【作者】 王波；

【导师】 张培军； 莫照兰；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2009， 博士

【摘要】 迟缓爱德华氏菌(Edwardsiella tarda)是重要的革兰氏阴性致病菌,可以感染包括人类在内的多种动物。由迟缓爱德华氏菌引发的爱德华氏菌病已经在许多水产养殖动物中被发现,每年给淡水和海水水产养殖业带来巨大的损失。目前为止对于迟缓爱德华氏菌病的防治以化学治疗为主,疫苗的研究还在进行中。III型分泌系统(T3SS)是E. tarda重要的致病因子,虽然T3SS基因簇的结构及部分基因的功能得到了初步研究,但其作用机制还未得到阐明。本研究着重开展了迟缓爱德华氏菌T3SS输送器蛋白EseC的分子伴侣的鉴定及功能研究,并对输送器蛋白及其免疫功能进行了初步探讨,希望进一步地了解T3SS在E. tarda致病中的功能及其在疫苗研制中的作用。一、迟缓爱德华氏菌III型分泌系统(T3SS)输送器蛋白EseC分子伴侣的鉴定和功能研究以前的工作表明,EseB、EseC和EseD蛋白是E. tarda T3SS输送器蛋白的组成成分,在分泌到细菌细胞外后可以组成输送器装置。分子伴侣对于输送器蛋白的稳定和分泌具有重要的作用,EscC已经被鉴定为EseB和EseD的分子伴侣,而EseC的分子伴侣还没有得到鉴定。在本实验中,我们以EseC作为研究对象,主要开展了EseC分子伴侣鉴定的研究。生物信息学分析表明,在E. tarda T3SS基因簇上的escA基因与eseC相邻,其编码的蛋白形成一个大的螺旋结构,为分子量较小(17.5kD)的酸性蛋白(pI 4.79),并与已鉴定的分子伴侣具有序列的同源性,这些符合细菌T3SS分子伴侣的特征。研究发现,EscA蛋白分布在细菌的细胞质和细胞膜上。在escA基因缺失后,大大降低了EseC分泌到细菌细胞外的量,同时EseC蛋白在细菌细胞质中的积聚量也减少,当escA基因缺失突变株得到escA基因互补后,EseC的分泌和在细胞质内的积聚恢复到了野生型菌株水平。氯霉素阻断蛋白质合成的实验发现,当细菌不表达EscA的情况下,EseC蛋白逐渐降解,说明了EscA可以影响EseC在胞质中的稳定。蛋白体外结合试验和免疫共沉淀实验发现,EseC和EscA在体外可以结合,在细菌细胞质中也可以相互结合,表明EseC和EscA可以相互作用。上述结果表明,EscA是EseC的分子伴侣。在确定了EscA是EseC的分子伴侣之后,我们进一步确定EscA对EseC表达的影响,以及两者相互作用的结构域。通过检测转录水平和翻译水平的EseC-LacZ融合蛋白表达情况,发现在EscA缺失的情况下,EseC的转录水平没有变化,而翻译水平下降,表明EscA对EseC的影响在转录后水平。通过构建含有部分结构域缺失的escA或eseC的体外共表达体系,并进行Western blot分析,确定了EseC的31-137氨基酸序列为与EscA结合的区域,而在EscA中并没有找到与EseC结合的区域。EseC的31-137氨基酸片段缺失后,EseC的分泌和在E. tarda细菌细胞中的积聚下降,其下降幅度与escA突变株相当,进一步表明EseC的31-137氨基酸为与EscA相互作用的区域。最后人工感染实验表明,分子伴侣EscA及其与EseC的相互作用对E. tarda的致病力有影响。二、迟缓爱德华氏菌T3SS输送器蛋白的研究一些研究表明,T3SS在细菌与宿主相互作用的过程中表达,在体外诱导的条件下也可表达。为了确定E. tarda T3SS体外诱导表达的条件,我们检测了不同培养温度、pH条件下,E. tarda T3SS输送器蛋白表达的情况。研究表明,37°C条件下,E. tarda生长快,T3SS的输送器蛋白表达较低;28°C条件下,T3SS的输送器蛋白表达最高,而在20°C条件下,没有检测到T3SS输送器蛋白的表达。在28°C和37°C的培养条件下,中性和碱性相对酸性来说适合细菌的生长和T3SS输送器蛋白的表达。我们分析了E. tarda野生型和输送器蛋白突变株中的输送器蛋白的细胞分布,并据此推测输送器形成的机制。单一输送器蛋白的缺失不影响其它两个输送器蛋白的积聚,而输送器蛋白的分泌之间存在一定的相互影响。通过检测输送器蛋白突变株?eseB, ?eseC, ?eseD生长、泳动、自凝聚和溶血能力的变化,发现在输送器蛋白基因缺失后,体外培养的E. tarda的生长速度变慢,泳动、自凝聚和溶血能力也变弱,说明了输送器蛋白在细菌的生长和功能行使中的重要作用。为了检测输送器蛋白的免疫保护效果,我们克隆了eseD基因,将其在表达菌株BL21(DE3)中进行表达,并将重组表达的EseD蛋白经Ni-NTA树脂进行纯化。以EseD纯化蛋白作为蛋白抗原对大菱鲆进行注射,EseD蛋白表现出了对鱼类的免疫原性,其抗体效价在第7周达到了最高,为1:5120。攻毒实验表明该蛋白对于保护大菱鲆免疫E. tarda的感染具有帮助作用,在105cfu攻毒浓度下大菱鲆的相对存活率(RPS)为62.5%。结果说明EseD蛋白可以作为蛋白抗原疫苗的候选,并能够在保护鱼类免疫爱德华氏菌病中发挥作用。更多还原

【Abstract】 Edwardsiella tarda is an important gram-negative pathogen affecting both animals and humans. Edwardsiellosis, caused by E. tarda, has been found in many commercially important cultured fish, and has led to extensive losses in both freshwater and marine aquaculture annually. Till now chemical methods are commonly used for prevention against edwardsiellosis, while related studies of vaccines are still on the road. This pathogen harbors a type III secretion system (T3SS) essential for bacterial pathogenesis. Although the structure of T3SS gene cluster together with the function of some genes in T3SS have been primarily studied, its mechanism hasn’t been illuminated yet. In this experiment, we focus our study on the identification and investigation of the chaperone for the E. tarda T3SS translocon protein EseC, and also carry out primary research for translocon proteins and their immunoprophylactic potential to fish, trying to learn more about the mechanism of T3SS in the pathogenesis of E. tarda and in the development of vaccine.1. Identification and investigation of the chaperone for the E. tarda T3SS translocon protein EseCAs previously reported, EseB, EseC, and EseD are the components of the T3SS translocon, and were shown to form this complex after secretion. Chaperones are important for the stabilization and secretion of T3SS translocon proteins. EscC functions as a T3SS chaperone for EseB and EseD, whereas the chaperone for EseC hasn’t been reported. In this experiment, we took EseC as the research object, and identified the chaperone of EseC.Bioinformatics assay showed that escA gene is located proximately to eseC. EscA is a small (molecular mass 17.5 kDa) an acidic (pI 4.79) protein, having a large helix configuration, and is homologous to the identified chaperones, all of which fulfill the common characteristics of a T3SS chaperone. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC; meanwhile the accumulation of EseC in the cytoplasm was also affected. While escA mutant was complemented by escA gene, the secretion and accumulation of EseC were resumed to the wild type secretion phenotype. As we used chloramphenicol to block the production of new proteins, we observed that EseC protein gradually degraded without the existence of EscA, showing EscA is important for the stabilization of EseC. Co-purification studies demonstrated a specific interaction between EscA and EseC in vitro, while co-immunoprecipitation studies proved that EscA could bind to EseC in vivo. Hereto we conclude that EscA functions as the chaperone for EseC.Having confirmed the chaperone role of EscA for EseC, we further studied the affection of EscA to the expression of EseC, and the binding region of these two proteins. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC by construction and measuration of an EseC-LacZ fusion protein in E. tarda. By constructing and Western blot analyzing in vitro co-expression systems containing in-frame mutations of partial sequence of EscA and EseC, we found that residues 31–137 of EseC are required for EseC-EscA interaction, while we couldn’t find a similar region in EscA particularly used for binding EseC. We constructed the ?eseC31-137 mutant and found that absence of EseC residues 31–137 reduced the secretion and accumulation of EseC at a similar level as escA deletion in E. tarda, attesting that 31-137 regions are the binding domain of EseC to EscA. Finally, artificial infection experiments showed that EscA and its interaction with EseC contribute to the virulence of E. tarda.2. Study of the E. tarda T3SS translocon proteinsAs previously reported, T3SS proteins could express when bacteria contact with their host, as well as bacteria are induced in vitro. We checked the expression of E. tarda T3SS translocon proteins in different temperatures and different pH values, in order to confirm the preferable induction condition. As the results, in 37°C, E. tarda grew fastest, but the expression of translocon proteins was lower; in 28°C, the expression of translocon proteins was highest; in 20°C, we couldn’t detect their expression. Meanwhile alkaline and neutral environments are better that acidic culture for growing and T3SS expressing. We analyzed the cellular fractionation of three Ese proteins in wild type strain as well as translocator mutants, and deduced the possible mechanism of the comformation of translocon. Absence of single translocon component protein doesn’t affect the expression of other two, while the secretion of translocon proteins could interact in some extent.We used the ?eseB, ?eseC and ?eseD mutants by measuring their growth conditions as well as their swimming motilities, autoaggregate abilities and hemolysis abilities, and found that in the deletion of translocon proteins, the growth of bacteria is slower than wild type, the abilities of motility, autoaggregate and hemolysis also reduced, demonstrating the important roles of the translocon proteins for the growth and function of E. tarda.To test the immunoprophylactic effect of translocon protein, the eseD gene was cloned, expressed in E. coli BL21 (DE3) strain, and recombinant EseD protein was purified by Ni-NTA resin. Scophthalmus maximus was immunized using the purified protein and developed antibodies with a climax titer of 1:5120 on the 7th week, showing that the protein is highly immunogenic in fish. Challenge experiment of E. tarda demonstrated that the protein is effective for preventing E. tarda infection, as the relative percent survival (RPS) of turtle fish was 62.5% when the concentration of E. tarda was 105cfu. The EseD protein shows promise as vaccine candidate in aquaculture and could probably be used for the protection of fish against edwardsiellosis.更多还原