TroR-mediated Resistance To Metal Toxicity Of Streptococcus Suis And Establishment Of A Two-step Markerless Gene Deletion Method

Streptococcus suis is an emerging zoonotic pathogen that causes severe swine and human infections.Metals are essential nutrients for life;however,excess metals can be toxic to bacteria.Therefore,maintaining intracellular metal homeostasis is crucial for bacterial survival.Here,we identified a metalloregulator in S.suis,TroR.RNA sequencing analysis of S.suis grown in the presence of excess manganese(Mn)showed that the expression of both troR and troABCD operons was significantly down-regulated.Bioinformatic analysis found that TroR of S.suis exhibited approximately 54%to 60%amino acid identity to the homologs from Streptococcus pyogenes,Streptococcus pneumoniae,Streptococcus gasseri,Streptococcus parasanguinis,and Streptococcus mutans.The growth curves showed that the growth of the troR gene deletion mutant(AtroR)was inhibited in the medium supplemented with zinc(Zn),cobalt(Co)and copper(Cu)compared with the wild strain(WT)and complemented(CΔtroR)strain;Mn supplementation alleviated the growth defect of ΔtroR in the presence of excess Zn or Co;the growth of ΔtroR was also inhibited by the addition of various concentrations of Mn in the medium treated with Chelex.Inductively coupled plasma optical emission spectrometry(ICP-OES)showed that in the medium supplemented with Mn,Co,Cu and Zn,the accumulation of Mn and Co in ΔtroR strains was higher than that in WT and CΔtroR strains,indicating that troR could maintain manganese and cobalt homeostasisin S.suis.RNA sequencing analysis of WT and ΔtroR revealed that compared with WT,a total of 20 genes were significantly differentially expressed in ΔtroR,among which the troABCD operon was significantly up-regulated,indicating that TroR negatively regulates the troABCD operon.Reverse transcription quantitative PCR analysis showed that ferrous[Fe(II)]and Co induced the expression of troA in WT,while Mn and Cu inhibited its expression;the inhibition of troA was mediated by TroR.The mouse intranasal infection model showed that compared with WT and CΔtroR,the ability of ΔtroR to colonize various tissues was attenuated,indicating that the deletion of troR attenuates the virulence of S.suis.Together,these data suggest that TroR is a negative regulator of the TroABCD system and is associated with resistance to metal toxicity and virulence in S.suis.The most commonly used method for the construction of S.suis gene deletion mutants is the homologous recombination method based on the pSET4s thermosensitive suicide plasmid.However,this method does not use counterselectable marker;thus it takes a lot of time to screen mutants.Some researchers have also constructed a gene deletion method independent of gene cloning based on the natural transformation of S.suis,but the resultant mutants carry resistance genes and cannot be used for subsequent vaccine research.A previous study found that inducing the expression of Vibrio parahaemolyticus YoeB toxin(YoeBvp)in Escherichia coli would lead to cell death,suggesting that YoeBvp has the potential to be a counterselectable marker for S.suis.In this study we first evaluated the counterselection potential of YoeBvp.The yoeBvp gene was overlapped with the copper-inducible promoter PcopA to form a DNA fragment,which was cloned into the S.suis-E.coli shuttle vector pSET2 and introduced into S.suis by natural transformation to evaluate the effect of YoeBvp expression on S.suis growth.Reverse transcription quantitative PCR analsyis showed that copper could significantly induce the expression of yoeBvp.Growth curve and spot dilution experiments showed that the expression of YoeBvp inhibited the growth of S.suis in both liquid medium and agar plates,revealing that YoeBVp has the potential to be a counterselectable marker for S.suis.Subsequently,we constructed a SCIY cassette,which consists of the spectinomycin resistance gene(spc)as the positive selection marker and the PcopA promoter-controlled yoeBVp gene(PcopA-yoeBvp)as the counterselection marker.Using this cassette,combined with the natural transformation of S.suis,a new two-step markerless gene deletion method was successfully established for S.suis.Finally,ΔpmtA,ΔperR and ΔlysR of S.suis were successfully and efficiently constructed by this method.This method does not require gene cloning and has high efficiency;in addition,this method can also be used for site-directed mutagenesis or deletion of a few bases in the S.suis genome.In conclusion,this study investigated the TroR mediated resistance to metal toxicity and it role in the pathogenicity of S.suis busing troR gene deletion strains and complementary strains.We showed that TroR is required for the resistance to metal toxicity and virulence in S.suis.The counterselection potential of YoeBvp toxin was evaluated in S.suis.Using the new constructed spc-PcopA-yoeBvp cassette and natural transformation,a new two-step markerless gene deletion method was successfully established for S.suis.