杀鱼爱德华氏菌1,6-二磷酸果糖醛缩酶的免疫原性及分泌调控
发布时间：2018-11-19 22:20
【摘要】：集约化的高密度海水养殖使养殖动物病害的发生几率增加。杀鱼爱德华氏菌(Edwardsiella piscicida)是水产养殖业中一种重要的病原菌,能感染多种鱼类,引起系统性的出血性败血病和肝、脾、肾及肌肉组织的坏死。为防控水产养殖业病害及减少抗生素使用,开发作用机制明确、安全有效的鱼用疫苗势在必行。课题组前期以E. piscicida EIB202基因组为基础的反向疫苗学亚单位疫苗筛选中,发现1,6-二磷酸果糖醛缩酶(Fructose 1,6-diphosphate aldolase, FBA)免疫斑马鱼后,对病原菌E. piscicida的侵染具有较好的免疫保护力。FBA是糖酵解途径中的一种酶,参与胞内的能量代谢。近些年多种病原菌中的糖酵解酶被鉴定为多功能蛋白,即在胞内行使能量代谢功能的同时,能分泌到胞外参与病原菌毒力相关的功能。虽有报道称,FBA在一些病原中具有一定的免疫原性并能分泌于细胞表面或细胞外参与病原菌的细胞黏附作用,但对于FBA在E. piscicida中的免疫原性、分泌途径与调控及如何参与病原菌的毒力过程等研究还未见报道。针对以上情况,本论文对E. piscicida的FBA进行了免疫原性、分泌途径与调控及毒力相关功能等研究,获得了如下结果：首先,基于多种病原菌的FBA序列分析发现,病原菌中的FBA在其亚分类下相当保守。在非胞内定位分析中,五种常见水产病原菌杀鱼爱德华氏菌、嗜水气单胞菌(Aeromonas hydrophila)、鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(V. harveyi)和溶藻弧菌(V. alginolyticus)的FBA均能分泌到胞外。在对多种病原菌的免疫保护评价中,FBA在斑马鱼上展示了较好的交叉免疫保护效果,相对免疫保护力达45～80%；在经济鱼种大菱鲆动物实验中针对E. piscicida具有68%的相对免疫保护力,经FBA免疫后的大菱鲆血清对抗原蛋白具有显著的特异性反应,且对上述五种海洋病原菌具有交叉特异性反应和显著的杀菌活力。免疫相关基因的上调表达进一步表明,FBA能够有效地激发鱼体的特异性免疫系统。杀鱼爱德华氏菌糖酵解途径中的持家酶FBA具有良好的免疫保护力,可用于广谱型候选疫苗的开发。其次,通过对杀鱼爱德华氏菌多种分泌途径缺失株胞外FBA分泌水平和外膜小泡(OMV)内FBA含量分析,证明T3SS、T4SS、T6SS和Tat分泌途径的缺失并不能阻止FBA的分泌,且OMV也不是FBA的主要分泌方式。在随后的Pull-down及验证实验中,FBA能与Sec途径中分子伴侣SecB相互作用,并有可能通过Sec途径进行转运。基于建立的ELISA筛选方法,在基因覆盖率为75.6%的插入失活突变体文库中,未能筛选到FBA分泌缺失菌株,很大的可能是FBA的分泌与菌体的生长及代谢密切相关。FBA可能不依赖于单一的分泌途径,而是通过复杂的多途径分泌,且其分泌可能是菌体生长必不可缺的。再次,在FBA分泌调控研究中,发现E. piscicida FBA的分泌不需要信号肽,分泌量受到菌体自身调控,在分泌后期处于分泌和降解的动态平衡状态。突变体文库的筛选和验证发现,esrC是调控FBA分泌的重要基因,基因esrC的缺失使FBA分泌量显著上调。胞内转录水平分析发现,随着培养时间的延长,缺失株△esrC中fbaA的表达水平与野生株没有显著性差异,然而secB表达水平相比于野生株呈明显下降趋势。野生株胞内FBA的转录水平与其胞外分泌量呈负相关,即在菌体生长过程中FBA胞外分泌量逐渐增加时,其转录水平反而降低。这些结果表明,FBA的分泌量并不受制于胞内表达水平,且转录调控因子EsrC并不直接调控fbα的表达,而是通过调控与FBA存在相互作用的secB或其他与分泌相关基因的表达来调控FBA的分泌,即EsrC通过未知的间接方式调控FBA的分泌。FBA的分泌调控是复杂的。最后,FBA胞外功能活性的分析发现,分泌到胞外及细菌表面的FBA依然具有糖酵解酶活性,FBA在胞质、周质空间和胞外组分中的结构是一致的,并未因为分泌行使非糖酵解功能而进行较大的结构修饰。随后的细胞侵染实验显示,FBA有助于爱德华氏菌对HeLa细胞的黏附。通过分析爱德华氏菌48株不同毒株FBA的胞外分泌量发现,各个菌株均能分泌FBA,但胞外分泌量与病原菌的毒力强弱并不呈正相关关系。综上所述,本工作不仅为鱼用广谱型疫苗的开发提供了理论依据,也为杀鱼爱德华氏菌糖酵解酶的胞外分泌调控机制研究奠定了一定基础。
[Abstract]:The intensive high-density seawater culture increases the occurrence probability of the disease of the cultured animals. Edwardsiella biscida is an important pathogen in the aquaculture industry, can infect various fishes, cause systemic hemorrhagic septicemia and necrosis of liver, spleen, kidney and muscle tissue. in ord to prevent and control that disease of the aquaculture industry and to reduce the use of the antibiotics, the development action mechanism is clear, and the safe and effective use of the fish vaccine is imperative. The first, 6-diphosphate aldolase (FBA) immunozebrafish was found in the screening of the anti-vaccine subunit vaccine based on the E. Piscida EIB202 genome. FBA is an enzyme in the glycolysis pathway and participates in the energy metabolism in the cell. In recent years, the glycolysis enzyme in a variety of pathogenic bacteria is identified as a multifunctional protein, that is, the function of energy metabolism is exercised in the cell, and the functions related to the virulence of the pathogenic bacteria can be secreted out of the cell. It has been reported that FBA has certain immunogenicity in some pathogens and can be secreted outside the cell surface or cell to participate in the cell adhesion of the pathogenic bacteria, but for the immunogenicity of FBA in E. Piscida, It is not reported that the way of secretion and regulation and how to take part in the virulence of the pathogenic bacteria. In this paper, the results of the study on the immunogenicity, the way of secretion and the related functions of the FBA of E. biscida were studied. The results showed that the FBA in the pathogenic bacteria was very conservative under the subcategory. In the non-intracellular localization analysis, the FBA of five common aquatic pathogens, such as Edwardsiella tarda, Aeromonas hydrophila, Vibrio anguillarium, Vibrio harveyi and V. alginolyticus, can be secreted out of the cell. In the evaluation of the immune protection of a plurality of pathogenic bacteria, the FBA has a good cross-immune protection effect on the zebrafish, and the relative immunization protection force is 45-80 percent; and the relative immune protection force of the E. biscida is 68 percent in the experiment of the large rhomborous animal of the economic species. The antigen protein has a remarkable specific reaction to the antigen protein after the immunization of the FBA, and has the cross-specific reaction and the remarkable bactericidal activity to the five marine pathogenic bacteria. The up-regulation of the immune-related genes further indicates that the FBA can effectively stimulate the specific immune system of the fish body. The enzyme-holding enzyme (FBA) in the glycolysis pathway of the Awardsiella tarda has a good immune protection force and can be used for the development of a broad-spectrum candidate vaccine. Secondly, by analyzing the FBA level and the content of FBA in the outer membrane vesicles (OMV), the loss of the secretion pathway of T3SS, T4SS, T6SS and Tat can not be prevented and the secretion of FBA can not be prevented, and the OMV is not the main secretory mode of FBA. In the subsequent Pull-down and validation experiments, the FBA can interact with the molecular chaperone SecB in the Sec pathway and may be transported through the Sec pathway. Based on the established ELISA screening method, the gene coverage was 75.6% of the inserted lost-activity mutant library, and the FBA secretion deletion strain could not be selected, and it was possible that the secretion of FBA was closely related to the growth and metabolism of the cells. FBA may not be dependent on a single secretory pathway, but is secreted by a complex, multi-pathway, and its secretion may be deficient in the growth of thalli. In the study of the secretion and control of FBA, it was found that the secretion of E. biscida FBA did not require a signal peptide, and the amount of secretion was regulated by the thalli itself, and it was in the state of the dynamic balance of secretion and degradation in the later stage of the secretion. The screening and validation of the mutant library found that esrC was an important gene for regulating the secretion of FBA, and the deletion of the gene esrC increased the secretion of FBA. The analysis of intracellular transcription level showed that, with the increase of the culture time, the expression level of fbaA in the deleted strain was not significantly different from that of the wild strain, but the expression level of the secB was significantly lower than that of the wild strain. The transcription level of FBA in the cell of the wild strain was negatively correlated with its extracellular secretion, that is, the level of the transcription of the FBA in the growth of the cell was gradually increased. These results show that the secretion of FBA is not subject to the intracellular level of expression, and the transcription regulatory factor EsrC does not directly regulate the expression of fb, but regulates the secretion of FBA by regulating the expression of the secB or other related genes that interact with FBA, That is, the EsrC regulates the secretion of the FBA in an unknown indirect manner. The secretion regulation of FBA is complicated. Finally, the analysis of the extracellular functional activity of the FBA found that the FBA secreted into the extracellular and bacterial surface still has glycolytic activity, and the structure of the FBA in the cytoplasm, periplasmic space and the extracellular component is consistent, and does not perform a large structural modification due to the secretion of the non-glycolysis function. The subsequent cell infection experiments show that the FBA is useful for the adhesion of the Edwardsiella tarda to the HeLa cells. By analyzing the extracellular secretion of different strains of FBA in 48 strains of Edwardsiella tarda, it was found that each strain can secrete FBA, but the amount of extracellular secretion and the virulence of the pathogenic bacteria are not in positive correlation. To sum up, this work not only provides a theoretical basis for the development of broad-spectrum vaccine for fish, but also lays a foundation for the study of the mechanism of extracellular secretion regulation and regulation of the glycolysis enzyme of Edwardsiella tarda.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S941.4
，
本文编号：2343605
[Abstract]:The intensive high-density seawater culture increases the occurrence probability of the disease of the cultured animals. Edwardsiella biscida is an important pathogen in the aquaculture industry, can infect various fishes, cause systemic hemorrhagic septicemia and necrosis of liver, spleen, kidney and muscle tissue. in ord to prevent and control that disease of the aquaculture industry and to reduce the use of the antibiotics, the development action mechanism is clear, and the safe and effective use of the fish vaccine is imperative. The first, 6-diphosphate aldolase (FBA) immunozebrafish was found in the screening of the anti-vaccine subunit vaccine based on the E. Piscida EIB202 genome. FBA is an enzyme in the glycolysis pathway and participates in the energy metabolism in the cell. In recent years, the glycolysis enzyme in a variety of pathogenic bacteria is identified as a multifunctional protein, that is, the function of energy metabolism is exercised in the cell, and the functions related to the virulence of the pathogenic bacteria can be secreted out of the cell. It has been reported that FBA has certain immunogenicity in some pathogens and can be secreted outside the cell surface or cell to participate in the cell adhesion of the pathogenic bacteria, but for the immunogenicity of FBA in E. Piscida, It is not reported that the way of secretion and regulation and how to take part in the virulence of the pathogenic bacteria. In this paper, the results of the study on the immunogenicity, the way of secretion and the related functions of the FBA of E. biscida were studied. The results showed that the FBA in the pathogenic bacteria was very conservative under the subcategory. In the non-intracellular localization analysis, the FBA of five common aquatic pathogens, such as Edwardsiella tarda, Aeromonas hydrophila, Vibrio anguillarium, Vibrio harveyi and V. alginolyticus, can be secreted out of the cell. In the evaluation of the immune protection of a plurality of pathogenic bacteria, the FBA has a good cross-immune protection effect on the zebrafish, and the relative immunization protection force is 45-80 percent; and the relative immune protection force of the E. biscida is 68 percent in the experiment of the large rhomborous animal of the economic species. The antigen protein has a remarkable specific reaction to the antigen protein after the immunization of the FBA, and has the cross-specific reaction and the remarkable bactericidal activity to the five marine pathogenic bacteria. The up-regulation of the immune-related genes further indicates that the FBA can effectively stimulate the specific immune system of the fish body. The enzyme-holding enzyme (FBA) in the glycolysis pathway of the Awardsiella tarda has a good immune protection force and can be used for the development of a broad-spectrum candidate vaccine. Secondly, by analyzing the FBA level and the content of FBA in the outer membrane vesicles (OMV), the loss of the secretion pathway of T3SS, T4SS, T6SS and Tat can not be prevented and the secretion of FBA can not be prevented, and the OMV is not the main secretory mode of FBA. In the subsequent Pull-down and validation experiments, the FBA can interact with the molecular chaperone SecB in the Sec pathway and may be transported through the Sec pathway. Based on the established ELISA screening method, the gene coverage was 75.6% of the inserted lost-activity mutant library, and the FBA secretion deletion strain could not be selected, and it was possible that the secretion of FBA was closely related to the growth and metabolism of the cells. FBA may not be dependent on a single secretory pathway, but is secreted by a complex, multi-pathway, and its secretion may be deficient in the growth of thalli. In the study of the secretion and control of FBA, it was found that the secretion of E. biscida FBA did not require a signal peptide, and the amount of secretion was regulated by the thalli itself, and it was in the state of the dynamic balance of secretion and degradation in the later stage of the secretion. The screening and validation of the mutant library found that esrC was an important gene for regulating the secretion of FBA, and the deletion of the gene esrC increased the secretion of FBA. The analysis of intracellular transcription level showed that, with the increase of the culture time, the expression level of fbaA in the deleted strain was not significantly different from that of the wild strain, but the expression level of the secB was significantly lower than that of the wild strain. The transcription level of FBA in the cell of the wild strain was negatively correlated with its extracellular secretion, that is, the level of the transcription of the FBA in the growth of the cell was gradually increased. These results show that the secretion of FBA is not subject to the intracellular level of expression, and the transcription regulatory factor EsrC does not directly regulate the expression of fb, but regulates the secretion of FBA by regulating the expression of the secB or other related genes that interact with FBA, That is, the EsrC regulates the secretion of the FBA in an unknown indirect manner. The secretion regulation of FBA is complicated. Finally, the analysis of the extracellular functional activity of the FBA found that the FBA secreted into the extracellular and bacterial surface still has glycolytic activity, and the structure of the FBA in the cytoplasm, periplasmic space and the extracellular component is consistent, and does not perform a large structural modification due to the secretion of the non-glycolysis function. The subsequent cell infection experiments show that the FBA is useful for the adhesion of the Edwardsiella tarda to the HeLa cells. By analyzing the extracellular secretion of different strains of FBA in 48 strains of Edwardsiella tarda, it was found that each strain can secrete FBA, but the amount of extracellular secretion and the virulence of the pathogenic bacteria are not in positive correlation. To sum up, this work not only provides a theoretical basis for the development of broad-spectrum vaccine for fish, but also lays a foundation for the study of the mechanism of extracellular secretion regulation and regulation of the glycolysis enzyme of Edwardsiella tarda.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S941.4
，
本文编号：2343605
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