Research On Identification And Function Of Type Ⅱ Toxinantitoxin System In Streptococcus Suis Serotype 2

Streptococcus suis is the major pathogen causing streptococcus suis disease,as a upper respiratory tract pathogens,leading to a variety of diseases of pig,such as meningitis,septicemia,arthritis,endocarditis,and even death,bringing significant economic losses in the pig industry worldwide.Among the known 33 serotypes,S.suis 2 is the most virulent and prevalent pathogen in the pig industry,and also a serious zoonotic agent that can cause meningitis and toxic shock syndrome.The toxin-antitoxin system(TAS)is a small genetic element widely found in the bacterial genome and initially found on low-copy plasmids to maintain the stability of the plasmid.TAS have been integrated into physiological framework,and give the bacteria a advantage coping with the stress environment.This study identified 7 pairs of Type II TAS which were predicted by a web,and focused on the effect of mutant strains lacking TAS on virulence.The main results are as follows:1.The co-transcription analysis of toxin antitoxin by RT-PCRThe total RNA of S.suis 2(SC19)was extracted,and c DNA was obtained by reverse transcription.c DNA was used for template by PCR amplification with specific primers to verify the co-transcription analysis of toxin and antitoxin.It was proved that toxin and antitoxin were regulated by the same operon.2.Study on the characteristics of toxin and antitoxinThe recombinant plasmid p BADHis A-toxin was constructed and transformed into Top10 competent cells,and arabinose was added to induce toxin expression,performing the growth curve and CFU counting.The results showed that the growth of harbouring p BADHis A-T7,p BADHis A-T5,p BADHis A-T7 and p BADHis A-T9 was significantly inhibited.Among them,the growth tendency of harbouring p BADHis A-T1,p BADHis A-T7 and p BADHis A-T9 were almost unchanged after 2h of toxin induction expression,but the growth of carrying p BADHis A-T5 has grown slowly;CFU results showed that induction of T1,T7,T9 exhibited significantly bactericidal effect,but the bacteria have not been all killed,and those survival bacteria beganed to resume growth slowly after 4h of induction,and expression of T5 did not significantly bactericidal effect.To further identify and validate the toxin-antitoxin system in S.suis,the recombinant plasmid p ET30a-antitoxin and p BADHis A-toxin were co-transformed into BL21 competent cells.IPTG and arabinose were added to induce the expression of antitoxin and toxin respectively.The growth curve was drawn to observe whether antitoxin can neutralize toxin and the bacteria of induction 5h was stained to observe the morphology of bacteria in the case of protein expression.The results showed that induction expression of A1,A7,A9 were able to neutralize corresponding toxin,so that the growth of bacterial returned to normal.The growth of alone inducing expression T1,T7,T9 were significantly limited,but the growth of T5 expression returned to normal level,indicating that the fifth pair is not TAS.Gram-stain results indicated that the induction erxpression of toxin can change the morphology of E.coli,and there is a clear filament formation,which may be a protective mechanism of bacteria.3.Construction and identification of toxin-antitoxin system mutant strains and complementary strainsAccording to the above experimental results,the 1,7,9 pairs of TAS were deleted to construct the corresponding gene deletion mutant.The recombinant plasmid were constructed successfully by temperature sensitive plasmid p SET4 s and transformed into SC19 competent cells by electric shock.The single exchange strain was obtained by screening temperature and spc resistance,and then the mutant was screened by the second homologous recombination.The recombinant plasmid by p SET2 were constructed for complementary strains,transformed into the corresponding mutant mutants,and screened by resistance.The mutant strains and complementary strains were successfully constructed by PCR and sequencing.4.Effect of toxin-antitoxin system mutant strains on S.suis 2The survival curves were drawn with SC19 and the mutant strain.The results showed that mutants had little effect on the virulence of S.suis 2.Erythromycin was used for treating wild strain and mutant to activate toxin-antitoxin system,and the growth curve of strains was measured,obtaining the viable count results.The result showed that there were some differences in SC19 and mutant strains,but the results were not significant.