Study On The Function And Assembly Of Diarizonae Toxin In Salmonella Diarizonae

Salmonella diarizonae is a common intestinal pathogen in reptiles.With the progress of evolution,S.diarizonae gradually expand the scope of its host invasion to warm-blooded animals,including humans,causing gastroenteritis and septicemia.Clarifying the pathogenesis of S.diarizonae is a prerequisite for the control of the pathogen.However,there is currently no in-depth research on it.Typhoid toxin,the typical A2B5 bacteria toxin,is composed of two subunit A(CdtB and PltA)and five subunit B(PltB),and is encoded by typhi islet on the genome of Salmonella Typhi.In this study,we find that there is also a similar typhi islet on the genome of S.diarizonae,and interestingly,two subunit B genes(pltBd1/pltBd2)on its typhi islet are adjacent to each other,while in S.Typhi,two subunit B genes(pltB and pltC)are on different typhi islets.We speculate that the pathogenesis of diarizonae toxin and typhoid toxin may be different.In clinical treatment,the traditional antibiotics against S.diarizonae are no longer effective.Therefore,it’s urgent to develop new anti-infective drugs against S.diarizonae.Clarifying the assembly mode and functional characteristics of the diarizonae toxin is an important way to develop new anti-infective drugs against it.We used the well-known typhoid toxin as a reference model to study the pathogenic mechanism of the diarizonae toxin.The main results of this study are as follows:1.The function of d iarizonae toxin(DT).S.diarizonae can infect NCM460 and it can only express toxin after penetrating into the infected cell.And we proved that the cell infection phenotype of S.diarizonae is similar with that of S.Typhi.Through the cell infection of mutant strains,we confirmed that diarizonae toxin is one of the key virulence factors of cytotoxicity induced by S.diarizonae,and that the active subunit of diarizonae toxin is CdtB and its active site is the His 160.2.The assemble pattern of diarizonae toxin(DT).By the heterologous expression in E.coli and the expression of diarizonae in situ,we found that there are three kinds of diarizonae toxin(PltBd1 holotoxin,PltBd2 holotoxin and heterozygous holotoxin).Western Blot analysis showed that PltBd1 and PltBd2 proteins could be normally expressed in S.diarizonae.We further demonstrated that PltBd1 and PltBd2 can form heterozygous toxin through pull-down assay.That is,S.diarizonae has the potential to assemble PltBd1/PltBd2 holotoxin and PltBd1PltBd2 heterozygous holotoxin.Then we compared the toxicity of diarizonae toxins with different assembly patterns by testing their concentration for 50%of maximal effect,and found that PltBd2 holotoxin and heterozygous toxin are more toxic to NCM460 than PltBd1 holotoxin.The EC50 values of PltBd1 holotoxin.PltBd2 holotoxin and heterozygous toxin are 14.16 pM,0.36 pM and 0.5042 pM,respectively.Finally,through the cell infection of subunit B deletion strains,we find that both PltBd1 and PltBd2 are necessary for the toxicity of S.diarizonae on NCM460 cells.3.The crystal structure of diarizonae toxin(DT).Typhoid toxin is the typical A2B5 toxin.which is composed of two subunit A and five subunit B.To understand the molecular mechanism of the assembly of diarizonae toxin,we purified PltBd1 holotoxin,PltBd2 holotoxin,PltBd1-PltBd2heterozygous holotoxin,PltBd1 pentamer,PltBd2 pentamer and PltBd1-PltBd2 heterozygous pentamer and tried to analyze their protein structure via X-Ray.Finally,we analyzed the atomic structure of PltBd2 pentamer successfully and demonstrated that the formation of PltBd2 pentamer is primarily determined by the interaction of surface potentials of adjacent subunits.This research work provides a research foundation for understanding the process of invasion of hosts by S.diarizonae and the molecular mechanism of toxin expression regulation,and also provides theoretical support for the development of new anti-infective drugs targeting S.diarizonae.