Research On Identification And Characterization Of The Chromosomal YefM-yoeB Toxin-antitoxin System In Streptococcus Suis Serotype 2

Toxin-antitoxin(TA)systems have attracted an increasing concern in recent years because of their abundance in the genomes of bacteria and archaea on the one hand,and the limitation of knowledge of their physiological functions on the other.TA systems were initially identified on low copy number plasmids,where they help to maintain plasmid stability by a mechanism called post-segregational killing or genetic addiction.The TA system was originally found on the low-copy plasmid to maintenance the plasmid stability.The TA systems play an important role in the bacterial growth control or biofilm formation or virulence and other stress response.These modules have been identified in Escherichia coli and various other bacteria,whereas their functions need to be further studied.In addition,the presence of TA systems in the genome of Streptococcus suis,an important zoonotic pathogen,has received little attention.S.suis 2 is considered the most virulent and the most frequently isolated serotype in association with diseases worldwide.Despite numerous studies having been carried out over the past few years,knowledge of the pathogenesis of S.suis remains very limited.In this study,we mainly carried out the identification and characterization of the chromosomal yefM-yoeB locus in S.suis to explore the relationship between TA system and the pathogenicity of S.suis.The details are as follows:1.Identification the chromosomal yefM-yoeB locus in S.suisBioinformatics analysis showed that the YefM-YoeB in S.suis were highly conserved with those of E.coli and Streptococcus pneumoniae,and the yefM-yoeB locus is present in the genome of most serotypes of S.suis.Overproduction of S.suis YoeB toxin inhibited the growth of E.coli,and the toxicity of S.suis YoeB could be alleviated by the antitoxin YefM from S.suis and S.pneumoniae,but not by that from E.coli YefM.The pull down and western blot experiments demonstrated that toxin YoeB interacts with its cognate antitoxin YefM in vivo.The promoter activity assays showed that YefM and YefM-YoeB could autoregulate negatively the TA operon transcription.These results demonstrated that the yefM-yoeB constitutes an active TA system.2.Deletion of the yefM-yoeB locus has no effect on S.suis 2 virulence in miceThe feature and function of yefM-yoeB remain to be further proved since the possibility of yefM-yoeB modules in the involvement in bacterial virulence has been largely neglected.To investigate the functions of the yefM-yoeB locus in S.suis 2,the isogenic yefM-yoeB and yoeB knockout mutants of S.suis 2 strain SC19,named ?yefM-yoeB and ?yoeB respectively.We also generated the complementation strain,designated C?yefM-yoeB,but failed to generated yefM knockout mutant.The growth kinetics and haemolytic activity of ?yefM-yoeB were essentially similar to those of the WT and C?yefM-yoeB strains.In a murine infection model,deletion of the yefM-yoeB locus had no role in S.suis 2 virulence.The mutant ?yefM-yoeB and WT strains have similar abilities to colonize the tissues.Collectively,our data suggested that the yefM-yoeB locus of S.suis is an active TA system without the involvement of virulence.3.Lys64 in YefM contributes to oxidative stress tolerance and pathogenicity of S.suis 2Based on SWISS-MODEL,selected amino acid residues were changed to assess which positions were crucial in the specificity of toxin–antitoxin interaction in the related YefM-YoeB complexes.The inactive potential forms of YefM were generated with point mutations,renamed C?yefM(A54S)-yoeB,C?yefM(N58A)-yoeB,C?yefM(L61A)-yoeB and C?yefM(K64A)-yoeB.Three strains(WT,?yefM-yoeB and C?yefM-yoeB)and four inactive potential forms of YefM were selected to explore their roles in growth curves under normal and oxidative stress conditions,oxidative stress tolerance,virulence of S.suis 2 and cytotoxicity to two different cells or cell lines(hBMEC and HEp-2).The results showed that C?yefM(K64A)-yoeB was weaker in growth curves under normal condition.The C?yefM(K64A)-yoeB was found to have diminished ability of oxidative stress tolerance,attenuated virulence in mice model.In addition,cytotoxicity to hBMEC(or HEp-2)of C?yefM(K64A)-yoeB was significantly attenuated,and cytotoxicity to HEp-2 of C?yefM(L61A)-yoeB was significantly attenuated,compared with those of the WT and C?yefM-yoeB strains.Our results revealed that Lys64 in YefM plays key roles in oxidative stress tolerance and virulence,and are crucial for the specificity of toxin-antitoxin interactions in the YefM-YoeB complex.The results of current study not only identified the yefM-yoeB system in S.suis 2 for the first time,but also enriched the understanding of TA systems.