殺魚愛德華氏菌1,6-二磷酸果糖醛縮酶的免疫原性及分泌調(diào)控
發(fā)布時間：2018-11-19 22:20
【摘要】：集約化的高密度海水養(yǎng)殖使養(yǎng)殖動物病害的發(fā)生幾率增加。殺魚愛德華氏菌(Edwardsiella piscicida)是水產(chǎn)養(yǎng)殖業(yè)中一種重要的病原菌,能感染多種魚類,引起系統(tǒng)性的出血性敗血病和肝、脾、腎及肌肉組織的壞死。為防控水產(chǎn)養(yǎng)殖業(yè)病害及減少抗生素使用,開發(fā)作用機制明確、安全有效的魚用疫苗勢在必行。課題組前期以E. piscicida EIB202基因組為基礎的反向疫苗學亞單位疫苗篩選中,發(fā)現(xiàn)1,6-二磷酸果糖醛縮酶(Fructose 1,6-diphosphate aldolase, FBA)免疫斑馬魚后,對病原菌E. piscicida的侵染具有較好的免疫保護力。FBA是糖酵解途徑中的一種酶,參與胞內(nèi)的能量代謝。近些年多種病原菌中的糖酵解酶被鑒定為多功能蛋白,即在胞內(nèi)行使能量代謝功能的同時,能分泌到胞外參與病原菌毒力相關的功能。雖有報道稱,FBA在一些病原中具有一定的免疫原性并能分泌于細胞表面或細胞外參與病原菌的細胞黏附作用,但對于FBA在E. piscicida中的免疫原性、分泌途徑與調(diào)控及如何參與病原菌的毒力過程等研究還未見報道。針對以上情況,本論文對E. piscicida的FBA進行了免疫原性、分泌途徑與調(diào)控及毒力相關功能等研究,獲得了如下結(jié)果：首先,基于多種病原菌的FBA序列分析發(fā)現(xiàn),病原菌中的FBA在其亞分類下相當保守。在非胞內(nèi)定位分析中,五種常見水產(chǎn)病原菌殺魚愛德華氏菌、嗜水氣單胞菌(Aeromonas hydrophila)、鰻弧菌(Vibrio anguillarum)、哈維氏弧菌(V. harveyi)和溶藻弧菌(V. alginolyticus)的FBA均能分泌到胞外。在對多種病原菌的免疫保護評價中,FBA在斑馬魚上展示了較好的交叉免疫保護效果,相對免疫保護力達45～80%；在經(jīng)濟魚種大菱鲆動物實驗中針對E. piscicida具有68%的相對免疫保護力,經(jīng)FBA免疫后的大菱鲆血清對抗原蛋白具有顯著的特異性反應,且對上述五種海洋病原菌具有交叉特異性反應和顯著的殺菌活力。免疫相關基因的上調(diào)表達進一步表明,FBA能夠有效地激發(fā)魚體的特異性免疫系統(tǒng)。殺魚愛德華氏菌糖酵解途徑中的持家酶FBA具有良好的免疫保護力,可用于廣譜型候選疫苗的開發(fā)。其次,通過對殺魚愛德華氏菌多種分泌途徑缺失株胞外FBA分泌水平和外膜小泡(OMV)內(nèi)FBA含量分析,證明T3SS、T4SS、T6SS和Tat分泌途徑的缺失并不能阻止FBA的分泌,且OMV也不是FBA的主要分泌方式。在隨后的Pull-down及驗證實驗中,FBA能與Sec途徑中分子伴侶SecB相互作用,并有可能通過Sec途徑進行轉(zhuǎn)運�；诮⒌腅LISA篩選方法,在基因覆蓋率為75.6%的插入失活突變體文庫中,未能篩選到FBA分泌缺失菌株,很大的可能是FBA的分泌與菌體的生長及代謝密切相關。FBA可能不依賴于單一的分泌途徑,而是通過復雜的多途徑分泌,且其分泌可能是菌體生長必不可缺的。再次,在FBA分泌調(diào)控研究中,發(fā)現(xiàn)E. piscicida FBA的分泌不需要信號肽,分泌量受到菌體自身調(diào)控,在分泌后期處于分泌和降解的動態(tài)平衡狀態(tài)。突變體文庫的篩選和驗證發(fā)現(xiàn),esrC是調(diào)控FBA分泌的重要基因,基因esrC的缺失使FBA分泌量顯著上調(diào)。胞內(nèi)轉(zhuǎn)錄水平分析發(fā)現(xiàn),隨著培養(yǎng)時間的延長,缺失株△esrC中fbaA的表達水平與野生株沒有顯著性差異,然而secB表達水平相比于野生株呈明顯下降趨勢。野生株胞內(nèi)FBA的轉(zhuǎn)錄水平與其胞外分泌量呈負相關,即在菌體生長過程中FBA胞外分泌量逐漸增加時,其轉(zhuǎn)錄水平反而降低。這些結(jié)果表明,FBA的分泌量并不受制于胞內(nèi)表達水平,且轉(zhuǎn)錄調(diào)控因子EsrC并不直接調(diào)控fbα的表達,而是通過調(diào)控與FBA存在相互作用的secB或其他與分泌相關基因的表達來調(diào)控FBA的分泌,即EsrC通過未知的間接方式調(diào)控FBA的分泌。FBA的分泌調(diào)控是復雜的。最后,FBA胞外功能活性的分析發(fā)現(xiàn),分泌到胞外及細菌表面的FBA依然具有糖酵解酶活性,FBA在胞質(zhì)、周質(zhì)空間和胞外組分中的結(jié)構(gòu)是一致的,并未因為分泌行使非糖酵解功能而進行較大的結(jié)構(gòu)修飾。隨后的細胞侵染實驗顯示,FBA有助于愛德華氏菌對HeLa細胞的黏附。通過分析愛德華氏菌48株不同毒株FBA的胞外分泌量發(fā)現(xiàn),各個菌株均能分泌FBA,但胞外分泌量與病原菌的毒力強弱并不呈正相關關系。綜上所述,本工作不僅為魚用廣譜型疫苗的開發(fā)提供了理論依據(jù),也為殺魚愛德華氏菌糖酵解酶的胞外分泌調(diào)控機制研究奠定了一定基礎。
[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.
【學位授予單位】：華東理工大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S941.4
，
本文編號：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.
【學位授予單位】：華東理工大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S941.4
，
本文編號：2343605
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