迟钝爱德华氏菌双精氨酸转运系统的功能
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摘要
迟钝爱德华氏菌(1Edwardsiella tarda)是一种革兰氏阴性、短杆状细菌,可感染包括人在内的多种宿主。它是鱼类爱德华氏菌病的病原菌,可引起经济鱼类如鲆鲽类的系统性出血性败血症,给世界范围的水产养殖业造成了巨大的经济损失和危害。分泌系统作为一个重要的毒力因子分泌途径,在细菌的致病性方面发挥重要作用,其中双精氨酸分泌(Tat)系统广泛存在于嗜盐古菌和病原菌中,以适应极端环境和分泌毒力因子。它可运输折叠好的蛋白到达周质空间。在迟钝爱德华氏菌EIB202基因组测序工作完成的基础之上,发现它存在Tat系统。由于它具有广耐盐性,甚至可在5%NaCl中生长,其Tat系统也备受关注,目前还没有关于迟钝爱德华氏菌Tat系统功能的报道。
E. tarda EIB202中Tat系统主要由5个组分TatA、TatB、TatC、TatD(?)口TatE组成,且tatABCD组成一个操纵子结构。实验室前期工作中从不同地区、不同物种中分离、搜集了19株迟钝爱德华氏菌和2株鲶鱼爱德华氏菌,发现tatABCD广泛存在于所研究的爱德华氏菌株中,而tatE则仅存在于强毒株中。同时利用生理生化和分子生物学的手段对21株细菌基因水平的差异性和多态性以及毒力因子的分布进行了研究。这就为研究爱德华氏菌致病性和开发针对爱德华氏菌病的疫苗具有重要的参考价值。
构建了Tat系统组分元件的无标记框内缺失突变株△tatA、△tatB、△tatC、△totD、△tatE、△tatAE、△tatABCD(?)口回补株,分析了它们对E. tarda EIB202多种生理功能的影响。Tat缺失株对该菌生长、形态和生物被膜形成几乎无影响。除△tatD和△tatE外,缺失株△tatA、△tatB、△tatC、△tatAAtatE和(?)△tatABCD均不能产H2S。△tatABCD在软平板上的泳动性减弱,对兔红血细胞凝集能力下降,在DMEM和TSB培养基中的细菌自聚集能力受损,对羊血细胞的溶血活性下调,对包括高温、高盐、SDS、乙醇和抗生素等压力应激更敏感,对EPC细胞和MDCK细胞黏附能力减小,在J774a巨噬细胞内寄生能力下降,对鱼模型毒力稍微减弱。
为了进一步阐明Tat系统在生理适应方面的作用机制,利用双向蛋白电泳技术结合基质辅助激光解吸附电离-飞行时间串联质谱技术对EIB202和(?)△tatABCD的全菌细胞裂解蛋白、周质空间蛋白和胞外蛋白进行了比较蛋白质组的研究。采用定性shotgun蛋白组学策略对胞外蛋白组分进行了鉴定。筛查到了与毒力或压力应激相关的蛋白EvpF、FklB、FtsZ、ClpB、GrpE、PstS、ETAE_2022、DsbA、GlmU和GapA等。实验发现,鉴定的这些蛋白大多数没有典型的Tat系统信号肽,且多与基础代谢和大分子生物合成相关,这为研究E. tarda Tat系统的分子作用机制提供了参考。
利用平板计数法比较EIB202和(?)△tatABCD的盐度敏感性,发现后者对盐度更敏感,提示可将△tatABCD用于开发盐度敏感型海水鱼类减毒活疫苗的生物安全限制系统。在E. tarda EIB202缺失tatABCD的基础上,对其TTSS相关毒力因子和内源性大质粒pEIB202进行敲除,构建了一株减毒活疫苗候选株WYM1,此候选疫苗株用于腹腔注射大菱鲆时表现出高度的减毒效果和抵抗野生毒株EIB202感染的免疫保护作用,此候选株一旦被释放到海水环境中能被迅速地清除,提高了疫苗的环境和生物安全性。
基于Tat系统运输折叠好的蛋白从而提高蛋白稳定性的特征,在实验室已经构建好的减毒活疫苗候选株E. tarda WED基础上,基于不同信号肽介导GFP分泌的检测结果筛选到了高效分泌表达GFP的信号肽,进一步利用这些信号肽介导鱼类病原菌嗜水气单胞菌(Aeromonas hydrophila) LSA34保护性抗原3-磷酸甘油醛脱氢酶(GapA)的分泌,Western blotting检测得到了3个可高效分泌抗原的菌株NapA-GA、Sufl-GA和DmsA-GA。对大菱鲆的相对免疫保护率结果显示,DmsA-GA。抵抗E. tarda(?)(?)A.hydrophila联合感染或单独感染时具有较佳的相对免疫保护率,为其在多效价载体活疫苗开发中的应用提供了新思路。
Edwardsiella tarda, a Gram-negative rod-shaped bacterium, infects a variety of host including human being. It is the aetiological agent of edwardsiellosis and responsible for systemic hemorrhagic septicemia in economic fish species such as turbot and flounder, leading to enormous economic losses in aquaculture worldwidely. Serection systems are competent for translocation of virulence fator and play a vital role in pathogenicity in bacteria. In addition, twin-arginine translocation (Tat) system is widely distributed in halophilic archaea and bacterial pathogens serving for extreme environments adaption and virulence factor secretion. It can translocate prefolded proteins into periplasm. Based on the completed work of E. tarda EIB202genomic sequencing, Tat system was detected on the genome of E. tarda EIB202. E. tarda Tat system gains more attention because of its adaptation to high-salt conditions (up to5%). Meanwhile, there is no report on E. tarda Tat system so far.
Tat system was composed by TatA, TatB, TatC, TatD, and TatE in E. tarda EIB202, and tatABCD was in one operon. In previous work, our lab has isolated or collected19E. tarda isolates and2E. ictaluri isolates from different area and host species. tatABCD was found to widely distribute in these tested Edwardsiella strains, while tatE was conserved in pathogenic strains. Meanwhile, the genetic relationship, polymorphism, and distribution of virulence genes of the21Edwardsiella strains were invesgated with biochemical characterization and molecular method. This will be helpful in the investigation of pathogenicity in Edwardsiella and the development of vaccines for edwardsiellosis.
Tat system component null in-frame deletion strains AtatA, AtatB, AtatC, AtatD, AtatE, AtatAE, AtatABCD, and complement strains were constructed, and their roles in pleiotropically physiological fitness were analyzed in EIB202. Tat mutant displayed no difference with EIB202on growth, morphology, and biofilm formation. Except for AtatD and AtatE, five mutants of AtatA, AtatB, AtatC, AtatAAtatE, and AtatABCD displayed deficient on H2S production. AtatABCD was defective in a serious of functions, including the mobility on soft agar, the haemagglutination against rabbit erythrocyte, the autoaggregation in DMEM and LB culture, the hemolytic activity against sheep erythrocyte, the stress response such as high temperature, high-salt condition, SDS, ethanol. and antibiotics, the adherence to EPC and MDCK, the intracellular survival in J774a, and the marginal in vivo virulence attenuation in fish models.
To further elucidate the underlining molecular mechanism for Tat system on physiological fitness, comparative proteomics analysis of whole cell lysated protein, periplasmic protein, and extracellular protein were implemented using two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry to identify proteins undergoing changes in expression levels when the Tat system was mutilated. In addition, a qualitative shotgun protein sequencing method was used for extracellular protein identification. The main abundance-different proteins were virulence-and stress-associated factors such as EvpF, FklB, FtsZ, ClpB, GrpE, PstS, ETAE_2022, DsbA, GlmU, and GapA. Interestingly, most of the identified proteins held non-classical Tat signal peptide and was associated with basal metabolism and biosynthesis of macromolecules. These would shed light on new mechanisms of the Tat system in E. tarda.
The results of plate counting confirmed that the Tat mutant was high-salt-sensitive than EIB202, indicating that Tat mutant had merits as a novel salt-sensitive biological containment system for live attenuated vaccine in marine fish vaccinology. To test this, we deleted virulence-associated TTSS genes and cured endogenous plasmid pEIB202to construct strain WYM1in the context of Tat abrogation in E. tarda. The results indicated that WYM1was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda EIB202in turbot (Scophthalmus maximus) while being rapidly eliminated in seawater. Therefore, the environmental biosafety of live attenuated vaccine was improved.
The Tat system translocated prefolded protein which would allow stabilization and prevent aggregation. Based on the live attenuated vaccine E. tarda WED, the Tat signal peptides mediated green fluorescent protein (GFP) secretion was analyzed with Western blotting. The signal peptides with optimized GFP translocation was further linked to gapA34, encoding a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from fish pathogen Aeromonas hydrophila LSA34. Western blotting analysis illustrated that DmsA-GA, NapA-GA, and Sufl-GA displayed an optimal efficiency for translocation of GapA. The relative percent survival of the three constructive strains on turbot was assayed and DmsA-GA showed optimized relative protection rate (RPS) against E. tarda and A. hydrophila LSA34co-infection or single infection. Therefore, this study demonstrated a new approach for multivalent attenuated carrier live vaccine development.
E. tarda EIB202中Tat系统主要由5个组分TatA、TatB、TatC、TatD(?)口TatE组成,且tatABCD组成一个操纵子结构。实验室前期工作中从不同地区、不同物种中分离、搜集了19株迟钝爱德华氏菌和2株鲶鱼爱德华氏菌,发现tatABCD广泛存在于所研究的爱德华氏菌株中,而tatE则仅存在于强毒株中。同时利用生理生化和分子生物学的手段对21株细菌基因水平的差异性和多态性以及毒力因子的分布进行了研究。这就为研究爱德华氏菌致病性和开发针对爱德华氏菌病的疫苗具有重要的参考价值。
构建了Tat系统组分元件的无标记框内缺失突变株△tatA、△tatB、△tatC、△totD、△tatE、△tatAE、△tatABCD(?)口回补株,分析了它们对E. tarda EIB202多种生理功能的影响。Tat缺失株对该菌生长、形态和生物被膜形成几乎无影响。除△tatD和△tatE外,缺失株△tatA、△tatB、△tatC、△tatAAtatE和(?)△tatABCD均不能产H2S。△tatABCD在软平板上的泳动性减弱,对兔红血细胞凝集能力下降,在DMEM和TSB培养基中的细菌自聚集能力受损,对羊血细胞的溶血活性下调,对包括高温、高盐、SDS、乙醇和抗生素等压力应激更敏感,对EPC细胞和MDCK细胞黏附能力减小,在J774a巨噬细胞内寄生能力下降,对鱼模型毒力稍微减弱。
为了进一步阐明Tat系统在生理适应方面的作用机制,利用双向蛋白电泳技术结合基质辅助激光解吸附电离-飞行时间串联质谱技术对EIB202和(?)△tatABCD的全菌细胞裂解蛋白、周质空间蛋白和胞外蛋白进行了比较蛋白质组的研究。采用定性shotgun蛋白组学策略对胞外蛋白组分进行了鉴定。筛查到了与毒力或压力应激相关的蛋白EvpF、FklB、FtsZ、ClpB、GrpE、PstS、ETAE_2022、DsbA、GlmU和GapA等。实验发现,鉴定的这些蛋白大多数没有典型的Tat系统信号肽,且多与基础代谢和大分子生物合成相关,这为研究E. tarda Tat系统的分子作用机制提供了参考。
利用平板计数法比较EIB202和(?)△tatABCD的盐度敏感性,发现后者对盐度更敏感,提示可将△tatABCD用于开发盐度敏感型海水鱼类减毒活疫苗的生物安全限制系统。在E. tarda EIB202缺失tatABCD的基础上,对其TTSS相关毒力因子和内源性大质粒pEIB202进行敲除,构建了一株减毒活疫苗候选株WYM1,此候选疫苗株用于腹腔注射大菱鲆时表现出高度的减毒效果和抵抗野生毒株EIB202感染的免疫保护作用,此候选株一旦被释放到海水环境中能被迅速地清除,提高了疫苗的环境和生物安全性。
基于Tat系统运输折叠好的蛋白从而提高蛋白稳定性的特征,在实验室已经构建好的减毒活疫苗候选株E. tarda WED基础上,基于不同信号肽介导GFP分泌的检测结果筛选到了高效分泌表达GFP的信号肽,进一步利用这些信号肽介导鱼类病原菌嗜水气单胞菌(Aeromonas hydrophila) LSA34保护性抗原3-磷酸甘油醛脱氢酶(GapA)的分泌,Western blotting检测得到了3个可高效分泌抗原的菌株NapA-GA、Sufl-GA和DmsA-GA。对大菱鲆的相对免疫保护率结果显示,DmsA-GA。抵抗E. tarda(?)(?)A.hydrophila联合感染或单独感染时具有较佳的相对免疫保护率,为其在多效价载体活疫苗开发中的应用提供了新思路。
Edwardsiella tarda, a Gram-negative rod-shaped bacterium, infects a variety of host including human being. It is the aetiological agent of edwardsiellosis and responsible for systemic hemorrhagic septicemia in economic fish species such as turbot and flounder, leading to enormous economic losses in aquaculture worldwidely. Serection systems are competent for translocation of virulence fator and play a vital role in pathogenicity in bacteria. In addition, twin-arginine translocation (Tat) system is widely distributed in halophilic archaea and bacterial pathogens serving for extreme environments adaption and virulence factor secretion. It can translocate prefolded proteins into periplasm. Based on the completed work of E. tarda EIB202genomic sequencing, Tat system was detected on the genome of E. tarda EIB202. E. tarda Tat system gains more attention because of its adaptation to high-salt conditions (up to5%). Meanwhile, there is no report on E. tarda Tat system so far.
Tat system was composed by TatA, TatB, TatC, TatD, and TatE in E. tarda EIB202, and tatABCD was in one operon. In previous work, our lab has isolated or collected19E. tarda isolates and2E. ictaluri isolates from different area and host species. tatABCD was found to widely distribute in these tested Edwardsiella strains, while tatE was conserved in pathogenic strains. Meanwhile, the genetic relationship, polymorphism, and distribution of virulence genes of the21Edwardsiella strains were invesgated with biochemical characterization and molecular method. This will be helpful in the investigation of pathogenicity in Edwardsiella and the development of vaccines for edwardsiellosis.
Tat system component null in-frame deletion strains AtatA, AtatB, AtatC, AtatD, AtatE, AtatAE, AtatABCD, and complement strains were constructed, and their roles in pleiotropically physiological fitness were analyzed in EIB202. Tat mutant displayed no difference with EIB202on growth, morphology, and biofilm formation. Except for AtatD and AtatE, five mutants of AtatA, AtatB, AtatC, AtatAAtatE, and AtatABCD displayed deficient on H2S production. AtatABCD was defective in a serious of functions, including the mobility on soft agar, the haemagglutination against rabbit erythrocyte, the autoaggregation in DMEM and LB culture, the hemolytic activity against sheep erythrocyte, the stress response such as high temperature, high-salt condition, SDS, ethanol. and antibiotics, the adherence to EPC and MDCK, the intracellular survival in J774a, and the marginal in vivo virulence attenuation in fish models.
To further elucidate the underlining molecular mechanism for Tat system on physiological fitness, comparative proteomics analysis of whole cell lysated protein, periplasmic protein, and extracellular protein were implemented using two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry to identify proteins undergoing changes in expression levels when the Tat system was mutilated. In addition, a qualitative shotgun protein sequencing method was used for extracellular protein identification. The main abundance-different proteins were virulence-and stress-associated factors such as EvpF, FklB, FtsZ, ClpB, GrpE, PstS, ETAE_2022, DsbA, GlmU, and GapA. Interestingly, most of the identified proteins held non-classical Tat signal peptide and was associated with basal metabolism and biosynthesis of macromolecules. These would shed light on new mechanisms of the Tat system in E. tarda.
The results of plate counting confirmed that the Tat mutant was high-salt-sensitive than EIB202, indicating that Tat mutant had merits as a novel salt-sensitive biological containment system for live attenuated vaccine in marine fish vaccinology. To test this, we deleted virulence-associated TTSS genes and cured endogenous plasmid pEIB202to construct strain WYM1in the context of Tat abrogation in E. tarda. The results indicated that WYM1was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda EIB202in turbot (Scophthalmus maximus) while being rapidly eliminated in seawater. Therefore, the environmental biosafety of live attenuated vaccine was improved.
The Tat system translocated prefolded protein which would allow stabilization and prevent aggregation. Based on the live attenuated vaccine E. tarda WED, the Tat signal peptides mediated green fluorescent protein (GFP) secretion was analyzed with Western blotting. The signal peptides with optimized GFP translocation was further linked to gapA34, encoding a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from fish pathogen Aeromonas hydrophila LSA34. Western blotting analysis illustrated that DmsA-GA, NapA-GA, and Sufl-GA displayed an optimal efficiency for translocation of GapA. The relative percent survival of the three constructive strains on turbot was assayed and DmsA-GA showed optimized relative protection rate (RPS) against E. tarda and A. hydrophila LSA34co-infection or single infection. Therefore, this study demonstrated a new approach for multivalent attenuated carrier live vaccine development.
引文
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