T3SS1和T3SS2影响副溶血弧菌生物学特性及细胞致病性的比较
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摘要
【目的】探究两套Ⅲ型分泌系统T3SS1和T3SS2影响副溶血弧菌生物学特性及细胞致病性的差异和相关性。【方法】以T3SS1和T3SS2主要结构基因vcrD1和vcrD2为研究对象,利用同源重组技术分别构建单基因和双基因缺失株ΔvcrD1、ΔvcrD2、ΔvcrD1-vcrD2,以及互补株CΔvcrD1和CΔvcrD2;分析各菌株的生长特性、生物被膜形成能力、运动性的差异;比较各菌株对细胞毒性以及对细胞炎性因子转录水平的影响。【结果】与野生株相比,各缺失株的生长速度无显著差异。缺失株ΔvcrD1生物被膜形成能力、运动性和细胞毒性均极显著下降;缺失株ΔvcrD2主要表现为细胞炎性因子IL-1β和IL-6转录水平的显著上调,同时对细胞毒性作用下降。双基因缺失株ΔvcrD1-vcrD2在缺失株ΔvcrD1的基础上,生物被膜形成能力、运动性、细胞毒性均进一步显著下降,但在细胞炎性因子的转录水平上,则与ΔvcrD1一致,与野生株相比均无显著差异。【结论】T3SS1和T3SS2对副溶血弧菌生物学特性和细胞致病性的影响存在差异。T3SS1主要影响细菌的生物被膜形成、运动性及细胞毒性作用;T3SS2不影响生物被膜形成、运动性等生物学特性,参与细菌对细胞炎性反应中的负调控作用,同时具有一定的细胞毒性作用。T3SS1有助于副溶血弧菌在环境中的生存,而T3SS2可有利于细菌在宿主体内免疫逃避的过程。T3SS1和T3SS2对副溶血弧菌生物学特性和细胞致病性的影响可能存在一定的相关作用,具体机制有待进一步研究。
[Objective] To elucidate differences of biological characteristics and cytopathogenicities between T3SS1 and T3SS2 in Vibrio parahaemolyticus. [Methods] With the main structural protein genes vcrD1 and vcrD2 as the research object, we used the homologous recombination technique to construct a series of deletion mutants ΔvcrD1, ΔvcrD2, ΔvcrD1-vcrD2, and complementary strains CΔvcrD1, CΔvcrD2 of V. parahaemolyticus SH112 strain, then analyzed the differences of growth, biofilm, motility, cytotoxicity to RAW264.7 macrophages and Caco-2 cells, and transcriptional regulation of pro-inflammatory cytokine IL-1β, IL-6 in RAW264.7 macrophages between mutant strain and wild strain. [Results] Compared to the wild strain, the mutant strains had no difference on growth rate, however, the vcrD1 mutant obviously weakened biofilm formation, motility, cytotoxicity of V. parahaemolyticus; the vcrD2 mutant positively upregulated the transcriptional levels of pro-inflammatory cytokines, and significantly attenuated cytotoxicity to cells. The ΔvcrD1-vcrD2 double deletion mutant showed weaker biofilm, motility, cell toxicity than vcrD1 mutant strain, and as similar to the vcrD1 mutant, in the mRNA levels of inflammatory cytokines when compared with the wild strain. [Conclusion] Contributions of two T3SSs to biological characteristics and cytopathogenicities of V. parahaemolyticus are different. The T3SS1 was mainly involved in biofilm formation and motility of V. parahaemolyticus, but also has a significant cytotoxic effect; T3SS2 did not affect bacterial biofilm formation and motility, but played a negative regulation on cell inflammatory reaction, potentially contributing to bacterial immune evasion in host. T3SS1 contributed to bacterial survival in the environment; T3SS2 may help V. parahaemolyticus to evade the host immune response. Moreover, T3SS1 and T3SS2 may play related functions on biological characteristics and cytopathogenicities of V. parahaemolyticus, but the specific mechanism still needs further study.
[Objective] To elucidate differences of biological characteristics and cytopathogenicities between T3SS1 and T3SS2 in Vibrio parahaemolyticus. [Methods] With the main structural protein genes vcrD1 and vcrD2 as the research object, we used the homologous recombination technique to construct a series of deletion mutants ΔvcrD1, ΔvcrD2, ΔvcrD1-vcrD2, and complementary strains CΔvcrD1, CΔvcrD2 of V. parahaemolyticus SH112 strain, then analyzed the differences of growth, biofilm, motility, cytotoxicity to RAW264.7 macrophages and Caco-2 cells, and transcriptional regulation of pro-inflammatory cytokine IL-1β, IL-6 in RAW264.7 macrophages between mutant strain and wild strain. [Results] Compared to the wild strain, the mutant strains had no difference on growth rate, however, the vcrD1 mutant obviously weakened biofilm formation, motility, cytotoxicity of V. parahaemolyticus; the vcrD2 mutant positively upregulated the transcriptional levels of pro-inflammatory cytokines, and significantly attenuated cytotoxicity to cells. The ΔvcrD1-vcrD2 double deletion mutant showed weaker biofilm, motility, cell toxicity than vcrD1 mutant strain, and as similar to the vcrD1 mutant, in the mRNA levels of inflammatory cytokines when compared with the wild strain. [Conclusion] Contributions of two T3SSs to biological characteristics and cytopathogenicities of V. parahaemolyticus are different. The T3SS1 was mainly involved in biofilm formation and motility of V. parahaemolyticus, but also has a significant cytotoxic effect; T3SS2 did not affect bacterial biofilm formation and motility, but played a negative regulation on cell inflammatory reaction, potentially contributing to bacterial immune evasion in host. T3SS1 contributed to bacterial survival in the environment; T3SS2 may help V. parahaemolyticus to evade the host immune response. Moreover, T3SS1 and T3SS2 may play related functions on biological characteristics and cytopathogenicities of V. parahaemolyticus, but the specific mechanism still needs further study.
引文
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[16]Mala W,Alam M,Angkititrakul S,Wongwajana S,Lulitanond V,Huttayananont S,Kaewkes W,Faksri K,Chomvarin C.Serogroup,virulence,and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.Infection,Genetics and Evolution,2016,39:212–218.
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[18]Wang ZF,Guo CM,Xu YN,Liu GJ,Lu CP,Liu YJ.Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression.Infection and Immunity,2014,82(6):2615–2625.
[19]Tagomori K,Iida T,Honda T.Comparison of genome structures of vibrios,bacteria possessing two chromosomes.Journal of Bacteriology,2002,184(16):4351–4358.
[20]Park KS,Ono T,Rokuda M,Jang MH,Jang MH,Okada K,Iida T,Honda T.Functional characterization of two type III secretion systems of Vibrio parahaemolyticus.Infection and Immunity,2004,72(11):6659–6665.
[21]Shafique M,Alvi IA,Abbas Z,Ur Rehman S.Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa.Acta Pathologica,Microbiologica et Iimmuologica Scandinavica,2017,125(6):579–584.
[22]Chaban B,Hughes HV,Beeby M.The flagellum in bacterial pathogens:for motility and a whole lot more.Seminars in Cell&Developmental Biology,2015,46:91–103.
[23]Josenhans C,Suerbaum S.The role of motility as a virulence factor in bacteria.International Journal of Medical Microbiology,2002,291(8):605–614.
[24]Martinez FO,Helming L,Gordon S.Alternative activation of macrophages:an immunologic functional perspective.Annual Review of Immunology,2009,27:451–483.
[25]Zhu Q,Li C,Yu ZX,Zou PF,Meng QX,Yao CL.Molecular and immune response characterizations of IL-6 in large yellow croaker(Larimichthys crocea).Fish&Shellfish Immunology,2016,50:263–273.
[26]Ding XY,Qu LY,Wang C,Tian XX,Sun CJ,Wang MQ.Effect of temperature on T3SS gene expression of Vibrio parahaemolyticus.Advances in Marine Science,2016,34(2):250–259.(in Chinese)丁晓燕,曲凌云,王琛,田欣欣,孙承君,王敏强.温度对副溶血弧菌T3SS中相关基因表达的影响.海洋科学进展,2016,34(2):250–259.
[27]Makino K,Oshima K,Kurokawa K,Yokoyama K,Uda T,Tagomori K,Iijima Y,Najima M,Nakano M,Yamashita A,Kubota Y,Kimura S,Yasunaga T,Honda T,Shinagawa H,Hattori M,Iida T.Genome sequence of Vibrio parahaemolyticus:a pathogenic mechanism distinct from that of V.cholerae.The Lancet,2003,361(9359):743–749.
[2]Wang RZ,Fang S,Wu DL,Lian JW,Fan J,Zhang YF,Wang SH,Lin WX.Screening for a single-chain variable-fragment antibody that can effectively neutralize the cytotoxicity of the Vibrio parahaemolyticus thermolabile hemolysin.Applied and Environment Microbiology,2012,78(14):4967–4975.
[3]Yu Y,Fang LH,Zhang Y,Sheng HX,Fang WH.Vgr G2 of type VI secretion system 2 of Vibrio parahaemolyticus induces autophagy in macrophages.Frontiers in Microbiology,2015,6:168.
[4]Matsuda S,Kodama T,Okada N,Okayama K,Honda T,Iida T.Association of Vibrio parahaemolyticus thermostable direct hemolysin with lipid rafts is essential for cytotoxicity but not hemolytic activity.Infection and Immunity,2010,78(2):603–610.
[5]Takahashi A,Kenjyo N,Imura K,Myonsun Y,Honda T.Cl–secretion in colonic epithelial cells induced by the Vibrio parahaemolyticus hemolytic toxin related to thermostable direct hemolysin.Infection and Immunity,2000,68(9):5435–5438.
[6]Raghunath P.Roles of thermostable direct hemolysin(TDH)and TDH-related hemolysin(TRH)in Vibrio parahaemolyticus.Frontiers in Microbiology,2015,5:805.
[7]Cornelis GR.The type III secretion injectisome.Nature Reviews Microbiology,2006,4(11):811–825.
[8]Ono T,Park KS,Ueta M,Iida T,Honda T.Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system.Infection and Immunity,2006,74(2):1032–1042.
[9]Zhou XH,Konkel ME,Call DR.Type III secretion system 1of Vibrio parahaemolyticus induces oncosis in both epithelial and monocytic cell lines.Microbiology,2009,155(3):837–851.
[10]Bhattacharjee RN,Park KS,Kumagai Y,Okada K,Yamamoto M,Uematsu S,Matsui K,Kumar H,Kawai T,Iida T,Honda T,Takeuchi O,Akira S.VP1686,a Vibrio type III secretion protein,induces toll-like receptor-independent apoptosis in macrophage through NF-κB inhibition.The Journal of Biological Chemistry,2006,281(48):36897–36904.
[11]Kodama T,Hiyoshi H,Okada R,Matsuda S,Gotoh K,Iida T.Regulation of Vibrio parahaemolyticus T3SS2 gene expression and function of T3SS2 effectors that modulate actin cytoskeleton.Cellular Microbiology,2015,17(2):183–190.
[12]Paranjpye R,Hamel OS,Stojanovski A,Liermann M.Genetic diversity of clinical and environmental Vibrio parahaemolyticus strains from the Pacific Northwest.Applied and Environmental Microbiology,2012,78(24):8631–8638.
[13]Ham H,Orth K.The role of type III secretion system 2 in Vibrio parahaemolyticus pathogenicity.Journal of Microbiology,2012,50(5):719–725.
[14]Hiyoshi H,Kodama T,Iida T,Honda T.Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity,enterotoxicity,and lethality in mice.Infection and Immunity,2010,78(4):1772–1780.
[15]Pi?eyro P,Zhou XH,Orfe LH,Friel PJ,Lahmers K,Call DR.Development of two animal models to study the function of Vibrio parahaemolyticus type III secretion systems.Infection and Immunity,2010,78(11):4551–4559.
[16]Mala W,Alam M,Angkititrakul S,Wongwajana S,Lulitanond V,Huttayananont S,Kaewkes W,Faksri K,Chomvarin C.Serogroup,virulence,and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.Infection,Genetics and Evolution,2016,39:212–218.
[17]Noh HJ,Nagami S,Kim MJ,Kim J,Lee NK,Lee KH,Park SJ.Role of vcr D1 protein in expression and secretion of flagellar components in Vibrio parahaemolyticus.Archives of Microbiology,2015,197(3):397–410.
[18]Wang ZF,Guo CM,Xu YN,Liu GJ,Lu CP,Liu YJ.Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression.Infection and Immunity,2014,82(6):2615–2625.
[19]Tagomori K,Iida T,Honda T.Comparison of genome structures of vibrios,bacteria possessing two chromosomes.Journal of Bacteriology,2002,184(16):4351–4358.
[20]Park KS,Ono T,Rokuda M,Jang MH,Jang MH,Okada K,Iida T,Honda T.Functional characterization of two type III secretion systems of Vibrio parahaemolyticus.Infection and Immunity,2004,72(11):6659–6665.
[21]Shafique M,Alvi IA,Abbas Z,Ur Rehman S.Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa.Acta Pathologica,Microbiologica et Iimmuologica Scandinavica,2017,125(6):579–584.
[22]Chaban B,Hughes HV,Beeby M.The flagellum in bacterial pathogens:for motility and a whole lot more.Seminars in Cell&Developmental Biology,2015,46:91–103.
[23]Josenhans C,Suerbaum S.The role of motility as a virulence factor in bacteria.International Journal of Medical Microbiology,2002,291(8):605–614.
[24]Martinez FO,Helming L,Gordon S.Alternative activation of macrophages:an immunologic functional perspective.Annual Review of Immunology,2009,27:451–483.
[25]Zhu Q,Li C,Yu ZX,Zou PF,Meng QX,Yao CL.Molecular and immune response characterizations of IL-6 in large yellow croaker(Larimichthys crocea).Fish&Shellfish Immunology,2016,50:263–273.
[26]Ding XY,Qu LY,Wang C,Tian XX,Sun CJ,Wang MQ.Effect of temperature on T3SS gene expression of Vibrio parahaemolyticus.Advances in Marine Science,2016,34(2):250–259.(in Chinese)丁晓燕,曲凌云,王琛,田欣欣,孙承君,王敏强.温度对副溶血弧菌T3SS中相关基因表达的影响.海洋科学进展,2016,34(2):250–259.
[27]Makino K,Oshima K,Kurokawa K,Yokoyama K,Uda T,Tagomori K,Iijima Y,Najima M,Nakano M,Yamashita A,Kubota Y,Kimura S,Yasunaga T,Honda T,Shinagawa H,Hattori M,Iida T.Genome sequence of Vibrio parahaemolyticus:a pathogenic mechanism distinct from that of V.cholerae.The Lancet,2003,361(9359):743–749.