Secretory Assays And Functional Identification Of YPK＿2589 In Yersinia Pseudotuberculosis

Bacterial type Ⅵ secretion system(T6SS)is widely distributed in gram-negative bacteria.These effectors play an important role in several crucial physiological processes such as pathogenicity,stresses combatting and competition.Thus,the investigation of these effectors exert great significance for thoroughly explore the bacterial physiology and functional mechanism.Here,we studied an unknown gene ypk＿2589 in T6SS gene cluster of Yersinia pseudotuberculosis YPⅢ strain.We aimed to identify the relationship of YPK＿2589 with T6SS and explored its biochemical activity and biological function by gene knockout,Western blot,GST pull-down,interspecies competition and enzyme activity assay,the following results were obtained:1.Our previous study found that YPⅢ contains four T6SS(T6SS-1 to T6SS-4).By using the mutation strains that lacking ClpV of different types T6SS,which are the core components of T6SS,we discovered that YPK＿2589 is a secreted protein depended on T6SS-3.2.Next,we over-pressed YPK＿2589 in E.coli BL21,DH5α and Pseudomonas aeruginosa PA14 and found that YPK＿2589 has cytotoxicity.3.Based on the previous reports that effectors and immunoproteins come in pairs,we confirmed the interaction between YPK＿2589 and YPK＿2588 by using bacterial two hybrid assay.In addition,we constructed mutants about related genes and conducted the intraspecies competition,the result confirmed YPK＿2588 can weaken the cytotoxicity of YPK＿2589,namely YPK＿2588 is the immunoprotein of YPK＿2589.4.Meanwhile,the result of biofilm formation and mice infection study on YPⅢ related strains and other strains showed: relative to wild and complementary strains,ypk＿2589 mutant hardly formed biofilm and the mortality rate of mice after ypk＿2589 mutant infected was significantly reduced.Therefore YPK＿2589 can affect the biofilm forming and the pathogenicity of YPⅢ.5.However,the result of interspecific competition indicated that YPK＿2589 hadn’t advantage in interspecific competition.Considering that receptors can help transport effectors into cells,we screened and confirmed YPK＿3409 and YPK＿3889 as two receptor proteins of YPK＿2589 via GST pull-down assay.Next,we constructed mutants of the related genes and other strains of over-expressing receptor proteins to performed interbacterial competition experiments.We found that that two receptors play an important role in the YPK＿2589 related interbacterial competition.6.Finally,to explore the possible mechanism,enzyme activity of YPK＿2589 was detected in vivo and in vitro,It was found that both 23 S rRNA and 16 S rRNA were missing in the sample stripe when YPK＿2589 protein was added separately or a small amount of YPK＿2588 protein was added at the same time in the extraneous reaction system.While 23 S rRNA and 16 S rRNA reappeared in the sample when more YPK＿2588 proteins were added.After expressed YPK＿2589 in Δypk＿2588-2589 mutant strains and PA14 bacteria,23 S rRNA and 16 S rRNA also disappeared.Therefore,YPK＿2589 can hydrolyze RNA including 23 S rRNA and 16 S rRNA,while YPK＿2588 can inhibit this activity.In conclusion,this study explored the property and function of YPK＿2589,which may provide clues to study the mechanisms and functions about similar T6SS effectors.Meanwhile,a new breakthrough point was found for the pathogenesis study of pathogenic bacteria.