Study On The New Mechanism Of Bifunctional Effector Tce2 In Yersinia Pseudotuberculosis Participating In Bacterial Competition

Type Ⅵ secretion system（T6SS）,a nano-transmembrane secretion system,is widely distributed in Gram-negative bacteria,which injects effectors into extracellular space and establishes the relationship between bacteria and bacteria,bacteria and environment,bacteria and eukaryotes.Yersinia pseudotuberculosis has four sets of T6SS（T6SS-1-T6SS-4）.Previous studies have shown that T6SS-1 is highly expressed at 37°C and is regulated by ferric uptake regulator（Fur）negatively,which suggests us that T6SS-1 may participate in nutrient acquisition and impact on host healthy.However,the specific functions and mechanisms of T6SS-1 and its corresponding effector are still poorly understood.In this study,we investigated the function and mechanism of T6SS-1 and its effector Tce2.The results are as follows:1.Firstly,Tce2（encoded by ypk＿0405）was identified as the effector of T6SS-1 by secretion assay.We focused on T6SS-1 which can be regulated by Fur negatively,and found that Tce2 was up-regulated significantly inΔfur by q RT-PCR.Then,Tce2 was found as a Fe³⁺binding protein using isothermal titration calorimetry（ITC）,atomic absorption assay and iron staining experiments.It was confirmed that Tce2 was involved in Fe³⁺transport by performing growth curve experiment and inductively coupled plasma mass spectrometry（ICP-MS）.In addition,it is clarified that Tce2 mediates nutrient competition in inter-bacteria using contact-independent competition assay.2.To find out if Tce2 has any other unknown feature,Clustal W and Espript were used to analyze,we found that Tce2 was identified as rearrangement hotspot（Rhs）.Subsequently,Tce2 was purified to perform DNase activity assay and was identified as a kind of non-specific DNase.Meanwhile,after expressing Tce2 in Escherichia coli,random mutation technology and GST-pull down experiment,Tce2 was verified as a toxin effector,and Tci2（encoded by ypk＿0406）as its immune protein.The toxic active site of Tce2 is the 17th glutamic acid residue.It was clarified that Tce2 can disrupt the genome of E.coli using flow cytometry technique and confocal.Next,the competition experiments were performed to confirm that Tce2mediates both contact-dependent and contact-independent competition.3.To explore the mechanism by which Tce2 plays the role in ion transport and contact-independent competition,GST-pull down was performed and the bacterial outer membrane receptor Tds R was screened through.The interaction between Tds R and Tce2 was verified by bacterial two-hybrid assay.It is clarified that Tds R is involved in ion transport using ICP-MS.Moreover,Tds R mediates contact-independent competition in intra-bacterial and inter-bacterial using Tce2 toxicity experiments,contact-independent competition experiments,and confocal technology based on Tce2-GFP and Tce2-AF488 labeling bacterial strains WT、Δtds R andΔtds R（tds R）.Subsequently,homology comparison analysis was performed to find the outer membrane receptor Tds R_EC in E.coli,which mediates the entry of Tce2.4.The functions of ion transport and bactericidal for Tce2 share the same receptor,which suggests us that they might have some relationship.It is verified that the efficiency of Tce2entering bacterial cells can be promoted by Fe³⁺by GST-pull down assay,the entry of Tce2experiments labeled by AF488,contact-independent competition experiments and Tce2toxicity assay in iron-free and iron-containing buffers.Thus,we propose a new model of bacterial competition:Tce2 with Fe³⁺masquerades as a"Trojan horse"to lure bacteria into the cell,and then acts as DNase to kill them.5.In order to explore whether the new model mentioned above is also applicable in physiological environment,the colonization of Yersinia pseudotuberculosis WT,Δtce2,andΔclp V1 was detected in cecum of antibiotic pretreated or untreated mice,respectively.As well as examining the gut microbiome of cecal contents using 16S r RNA sequencing technology,T6SS-1 and Tce2 were performed to mediate Yersinia pseudotuberculosis colonization by influencing a variety of intestinal flora.Subsequently,the competition experiments between Yersinia pseudotuberculosis and other bacteria（Escherichia coli,Salmonella,Klebsiella,Staphylococcus aureus and Enterococcus faecalis）were performed in cecum and environment,which suggested that the participation of Tce2 in bacterial competition was extensive.6.To explore the potential value of Tce2 in advocating the treatment of drug-resistant pathogens,Tce2 was expressed in Bacillus subtilis.Mice pre-infected with different pathogens（Escherichia coli,Salmonella,and Klebsiella）were orally gavaged with PBS buffer,Bacillus expressing empty vector or Tce2 respectively.And then CFU of intestinal pathogenic bacteria at different time points were detected.It is clarified that Tce2 can reduce intestinal pathogenic bacteria effectively,which provides a new idea for treatment of highly resistant bacteria.Summary:This study found that the T6SS-1 effector Tce2 of Yersinia pseudotuberculosis is a bifunctional protein which can transport Fe³⁺and hydrolyze DNA,and participate in nutrient competition and killing competition in a"Trojan horse"manner.Because of its universality receptor in bacteria,it can be used as one of the means to treat drug-resistant intestinal bacteria.