Regulation Of (p)ppGpp And Their Synthetases On The Stringent Response And Virulence Of Streptococcus Suis

The mechanism of adapting to environment and pathogenesis of bacteria is important for the animal pathogens research,in which stringent response(SR)is one of the most important mechanism of adapting to environment.SR is mediated by(p)ppGpp,which is the crucial signal molecules to response the different environment.In our previous study,there were two(p)ppGpp synthetases,RelA and RelQ in Streptococcus suis S.suis).RelA was an bifunctional enzyme,which could not only synthetase(p)ppGpp,but also hydrolase(p)ppGpp while RelQ only had the(p)ppGpp synthetase domain.RelA played an important role in SR induced by amino acid starvation and glucose starvation,but it was not clear of the mechanism of SR induced by glucose starvation so far.In this study,we researched on the mechanism of SR induced by glucose starvation and pathogenesis in S.suis.The main results were as follows:1.The molecular mechanism of stringent response under glucose starvation in S.suisIn S.suis,it was found that RelA did not interact with acyl carrier protein(ACP)to regulate SR induced by carbon starvation in our previous study.In this study,Bacterial Two-Hybrid,Pull-down and ELISA assays were utilized to find HPr serine kinase/phosphorylase(HPrK)could interact with RelA,and HPrK N interacted with RelA N significantly.RelA and RelA N could synthesize(p)ppGpp while HPrK and HPrK C had kinase activity by isotope labeling detection.RelA N could enhance the kinase activity of HPrK while HPrK could inhibit synthetic activity of RelA and RelA N in vitro.Comparing the phosphorylated product between SC-19 and the ArelA mutant,we found HPr-Ser-Pi amout of the ArelA mutant was less than that of SC-19 in vivo.This study initially revealed a certain link between SR and CCR/CCA and established a foundation in SR of gram-positive bacteria under glucose starvation.2.(p)ppGpp synthetases regulate the growth and pathogenesis of S.suis(p)ppGpp-mediated stringent response is one of the main adaption mechanism in bacteria,and the ability to adapt to environment is linked to the pathogenesis of bacterial pathogens.To investigate the regulatory functions of(p)ppGpp/(p)ppGpp synthetases on the pathogenesis of S.suis,the phenotypes of the[(p)ppGpp0]mutant ?relA?relQ and its parental strain were compared.Light and electron microscopy observation showed that the mutant strain had a longer chain-length than its parental strain.Disruption of relA and relQ led to decreased adhesive and invasive ability to HEp-2 cells,and increased sensitivity to the blood killing and phagocytosis.Mouse infection experiments showed that the mutant strain was attenuated and easier to be cleaned up in vivo.Quantitative reverse transcription PCR(qRT-PCR)analysis indicated that the expressions of some genes involving in morphology(cps2A,gpsB)and virulence(gapdH,fbps,arcA,vicR,virD4,sod,epf)were down-regulated in the mutant strain.Our study demonstrated that the(p)ppGpp synthetases or(p)ppGpp can regulate the pathogenesis of this important zoonotic pathogen.3.(p)ppGpp synthetases and CodY co-mediated regulate the growth and pathogenesis of S.suisIn some bacteria,pleiotropic regulator CodY and(p)ppGpp synthetases were required to co-mediate SR and pathogenesis.It was reported that lack of codY could restore some phenotypes in(p)ppGpp deletion mutant in Listeria monocytogenes,Staphylococcus aureus and so on.Therefore,we constructed the ?relA?relQ?codY mutant in the[(p)ppGpp0]mutant ?relA?relQ.The phenotypes of ?relA?relQ,?codY,?relA?relQ?codY and their parental strain were compared.Our study showd that the capsular thickness and chain length was similar to SC-19 while the pathogenesis was not restored after deleting codY in ?relA?relQ.In EMSA assay,CodY could interact with the promoter of relA directly,indicating that CodY might control the expression of RelA to regulate bacterial virulence and SR indirectly.It maybe a new mechanism of CodY mediating virulence and SR.