| Streptococcus suis(Streptococcus,SS)is an important zoonotic pathogen that can cause bacterial meningitis,arthritis,sepsis and acute shock death.It is also reported worldwide.The frequently nucleic acid exchange results to diversity of chromosome genomics and serotypes,that potentially confers them with the ability to adapt to changing environmental conditions,like antibiotic resistance or transmission between different hosts.Natural transformation,an important mechanism for genes horizontal transfer in streptococcal strains,play a significant role in bacterial evolutionary variation.There are few studies on the natural transformation of SS until now,and the related to functional proteins is not clear.In this study,we develop a rapid genetic edit method with the natural transformation ability,which facilitated molecular research and the contribution of diversity ComRS system.Meanwhile,we deeply explored the function of core components ComRS system and competence regulated murein hydrolase CrfP in the process of competent state and bacterial virulence.Subsequently,evolutionary analysis was carried out to clarify the virulence genotyping,defense system and ComRS system phenotypic diversity among SS2 strains,that caused by switch of SS2,SS3 and SS7 capsular gene clusters.This study provide a new understanding and theoretical basis for further study of the natural transformation mechanism in SS and the variability of capsular gene clusters on bacterial biology.1 Utilization of the ComRS system for the rapid markerless deletion of chromosomal genes in Streptococcus suisThe diversity of SS genomes result to complex in pathogenic mechanism.So genomic approaches are important to determine virulence mechanisms;however,conventional electrotransformation to deliver plasmids for gene deletion is not effective in many clinical isolates,particular serotype Chz strains.Based on a phylogenetic analysis,we identified a three ComRS system in S.suis strains,designated type A,B and C systems.All three ComRS systems were functional for natural transformation by specific recognition between ComR and comX-inducing peptide.The transformation efficiency varied substantially among isolates but was uncorrelated with virulence.Brief based on these findings,we established a simple,markerless gene deletion method using accessible DNA fragments obtained by PCR amplification.Fusion PCR was used to construct a sacB-spc cassette between the upper and lower homologous fragments,followed by spectinomycin-positive and sucrose-negative selection.The efficient replacement of the sacB-spc cassette could be achieved by a second transformation step based on sucrose sensitivity.The efficiency of natural transformation using various mutants was dependent on the length of the homologous fragment and the concentration of donor DNA.This rapid gene editing technique may facilitate genetic studies of S.suis.2 Evaluation of ComR involved in virulence of Streptococcus suis serotype 2 strainStreptococcus suis serotype 2 strains have received increasing attention for its involvement in both human and swine infections worldwide.ComR,the Rgg-like transcription regulator,was characterized by an N-terminal HTH type DNA-binding domain and C-terminal peptide-binding domain.It controls competence for natural DNA transformation in S.suis via directly activate competence master sigma factor ComX and pheromone peptide ComS.In this study,we identified the type A ComR present unique to virulent strains of SS2,which may a potential virulence related factor.The gene deletion mutant(?comR)was constructed in ZY05719 to assessment the contribution of ComR in SS2 pathogenicity.Our results demonstrated that type A ComR factor involved in adhesion for HEp-2 and HBMEC cells.In addition,it also participated in ZY05719 resistance to swine whole blood killing in vitro.Further analysis showed that comR inactivation significantly attenuated SS2 virulence in BALB/c mice model,both for the abilities to colonize the tissues and survive rate,which couple with the significant expression observed in mice blood in vivo.The qRT-PCR analysis for cytokines mRNA expression indicated ComR help ZY05719 escape the immunity recognition.Taken together,these results suggested that ComR play important role in virulence of SS2 during infection.3 CrfP,the fratricide protein in Streptococcus suis contributes to natural transformation and virulenceRecently it has been shown that Streptococcus suis is competent for natural genetic transformation.It was an important mechanism for DNA horizontal transfer,but some puzzling problems remains to be solved.Upon entering the competent state,streptococcus stimulate the transcription of core competence essential genes which were responsible for exogenous DNA binding,uptake and processing.In addition to the competence specific genes,some accessory element for transformation process were expressed and played a major effect.CbpD,the competence related murein hydrolase in Streptococcus pneumoniae,which could lyses non-competent sister cells or members of closely related species in order to release homologous DNA that can be taken up by the competent attacker cells.In our study,we identified an CbpD-like protein,CrfP,in SS2 ZY05719 model,which also present in the whole S.suis species.This protein was controlled by ComX with analysis of qRT-PCR and promoter conversed motif.Indeed,we characterized CrfP as a murein hydrolase could secret out of cell and lytic all of serotype SS with specific cell surface binding domain.The TEM results confirmed this lytic ability.To investigate the functions of CrfP in vivo,the gene deletion mutant(?crfP)was constructed,we found inactive of crfP can not stop transformations,but will reduce the natural transformation effective rate significantly.Furthermore,to assess the phenotypic changes between the parental strain and?crfP in virulence,BALB/c mouse and zebrafish were chosen for infection experiments.The results demonstrated that the abilities of ?crfP to death were significantly attenuated in both infection models.Totally,these results indicated this CrfP is involved in competence and virulence of SS2.4 Comparative Genome Analyses provides clues to capsule switch in Streptococcus suisThe variation of capsule cluster lead to the different serotypes in Streptococcus suis.Even now little understood about the molecular mechanism in epidemiology and capsule types.SS2 strains are the most prevalent strains worldwide,but the huge difference in virulence between SS2 subgroups were reported in our previously study.And aviruelnt SS2 strains share the same ComRS system with SS3 and SS7 strains.In this way,the virulence genotyping assay was performed by PCR in totally 88 isolates to detect the distribution of virulence related genes among SS2,SS3 and SS7.Two clusters were apparent,the virulent SS2 strains belong to cluster ? and the avirulent SS2 strains,SS3,SS7 strains belong to cluster ?,which was consistent with MLST analysis results.The hypothesis of capsular switching may occurred between these serotypes isolates was proposed,whose golden standard is that strains of different serotypes sharing the same ST.Indeed,phylogenetic analysis of 25 complete genomes of different serotype strains deciphered two distinct clades in SS2,and Clade ? SS2 had a higher genomic similarity with SS3 and SS7 than Clade ? SS2.Moreover,Clade ? strains also share the same competence and defense systems.cps gene locus analysis brought evidence for strongly supports the hypothesis of capsular switch between SS2 and SS3,and favors the proposal that SS3 to SS2 capsular switch results from a recombinational crossover flanking sequence.This is the first time explain the capsular switch in SS,revealed the source of avirulent SS2 strains. |
PDF Full Text Request