Preliminary Study On C-di-GMP Signaling Of Leptospira Interrogans And Development Of Multiple Chimeric Epitope Vaccine

Leptospirosis is a globally widespread zoonotic disease caused by pathogenic Leptospira speices.The most common species,Leptospira interrogans,can transfer from contaminated soil or water to human bodies,it survives through sensing and responding to the changes of environmental cues.Therefore,Leptospira were supposed to harbor intact signaling transduction which can regulate bacterial behaviors in response to enviramental changes.Cyclic di-GMP(c-di-GMP)is a special secondary messenger in bacteria,which can respond to the environment and regulate diverse bacterial behaviors,such as cell cycles,virulence,biofilm formation,motility and chemotaxis.The c-di-GMP level was regulated by diguanylatecyclases(DGC)and phosphodiesterases(PDE),they are responsible for synthesizing or hydrolyzing c-di-GMP,respectively.c-di-GMP triggers the expression of downstream genes through binding with recepors,resulting in regulating bacterial behaviours.In this study,we systematically analyzed the distribution and phylogenetics of c-di-GMP metabolic genes among 15 Leptospiral species using bioinformatics methods.We found that each individual species contains lots of c-di-GMP metabolic genes,suggesting that c-di-GMP is an important second messager among Leptospira.Since L.interrogans is the most common pathogenic speices that causes leptospirosis,we further analyzed the c-di-GMP signaling system in L.interrogans Lai strain 56601.25 c-di-GMP metabolic genes were found in its genome,including 13 GGDEF domain-containg proteins,three GGDEF-EAL domain-containg prtoeins,five EAL domain-containg proteins and four HD-GYP domain-containing proteins.Among them,most contain multiple domains including sensory domains.Most of these putative DGCs and PDEs possess enzymatic activities,which were determined via riboswitch-based dual-fluorescence reporters in vivo.Furthermore,when deleted GAF domain from LA1483 and PAS domain from LA2932,two GGDEF-containing proteins,they lose the ability of synthesis c-di-GMP,while deleted REC domain from LA2528,another GGDEF protein,can't affect the ability of synthesize c-di-GMP.High temperature resulted in low c-di-GMP concentration in L.interrogans and most of c-di-GMP metabolic genes exhibit differential temperature regulation.Determination of the comparation expression of c-di-GMP metabolic genes showed that the majority genes exhibited relative low expression,while LA1483 surprisingly was found extraordinarily high expression compared to others,even hundreds of times as high expression as others.Infection of murine J774A.1 cells resulted in reduction of c-di-GMP levels,while there is no significantly change of the mRNA levels of c-di-GMP metabolic genes during the infection of J774A.1 cells.In addition,we identified two PilZ domain-containing proteins can interact with c-di-GMP in vitro.Leptospirosis is an important reemerging zoonosis,with more than half a million cases reported annually,and is caused by pathogenic Leptospira species.Vaccination is the most effective method to prevent and control the prevalence of leptospirosis.Currently,the widely used vaccine against leptospirosis is still the killed leptospires.However,these vaccines are associated with high rates of side-effects,and they confer only short-term serovar-specific immunity,which strongly limits their widely applications.Therefore,it is urgent to develop a universal leptospirosis vaccine with high efficiency and low toxicity.Outer membrane proteins expose on the cell surface,involving in interacting with host,beside,they are conserved across different serotypes,so they become the one of best vaccine candidates.In order to improve the protective efficacy and wide cross-protection,some researchers developed chimeric multiple epitope vaccine to immune animals.In our previous research,we identified six B-and T-cell combined immunodominant epitopes in the OMPs OmpLl,LipL32 and LipL21 from Leptospira.According to the six epitopes sequences,we synthesized a gene fragment that encoding all six epitopes.This gene fragment was cloned as four consecutive copies in an expression vector pET28a,named r4R.After induction with IPTG,result of SDS-PAGE showed that this recombinant protein was successfully expressed in the soluble fraction with 64 kD.Western bolt assays demonstrated that r4R recombinant protein can interact with antisera.Through MAT(Microscopic agglutination tests)assays,we found that r4R antisera from the r4R-immunized guinea pigs were able to cross-agglutinate each of the L.interrogans serogroup-specific standard reference strains,indicating that r4R vaccine can provide cross-protection.The results of ELISA showed that the levels of IgG1 and IgG2a were significantly increased in r4R antisera compared to PBS antisera,the level of IgG2a was higher than that of IgG1.Lymphocyte proliferation and cytokine production in response to chimeric r4R protein were measured by CCT-8 and ELISA respectively.Lymphocytes stimulated with r4R showed a significantly higher proliferation response compared with the controls.The IFN-y secreted by lymphocytes was also increased after stimulating with r4R compared to PBS treatments.These results suggested that r4R vaccine elicits Thl-bised cell-mediated immunity.Guinea pigs immunized r4R showed increased survival compared with unvaccinated guinea pigs.Histological analysis showed that r4R-immunized guinea pigs didn't elicit obviously tissue damage,while PBS-immunized guinea pigs happened to severe tissue damage.