LIAN Lele,XUE Jiao,LI Wanjun,et al.Functional analysis of the inner rod protein gene vscI in Vibrio parahaemolyticus Type Ⅲ secretion system 1[J].Journal of Nanjing Agricultural University,2021,44(3):506-513.[doi:10.7685/jnau.202003056]





Functional analysis of the inner rod protein gene vscI in Vibrio parahaemolyticus Type Ⅲ secretion system 1
廉乐乐1 薛娇1 李婉君1 任建鸾1 诸葛祥凯1 汤芳1 薛峰1 戴建君12
1. 南京农业大学教育部动物健康与食品安全国际合作联合实验室/动物医学院, 江苏 南京 210095;
2. 中国药科大学, 江苏 南京 211199
LIAN Lele1 XUE Jiao1 LI Wanjun1 REN Jianluan1 ZHUGE Xiangkai1 TANG Fang1 XUE Feng1 DAI Jianjun12
1. Joint Laboratory of Animal Health and Food Safety International Cooperation, Ministry of Educations/College of Veterianry Medicine, Nanjing Agricultural University, Nanjing 210095, China;
2. China Pharmaceutical University, Nanjing 211199, China
Vibrio parahemolyticusT3SS1inner rod proteinvscI
[目的] Ⅲ型分泌系统(Type Ⅲ secretion systems,T3SS)在副溶血弧菌(Vibrio parahemolyticus)致病过程中发挥重要作用。本试验旨在探讨T3SS1内杆状蛋白基因vscI的功能。[方法] 以副溶血弧菌POR-1菌株为研究对象,构建vscI缺失株ΔvscI与回补株CΔvscI,检测野生株、缺失株与回补株的细胞毒性、细胞黏附等生物学特性,分析vscI缺失对副溶血弧菌感染细胞能力的影响;通过Western blot与ELISA检测HeLa细胞内效应蛋白的易位量。[结果] 与POR-1菌株相比,缺失菌株ΔvscI在生长速度上并无显著差异,但对HeLa细胞的黏附能力下降85%。细胞毒性检测结果显示:与POR-1菌株相比,缺失株ΔvscI感染细胞组释放LDH量显著下降60%。同时与野生株POR-1感染HeLa细胞组相比,ΔvscI感染HeLa细胞组发生凋亡的细胞数量显著下降了85%。腺苷酸环化酶报告系统研究结果显示:与POR-1菌株感染组相比,ΔvscI菌株感染HeLa细胞内的cAMP含量显著下降,CyaA融合蛋白的易位量显著下降,而在回补株CΔvscI中上述生物性状均恢复。[结论] vscI提高了副溶血弧菌对细胞的黏附能力和毒力,促进细胞凋亡,同时对于T3SS1效应子的易位是必需的。
[Objectives] The structural components and the biological functions of Vibrio parahemolyticus T3SS1 have not been fully described. In this study we investigated the role of gene vscI. [Methods] To do some research about the vscI,we constructed the deletion strain ΔvscI and complementation strain CΔvscI to analyze the cytotoxicity,adhesion and other biological characteristics of wild strain(POR-1),ΔvscI and CΔvscI when infected HeLa. Further,Western blot analysis and ELISA kits were used to research the translocation of effector protein in Hela cell. [Results] Compared with POR-1 infection group,the results showed that the number of apoptosis decreased significantly by about 80%(P<0.01),the adhesion decreased significantly by 85%(P<0.01),and the cytotoxicity decreased significantly by 60%(P<0.01) in ΔvscI infection group. In addition,Western blot and ELISA data indicated that the intracellular cAMP levels of the ΔvscI group was significantly lower than those of the POR-1,CΔvscI. [Conclusions] vscI improves the adhesion ability of V.parahaemolyticus to cells,and produces cytotoxicity,promotes cell apoptosis,and it is necessary for the translocation of T3SS1 effector.


[1] Daniels N A,Mackinnon L,Bishop R,et al. Vibrio parahaemolyticus infections in the United States,1973-1998[J]. J Infect Dis,2000,181(5):1661-1666.
[2] 付萍,王连森,陈江,等. 2015年中国大陆食源性疾病暴发事件监测资料分析[J]. 中国食品卫生杂志,2019,31(1):64-70. Fu P,Wang L S,Chen J,et al. Analysis of surveillance data of foodborne disease outbreaks in mainland China in 2015[J]. Chinese Journal of Food Hygiene,2019,31(1):64-70(in Chinese with English abstract).
[3] Park K S,Ono T,Rokuda M,et al. Functional characterization of two type Ⅲ secretion systems of Vibrio parahaemolyticus[J]. Infect Immun,2004,72(11):6659-6665.
[4] Cao S Y,Liu W B,Tan Y F,et al. An interaction between the inner rod protein YscI and the needle protein YscF is required to assemble the needle structure of the Yersinia type three secretion system[J]. Journal of Biological Chemistry,2017,292(13):5488-5498.
[5] Sal-Man N,Deng W,Finlay B B. EscI:a crucial component of the type Ⅲ secretion system forms the inner rod structure in enteropathogenic Escherichia coli[J]. Biochem J,2012,442(1):119-125.
[6] Lefebre M D,Galan J E. The inner rod protein controls substrate switching and needle length in a Salmonella type Ⅲ secretion system[J]. Proc Natl Acad Sci USA,2014,111(2):817-822.
[7] Hu M,Zhao W,Li H,et al. Immunization with recombinant Salmonella expressing SspH2-EscI protects mice against wild type Salmonella infection[J]. BMC Vet Res,2018,14(1):79.
[8] Wood S E,Jin J,Lloyd S A. YscP and YscU switch the substrate specificity of the Yersinia type Ⅲ secretion system by regulating export of the inner rod protein YscI[J]. Journal of Bacteriology,2008,190(12):4252-4262.
[9] Shimohata T,Mawatari K,Iba H,et al. VopB1 and VopD1 are essential for translocation of type Ⅲ secretion system 1 effectors of Vibrio parahaemolyticus[J]. Can J Microbiol,2012,58(8):1002-1007.
[10] Sreelatha A,Bennett T L,Zheng H,et al. Vibrio effector protein,VopQ,forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux[J]. Proc Natl Acad Sci USA,2013,110(28):11559-11564.
[11] Sreelatha A,Bennett T L,Carpinone E M,et al. Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes[J]. Proc Natl Acad Sci USA,2015,112(1):100-105.
[12] Yarbrough M L,Li Y,Kinch L N,et al. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling[J]. Science,2009,323(5911):269-272.
[13] Broberg C A,Zhang L,Gonzalez H,et al. A Vibrio effector protein is an inositol phosphatase and disrupts host cell membrane integrity[J]. Science,2010,329(5999):1660-1662.
[14] Waddell B,Southward C M,Mckenna N,et al. Identification of VPA0451 as the specific chaperone for the Vibrio parahaemolyticus chromosome 1 type Ⅲ-secreted effector VPA0450[J]. FEMS Microbiol Lett,2014,353(2):141-150.
[15] Miller V L,Mekalanos J J. A novel suicide vector and its use in construction of insertion mutations-osmoregulation of outer-membrane proteins and virulence determinants in Vibrio-Cholerae requires toxr[J]. Journal of Bacteriology,1988,170(6):2575-2583.
[16] Makino K,Oshima K,Kurokawa K,et al. Genome sequence of Vibrio parahaemolyticus:a pathogenic mechanism distinct from that of V cholerae[J]. Lancet,2003,361(9359):743-749.
[17] Park K S,Ono T,Rokuda M,et al. Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus[J]. Microbiology and Immunology,2004,48(4):313-318.
[18] Kodama T,Akeda Y,Kono G,et al. The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with alpha-catenin[J]. Cellular Microbiology,2002,4(4):213-222.
[19] Ono T,Park K S,Ueta M,et al. Identification of proteins secreted via Vibrio parahaemolyticus type Ⅲ secretion system 1[J]. Infection and Immunity,2006,74(2):1032-1042.
[20] Hueck C J. Type Ⅲ protein secretion systems in bacterial pathogens of animals and plants[J]. Microbiol Mol Biol Rev,1998,62(2):379-433.
[21] Navarre W W,Schneewind O. Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope[J]. Microbiol Mol Biol Rev,1999,63(1):174-229.
[22] Jiang W,Han X,Wang Q,et al. Vibrio parahaemolyticus enolase is an adhesion-related factor that binds plasminogen and functions as a protective antigen[J]. Appl Microbiol Biotechnol,2014,98(11):4937-4948.
[23] Bhattacharjee R N,Park K S,Kumagai Y,et al. VP1686,a Vibrio type Ⅲ secretion protein,induces toll-like receptor-independent apoptosis in macrophage through NF-kappaB inhibition[J]. J Biol Chem,2006,281(48):36897-36904.
[24] Marlovits T C,Kubori T,Lara-Tejero M,et al. Assembly of the inner rod determines needle length in the type Ⅲ secretion injectisome[J]. Nature,2006,441(7093):637-640.
[25] Sory M P,Cornelis G R. Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells[J]. Mol Microbiol,1994,14(3):583-594.


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更新日期/Last Update: 1900-01-01