Research On Type ? Toxin-Antitoxin System In Streptococcus Suis ZY05719

Streptococcus suis(S.suis)is an important zoonotic pathogen and causes swine streptococcosis.Human infection with S.suis occurs by direct contacting with sick pigs or sick pork by-products with acute toxic shock syndrome.The virulent strain ZY05719 of S.suis serotype 2 was isolated from diseased pigs in Sichuan in 2005.S.suis strains are divided into more than 33 serotypes according to differences of the surface antigen of capsular polysaccharide.S.suis serotype 2 delivered a high clinical isolates and more frequently reports,often associated with highly virulence for human beings and pigs.Persister cells refer to the special forms of bacteria in the stress environment,which constitute subgroups of dormant cells in the microbial community with resistance to antibiotics.The toxin-antitoxin(TA)system has been identified to be involved in the formation of bacteria persistence in a variety of bacteria.In recent years,researching on bacterial TA system,bacterial virulence and physiological characteristics have gradually increased,but there are few studies on the relation between persister of S.suis and TA system.In this study,we constructed the TA modules mutant strains and verification system of toxins and antitoxins.It aims at providing experimental systems and theoretical basis for the interaction of persister formation and TA systems in S.suis.The toxin-antitoxin(TA)system is composed of a stable toxin and its homologous unstable antitoxin.In this study,9 pairs of type ? TA modules in ZY05719 were predicted by bioinformatics analysis.7 TA deletion mutants were constructed by natural DNA transformation and identified by targeted sequencing alignment.There is no obvious difference of morphological feature and growth curve between the TA mutants and wild-type strain.Recombinant plasmids pBADHisA-toxin and pBADHisA-toxin-antitoxin were transformed into Top 10 competent cells to draw growth curve by inducing expression of arabinose.The results showed that the growth of bacteria containing pBADHisA-T1 plasmid was significantly inhibited as soon as the toxin1 was induced.The growth trend had been unchanged in 4 hours until the anti-toxin protein 1 was able to neutralize the corresponding toxin protein.Persister cells mean the shock forms of bacteria under the external pressure,which constitute subgroups of dormant cells in the microbial community.Persister cells can form resistance to antibiotics.In this study,the sensitive drugs including rifampicin and ampicillin were used to determine the minimal inhibitory concentration(MIC).Then 100-fold MIC antibiotics prepared in cultured were inoculated exponential grown bacteria,and the colony forming unit(CFU)was counted for monitoring the persistence level.The persistence of the TA nodules mutants were measured under ampicillin resistance.As a result,S.suis strain ZY05719 can form persister cells in cultured with 100-fold MIC of rifampicin and ampicillin and TA modules deficient strains could form persister bacteria in a 100-fold MIC ampicillin concentration.RT-qPCR was used to analyze the transcription level of regulatory proteins RelA/RelQ associated with stress.Results showed RelA/RelQ has significant related to the regulation of S.suis persister formation,but the relationship of formation of persister bacteria needs further experimental verification.However the regulation of transcription of RelA/RelQ in TA1 mutant strain does not directly affect the formation of persister cells.As a conclusion,we speculate that the formation of persister cells in S.exposed antibiotics is not a single signaling pathway,but a complex process that needs more insights in this topic.