【作者】 潘君风；

【导师】 王瑶；

【作者基本信息】 西北农林科技大学 ， 生化与分子生物学， 2021， 博士

【摘要】 细菌VI型分泌系统（Type VI Secretion System,T6SS）是一种广泛存在于革兰氏阴性菌中的结构复杂的细菌分泌系统。作为一种重要的接触依赖型细菌竞争武器,一般认为,在环境中相邻的细菌间,攻击者（供体菌）利用T6SS直接刺穿相邻的被攻击者（受体菌）的细胞,从而直接将效应蛋白注射到被攻击者细胞的特定位点,发挥杀菌活性,以获得竞争优势。目前,有关T6SS杀菌功能的研究报道都是接触依赖型的,T6SS是否具有非接触依赖型的杀菌方式是该领域一个未解之谜。假结核耶尔森氏菌（Yersinia pseudotuberculosis）是一种重要的人畜共患致病菌,其基因组中含有4套完整的T6SS,其中第三套T6SS（T6SS-3）基因簇中包含有多对功能未知的效应物-免疫蛋白基因对（E-I,effector-immunity pair）。T6SS-3是否可以分泌具有杀菌活性的效应蛋白,以及T6SS-3是否在假结核耶尔森氏菌细菌间竞争中发挥功能尚不清楚。本研究以假结核耶尔森氏菌T6SS-3分泌的一对功能未知的候选效应物-免疫蛋白对:YPK＿0954-YPK＿0955（本研究将YPK＿0954命名为:Tce1,T6SS contact-independent antibacterial effector 1,而将YPK＿0955命名为:Tci1,T6SS contact-independent antibacterial immunity 1）作为研究对象,利用生物信息学预测分析Tce1的生化功能,采用原核表达系统纯化获得Tce1重组蛋白对其进行酶学活性分析;构建tce1-tci1突变体进行细菌间竞争试验,揭示tce1参与细菌竞争的作用方式;利用Tce1重组蛋白检测其杀菌活性、以及采用GST pull-down检测与Tce1相互作用的受体菌蛋白,以期揭示Tce1参与T6SS细菌竞争作用的分子机制,试验获得了以下结果:1.确定了tce1-tci1是假结核耶尔森氏菌T6SS-3基因簇中一对具有功能的效应物-免疫蛋白对,并且效应蛋白Tce1具有Ca²⁺和Mg²⁺依赖的DNA水解酶活性。分泌检测试验发现Tce1是T6SS-3所特异分泌的效应蛋白,Tce1毒性检测、体外相互作用试验证明tce1-tci1是一对效应物-免疫蛋白基因对。通过对原核表达纯化获得的Tce1重组蛋白进行体外酶活检测,发现效应蛋白Tce1具有Ca²⁺和Mg²⁺依赖的DNA水解酶活性,但不具有RNA水解酶活性。Tce1不仅能够水解线性Lambda DNA分子,而且还可以水解环状质粒DNA分子。异源表达效应蛋白Tce1发现其对大肠杆菌具有毒性,流式细胞术检测及激光共聚焦显微观察发现Tce1在细菌胞内能破坏其DNA的完整性,导致细菌死亡。生物信息学分析、毒性及酶活检测结果说明,Tce1是一个全新的小分子量（67个氨基酸残基组成）DNA水解酶类效应蛋白,Tci1是其特异的免疫蛋白。2.首次发现假结核耶尔森氏菌T6SS-3借助效应蛋白Tce1发挥非接触依赖型的杀菌功能。通过对供体菌（假结核耶尔森氏菌野生型、T6SS-3的功能性突变体Δclp V3及其功能回补菌株、Δtce1菌株）与受体菌（WT野生型菌株、Δtce1Δtci1突变体及其tci1回补菌株）间,在固体培养基表面进行细菌-细菌间紧密接触的生长竞争试验、以及在液体培养基中供体菌与受体菌无紧密接触时的竞争试验结果分析发现,Tce1不但在细菌间紧密接触时发挥了杀菌功能,使得供体菌获得了竞争优势,而且在细菌间无紧密接触时也能发挥杀菌功能;Tce1参与的这种细菌间接触与非接触的生长竞争作用,依赖于T6SS功能的发挥。进一步,在固体培养基表面利用0.22μm滤膜将供体菌与受体菌物理性隔离开进行细菌间竞争试验,结果显示,Tce1仍可借助T6SS的分泌发挥杀菌功能,使得供体菌获得竞争优势。以上结果表明假结核耶尔森氏菌T6SS-3能够利用效应蛋白Tce1发挥接触依赖型与非接触依赖型双模式杀菌功能。重组效应蛋白杀菌试验以及AF-488荧光标记蛋白对靶细胞的结合试验结果均表明,Tce1能够识别并进入到靶细胞内发挥杀菌功能。3.初步揭示了效应蛋白Tce1通过识别并结合受体菌外膜上的受体蛋白Btu B和Omp F进入受体菌细胞的分子进入途径。为了研究Tce1是如何识别并进入到靶细胞内发挥杀菌功能,本研究通过GST-Tce1与假结核耶尔森野生型全细胞裂解液进行pull-down,经质谱检测、体外蛋白互作及细菌双杂交试验,发现了与Tce1相互作用的靶细胞外膜受体分子Btu B和Omp F,以及细胞周质空间蛋白Tol B。通过Tce1的荧光标记蛋白对假结核耶尔森氏菌WT菌株、Δbtu B、Δomp F、Δbtu BΔomp F等细菌突变体及其回补菌株的细胞结合试验,以及重组蛋白对受体突变体的杀菌试验表明,Tce1通过识别并结合受体菌外膜受体Btu B和Omp F,进入胞内发挥杀菌功能。4.靶细胞外膜受体蛋白Btu B和Omp F介导了Tce1非接触依赖的杀菌作用,在T6SS接触依赖型的杀菌作用中Btu B和Omp F不发挥作用。通过对供体菌（假结核耶尔森氏菌WT菌株和Δtce1突变体菌株）与外膜受体btu B和omp F的受体突变体及其基因回补菌株间进行液体培养条件下与固体培养基表面进行细菌竞争试验发现,细菌间非接触竞争作用依赖于靶细胞外膜受体分子Btu B和Omp F发挥作用,而在细菌间紧密接触竞争中则不依赖此类受体分子的参与。本研究首次发现了假结核耶尔森氏菌T6SS-3通过分泌一个新型Ca²⁺和Mg²⁺依赖性的DNA水解酶类效应蛋白Tce1,发挥非接触依赖型杀菌功能的分子机制,拓展了当前对于T6SS杀菌功能的认识,初步揭示了T6SS非接触依赖型杀菌作用中Tce1靶向并进入受体细胞依赖于外膜受体Btu B和Omp F进入细胞的途径,完善了对于效应蛋白靶向受体细胞的机制,为此类效应蛋白在临床耐药菌生物防控、农业病原菌的防治与环境生态治理中的发挥重要作用奠定了理论基础。更多还原

【Abstract】 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 withλ-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 theΔtce1Δtci1recipient.The Tce1-mediated growth advantage requires a functional T6SS-3.The WT donor exhibited a 3-fold growth advantage in competition with theΔtce1Δtci1 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 theΔtce1Δtci1 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 andΔbtu B orΔomp F mutants yielded fluorescent bacteria,theΔbtu BΔomp 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 theΔbtu BΔomp F mutant.Deletion of btu B or omp F reduced the sensitivity of theΔtce1Δtci1 mutant to exogenously supplied Tce1 protein,while theΔtce1Δtci1Δbtu BΔomp 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 ofΔtce1Δtci1,it failed to inhibit the growth of theΔtce1Δtci1Δbtu BΔomp F mutant.However,the reduced sensitivity in theΔtce1Δtci1Δbtu BΔomp 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 theΔtce1Δtci1Δbtu BΔomp F mutant compared with theΔtce1Δtci1 mutant.We found that WT Yptb exhibited stronger inhibition of the growth of the E.coliΔbtu BΔomp 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.更多还原