Study On The Bactericidal Mechanism Of Yersinia Pseudotuberculosis T6SS Effector Protein Tce1

The type VI secretion system(T6SS)iswidely distributed transmembrane complexes used by many gram-negative bacteria to translocate effectors into adjacent cells in a contact-dependent manner.Although some T6SSs are involved in bacterial pathogenesis through delivery of anti-eukaryotic effectors into host cells,T6SS is primarily considered an antibacterial weapon to compete against rival bacteria in polymicrobial environments.The antibacterial function of T6SSs relies on injection of antibacterial effectors that target conserved,essential features of the bacterial cell.All contact-dependent T6SS antibacterial weapons characterized to date do not require specific receptors in target cells for delivery of effectors or recognition of prey cells.Whether T6SS can similarly secrete toxic effectors into the extracellular milieu that recognize and enter target cells,thereby mediating contact-independent killing.This study focused on the T6SS-3 gene cluster in a potential function of the unknown effector of the Yersinia pseudotuberculosis ypk?0954,was named as Tce1(T6SS contact-independent antibacterial effector 1),and its immunity protein YPK?0955 as T6SS contact-independent antibacterial immunity 1(Tci1).Through bioinformatics function prediction analysis,effects of protein expression and purification for recombination in vitro biological enzyme activity detection and identification of its biochemical function.Further,the tce1 mutant was constructed for bacterial competition test to reveal the mode of action of tce1 participating in bacterial competition.The following results were obtained:1.Tce1-Tci1 is a T6SS-3 nuclease effector-immunity pair.When VSVG-tagged tce1 was produced in YPIII,the secreted protein was readily detected in the supernatant,further indicating that tce1 is a T6SS effector mainly associated with T6SS-3.To confirm the toxic activity of Tce1,we performed toxicity assays in Escherichia coli.Expression of Tce1significant growth inhibition.The growth inhibition was relieved by co-expression of the gene Tci1.To assess whether the immunity results from direct protein-protein interaction,we performed glutathione S-transferase(GST)pull-down,bacterial two-hybrid assays,and Isothermal titration calorimetry(ITC),the results showed specific interactions between Tce1and Tci1.These results demonstrate that Tce1 is a T6SS-3 secreted antibacterial effector and that its toxicity is neutralized by the Tci1 immunity protein.2.Tce1 exhibits Ca²⁺,Mg²⁺-dependent DNase activity.Using HHpred revealed similarity of Tce1 with the DNA-binding proteins,implying its potential role as a nuclease toxin.Incubation of purified Tce1 with?-DNA or the circular plasmid p UC19 DNA as substrates led to dramatic DNA degradation.The DNase activity of Tce1 critically relies on the co-existence of Ca²⁺and Mg²⁺in the reaction buffer.However,Tce1 did not display detectable RNase activity in vitro.Based on random mutant library screening,a mutant that lost toxicity to E.coli(Tce1^S8A/A16E)was identified.The DNase activity of Tce1 was also confirmed in vivo using the terminal deoxynucleotidyl transferase d UTP nick-end labelling(TUNEL)assay and DAPI staining.Results establish that Tce1 is an actual DNase in E.coli.3.Tce1 mediates contact-independent T6SS killing.To assess the contribution of the Tce1-Tci1 effector-immunity pair to bacterial antagonism,we performed growth competition assays using labelled derivatives of Yptb co-cultured under conditions promoting cell contact.The WT donor exhibited a 3-fold growth advantage in competition with the?tce1?tci1recipient.The Tce1-mediated growth advantage requires a functional T6SS-3.The WT donor exhibited a 3-fold growth advantage in competition with the?tce1?tci1 recipient.Similar results were obtained when the assay was repeated with a cell-impermeable membrane separating the donor and recipient cells on the surface of solid medium.We performed a fluorescence-based assay using Alexa Fluor 488-conjugated Tce1 to probe protein importation.Addition of AF488-Tce1 to WT bacteria cells yielded fluorescent bacteria.The contact-independent killing activity of Tce1 was also verified by examining its toxicity to target cells in liquid medium.While addition of purified Tce1 protein to the liquid medium had little effect on WT survival,it greatly reduced the survival rate of the?tce1?tci1 mutant.These results demonstrate that Yptb T6SS-3 follows a non-canonical contact-independent killing mechanism mediated by secretion of Tce1,a unique antibacterial effector with an intrinsic cell-entry mechanism.4.Tce1 interacts with the outer membrane receptors Btu B and Omp F.We performed GST pull-down assays using GST-Tce1 coated beads against total cell lysates of Yptb WT cells.Mass spectrometric analysis identified several potential Tce1 partners:Btu B,Omp F,Tol B,Omp A and Acn B.The specific interactions of Tce1 with Btu B,Omp F,and Tol B were confirmed based on bacterial two-hybrid and in vitro binding assays.Interactions of Tce1with OM receptors Btu B and Omp F were further confirmed using ITC,the result indicated that Tce1 binds Btu B more tightly than Omp F.5.Tce1 requires Btu B and Omp F for target cell entry.We performed a fluorescence-based assay using Alexa Fluor 488-conjugated Tce1 and GFP-Tce1 protein to probe its import in vivo.WT Yptb and?btu B or?omp F mutants yielded fluorescent bacteria,the?btu B?omp F mutant was not labelled.The Btu B/Omp F-dependent entry of Tce1 into cytosol was further confirmed based on a cell fractionation experiment.Tce1 was detected in the cytosol of WT Yptb but not the?btu B?omp F mutant.Deletion of btu B or omp F reduced the sensitivity of the?tce1?tci1 mutant to exogenously supplied Tce1 protein,while the?tce1?tci1?btu B?omp F quadruple mutant was not sensitive to Tce1 protein.These observations indicate that Btu B or Omp F must be present for Tce1 to enter target cells.And Tol B plays an important role in facilitating Tce1 translocation across the OM.To investigate whether Btu B and Omp F are involved in Tce1-mediated contact-independent T6SS killing,intra-species competition assays were performed in liquid medium,the WT strain strongly inhibited the growth of?tce1?tci1,it failed to inhibit the growth of the?tce1?tci1?btu B?omp F mutant.However,the reduced sensitivity in the?tce1?tci1?btu B?omp F mutant was substantially restored by complementation of omp F or btu B.Similar results were obtained when the assay was repeated with a cell-impermeable membrane separating the donor and recipient cells on the surface of solid medium.We also examined the roles of Btu B and Omp F in Tce1-mediated contact-dependent T6SS killing on the surface of solid medium.The WT Yptb caused stronger inhibition of the growth of the?tce1?tci1?btu B?omp F mutant compared with the?tce1?tci1 mutant.We found that WT Yptb exhibited stronger inhibition of the growth of the E.coli?btu B?omp F mutant compared to WT E.coli during contact-dependent competition.These results demonstrated that although Tce1 mediates both the contact-dependent and-independent T6SS killing pathways,only the contact-independent pathway requires the Btu B and Omp F receptors.The discovery of the contact-independent,receptor-dependent long-range T6SS delivery mechanism provides a new perspective for understanding the physiological roles of T6SS in competition,opens a new avenue for understanding the ecological consequences of T6SSs and may lead to novel microbiota intervention strategies in medical,agricultural,and industrial settings in the future.