variation and affects the pathogenicity of S.pneumoniae during infection in vivo.Encapsulated ?fabT mutant show reduced colonization in mice nasopharynx,and the slightly lower virulence also presents in bacteremia model.The abilities of JH1900 and JH0004 to adhere to A549 human alveolar epithelial cells were also determined.We found that adherence of the ?fabT mutant to the epithelial A549 cells was significantly decreased compared to WT levels.?fabT mutant showed an increase in antiphagocytosis against macrophages may be due to their reduced ability to adhere to the cell surface.These results suggest that,in addition to the reduction of the capsule caused by the phase variable ?fabT mutant,it may also affect other components of cell wall surface related to adhesion and invasion ability,such as teichoic acids or surface proteins,and the influence await further investigation.A whole-genome transcriptome sequencing analysis was conducted to compare the m RNA levels in the ?fabT mutant to those in WT.The absence of FabT led to upregulation of 67 genes and downregulation of 66 genes(padj<0.05).Of particular interest,the ?fabT mutant showed significantly upregulated expression of psrA(a site-specific recombinase,also known as cre X)as well as hsdS',with downregulated hsdS gene,from the SpnD39?(Spn556II)type I restriction modification system.The transcriptional upregulation of psrA was verified by qRT-PCR.The genetic variation due to recombination within the hsdS locus between pairs of inverted repeats(IRs)catalyzed by recombinase psrA,resulting in six specific allelic variants which are associated with pneumococcal phase variation in colony opacity.we constructed frameshift psrA mutants by truncating the PsrA protein at amino acid 6 without altering the size of the gene,the increased ratio of transparent colonies in ?fabT mutant was completely reversed,suggesting that the inactivation of PsrA directly caused the occurrence of opaque phase.In order to investigate whether the increased T phase in ?fabT mutant was mediated by PsrA mediated inversion,the inversion of each IRs and the frequency of hsdS inversion in SpnD39? locus was assessed by qRT-PCR.The results show that in the absence of PsrA completely abrogated the inversion of IR1 and IR3,indicating that PsrA completely catalyzes the sitespecific recombination mediated by these two pair of IRs.In encapsulated strains,the inversion mediated by IR3 dramatically changed the configuration in the ?fabT mutant.Moreover,IR2 sequence was lost in?fabT mutant and the ?fabT?psrA mutant in the hsdS locus,indicating that there are unknown mechanisms or specific recombination systems regulated by FabT playing an important role in SpnD39? locus inversion.The results of hsdS allele detection showed that the six specific allelic variants mediated by the three IRs were existed in different psrA mutants,since they all showed consistent opacity in the observation of phase phases.We hypothesis that the generation of phase variation is just related to the activity of PsrA,not to a specific allele.Conclusion The transcriptional regulator FabT affects the growth stability of Streptococcus pneumoniae,variating the morphology of colony phenotype,and causes a decrease in bacterial capsule synthesis.Transparent colony phenotype causes the weakening of the bacterial systemic virulence.Transcriptome sequencing showed that hundreds of genes were regulated FabT mutations,which play a global transcriptional regulatory effect in bacteria.?fabT mutant was correlated with the DNA invertase PsrA expression level,which catalyzes the site-specific recombination of a type I restriction modification(RM)system SpnD39?.FabT affects the recombination of SpnD39 III locus in a PsrA-dependent manner,and controlling the phase variation of colony morphology.Importantly,there is a PsrA-independent way for FabT to regulate the phase variation in S.pn.