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。
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 EseC
As 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 proteins
As 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.
引文
蔡完其,孙佩芳,刘至治等.中华鳖爱德华氏菌病病原和组织病理研究.水产学报1997(2): 428-433.
陈翠珍.爱德华氏菌及鱼类爱德华氏菌病.河北科技师范学院学报2004(8): 70-76.
李杰,莫照兰,茅云翔等.两株养殖大菱鲆体表出血病原菌的分离鉴定.海洋科学2008(12): 1-5.
李筠,颜显辉,陈吉祥等.养殖大菱鲆腹水病病原的研究.中国海洋大学学报(自然科学版) 2006(4): 649-654.
李正花. III型分泌系统的研究进展.医学信息2008(2): 2119-2122.
欧阳志明,陈怀青,陆承平等.迟缓爱德华氏菌的粘附特性.南京农业大学学报1997(10): 87-91.
唐电明,郑雅卡,陆红玉等.进境海狮迟钝爱德华氏菌的分离与鉴定.中国兽医科技2003(3): 71-72.
王波,莫照兰.迟缓爱德华氏菌及其致病机理.海洋科学集刊2007(5): 133-139.
王健,赵立平.细菌III型分泌系统.生命的化学2001(2): 147-149.
王蔚淼,潘玲.细菌III型分泌系统的研究进展.畜牧与兽医2006(8): 58-60.
王印庚,秦蕾,张正等.养殖大菱鲆的爱德华氏菌病.水产学报2007(3): 487-495.
汪莉,王玉民,岳俊杰等. III型分泌系统分子伴侣研究进展.微生物学报2004(4): 840-844.
肖鹏,莫照兰,邹玉霞等.鳗弧菌油乳化二价口服疫苗免疫养殖大菱鲆的免疫应答及免疫效果的研究.高技术通讯2007(7): 979-985.
许龙,周晓巍,黄培堂等.致病岛及其分泌系统.生物技术通讯2006(1): 614-617.
薛淑霞,冯守明,孙金生等.海水工厂化养殖大菱鲆(Scophthalmus maximus)和褐牙鲆(Paralichthys olivaceus)腹水病病原菌的分离与鉴定.海洋与湖沼2006(3): 548-554.
杨佳银,莫照兰,茅云翔等.从迟缓爱德华氏菌基因组fosmid文库克隆编码寡肽透过酶的opp基因簇.海洋科学2008(12): 13-19.
张元兴,孙修勤.海洋鱼病疫苗开发与生物反应器大规模生产.高技术通讯2000(10): 1-5.
周常义,陈晓凤,何仲京等.牛蛙迟缓爱德华氏菌变异株K的分离与鉴定.集美大学学报2004(9): 26-31.
Acharya, M., Maiti, N. K., Mohanty, S., Mishra, P. & Samanta, M. Genotyping of Edwardsiella tarda isolated from freshwater fish culture system. Comp Immunol Microbiol Infect Dis 2007(3): 33-40.
Akeda, Y. & Galan, J. E. Chaperone release and unfolding of substrates in type III secretion. Nature 2005(7): 911-915.
Backert, S. & Meyer, T. F. Type IV secretion systems and their effectors in bacterial pathogenesis. Curr Opin Microbiol 2006(9): 207-217.
Bartlett, K. H. & Trust, T. J. Isolation of Salmonellae and other potential pathogens from the freshwater aquarium snail Ampullaria. Appl Environ Microbiol 1976(3) 635-639.
Bennett, J. C. & Hughes, C. From flagellum assembly to virulence: the extended family of type III export chaperones. Trends Microbiol 2000(8): 202-204.
Beuzon, C. R., Banks, G., Deiwick, J., Hensel, M. & Holden, D. W. pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium. Mol Microbiol 1999(3): 806-816.
Birtalan, S. C., Phillips, R. M. & Ghosh, P. Three-dimensional secretion signals in chaperone-effector complexes of bacterial pathogens. Mol Cell 2002(9): 971-980.
Bladergroen, M. R., Badelt, K. & Spaink, H. P. Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant Microbe Interact 2003(16): 53-64.
Boyd, A. P., Lambermont, I. & Cornelis, G. R. Competition between the Yops of Yersinia enterocolitica for delivery into eukaryotic cells: role of the SycE chaperone binding domain of YopE. J Bacteriol 2000(8): 4811-4821.
Brandt, L., Skeiky, Y. A., Alderson, M. R. & other authors. The protective effect of the
Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs. Infect Immun 2004(7): 6622-6632.
Braun, V., Schonherr, R. & Hobbie, S. Enterobacterial hemolysins: activation, secretion and pore formation. Trends Microbiol 1993(1): 211-216.
Broms, J. E., Edqvist, P. J., Forsberg, A. & Francis, M. S. Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion. FEMS Microbiol Lett 2006(6): 57-66.
Bronstein, P. A., Miao, E. A. & Miller, S. I. InvB is a type III secretion chaperone specific for SspA. J Bacteriol 2000(2): 6638-6644.
Buttner, C. R., Sorg, I., Cornelis, G. R., Heinz, D. W. & Niemann, H. H. Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD. J Mol Biol 2008(5): 997-1012.
Castro, N., Toranzo, A. E., Nunez, S. & Magarinos, B. Development of an effective Edwardsiella tarda vaccine for cultured turbot (Scophthalmus maximus). Fish Shellfish Immunol 2008(2): 208-212.
Cendron, L., Seydel, A., Angelini, A., Battistutta, R. & Zanotti, G. Crystal structure of CagZ, a protein from the Helicobacter pylori pathogenicity island that encodes for a type IV secretion system. J Mol Biol 2004(3): 881-889.
Chen, J. D. & Huang, S. L. Hemolysin from Edwardsiella tarda strain ET16 isolated from eel Anguilla japonica identified as a hole-forming toxin. Fish Science 1996(6): 638-542.
Chen, J. D., Lai, S. Y. & Huang, S. L. Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda. Arch Microbiol 1996(5): 9-17.
Chen, L., Chen, Y., Wood, D. W. & Nester, E. W. A new type IV secretion system promotes conjugal transfer in Agrobacterium tumefaciens. J Bacteriol 2002(1): 4838-4845.
Cheng, L. W. & Schneewind, O. Yersinia enterocolitica type III secretion. On the role of SycE in targeting YopE into HeLa cells. J Biol Chem 1999(4): 22102-22108.
Cheng, L. W. & Schneewind, O. Type III machines of Gram-negative bacteria: delivering the goods. Trends Microbiol 2000(8): 214-220.
Chiu H. J. & Syu W. Jr. Functional analysis of EspB from enterohaemorrhagic Escherichia coli. Microbiology 2005(151): 3277–3286
Christie, P. J. Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim Biophys Acta 2004(9): 219-234.
Cianciotto, N. P. Type II secretion: a protein secretion system for all seasons. Trends Microbiol 2005(13): 581-588.
Cook, R. A. & Tappe, J. P. Chronic enteritis associated with Edwardsiella tarda infection in Rockhopper penguins. J Am Vet Med Assoc 1985(1): 1219-1220.
Cornelis, G. R. & Van Gijsegem, F.. Assembly and function of type III secretory systems. Annu Rev Microbiol 2000(4): 735-774.
Creasey, E. A., Friedberg, D., Shaw, R. K., Umanski, T., Knutton, S., Rosenshine, I. & Frankel, G. CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB. Microbiology 2003(9): 3639-3647.
Dai, S. & Zhou, D. Secretion and function of Salmonella SPI-2 effector SseF require its chaperone, SscB. J Bacteriol 2004(6): 5078-5086.
Darwin, K. H. & Miller, V. L. Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium. EMBO J 2001(2): 1850-1862.
Das, S. & Chaudhuri, K. Identification of a unique IAHP (IcmF associated homologous proteins) cluster in Vibrio cholerae and other proteobacteria through in silico analysis. In Silico Biol 2003(3): 287-300.
Delepelaire, P. Type I secretion in gram-negative bacteria. Biochim Biophys Acta 2004(1): 149-161.
Dorson, M. Applied immunology of fish. OIE Press 1984: 39-74.
Du, M., Chen, J., Zhang, X., Li, A., Li, Y. & Wang, Y. Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate. Appl Environ Microbiol 2007(7): 1349-1354.
Dudley, E. G., Thomson, N. R., Parkhill, J., Morin, N. P. & Nataro, J. P. Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli. Mol Microbiol 2006(6): 1267-1282.
Earlix, D. J. Host Pathogen and environmental interactions of enteric septicaemia ofcatfish. PhD desertation, Auburn University, Auburn University, Auburn, Alabama 1995. 102.
Edqvist, P. J., Broms, J. E., Betts, H. J., Forsberg, A., Pallen, M. J. & Francis, M. S. Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion. Mol Microbiol 2006(9): 31-44.
Edwards, R. A., Keller, L. H. & Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 1998(7): 149-157.
Eko, F. O., Szostak, M. P., Wanner, G. & Lubitz, W. Production of Vibrio cholerae ghosts (VCG) by expression of a cloned phage lysis gene: potential for vaccine development. Vaccine 1994(12): 1231-1237.
Elliott, S. J., Wainwright, L. A., McDaniel, T. K., Jarvis, K. G., Deng, Y. K., Lai, L. C., McNamara, B. P., Donnenberg, M. S. & Kaper, J. B. The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 1998(8): 1-4.
Elliott, S. J., Hutcheson, S. W., Dubois, M. S., Mellies, J. L., Wainwright, L. A., Batchelor, M., Frankel, G., Knutton, S. & Kaper, J. B. Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol Microbiol 1999(3): 1176-1189.
Enninga, J., Mounier, J. & Sansonetti, P. Secretion of type III effectors into host cells in real time. Nat Methods 2005(2): 959.
Evans, L. D., Stafford, G. P., Ahmed, S., Fraser, G. M. & Hughes, C. An escort mechanism for cycling of export chaperones during flagellum assembly. Proc Natl Acad Sci U S A 2006(1): 17474-17479.
Evdokimov, A. G., Tropea, J. E., Routzahn, K. M. & Waugh, D. S. Three-dimensional structure of the type III secretion chaperone SycE from Yersinia pestis. Acta Crystallogr D Biol Crystallogr 2002(5): 398-406.
Ewing, W. H. & McWhorter, A. C. Edwardsiella, a genus of Enterobacteriaceae based on a new species, E tarda. Int Bul Bact Nomencl Toxon 1965(15): 33-38.
Feldman, M. F., Muller, S., Wuest, E. & Cornelis, G. R. SycE allows secretion of YopE-DHFR hybrids by the Yersinia enterocolitica type III Ysc system. Mol Microbiol 2002(4): 1183-1197.
Feldman, M. F. & Cornelis, G. R. The multitalented type III chaperones: all you can do with 15 kDa. FEMS Microbiol Lett 2003(9): 151-158.
Femandez-Alonso, M., Alvarez, F. & al., A. E. e. A model to study fish DNA immersion vaccination by using the green fluorescent protein. J Fish Dis 1999(22): 237-241.
Feng, S. M., Zhan, W. B., Sheng, X. Z., Yang, K., Han, J. G., Wei, J. L., Li, J. & Qiao, X. T. Response of mucosal and systemic sIgM-positive cells in turbot (Scophthalmus maximus L.) immunization with Edwardsiella tarda. Vet Immunol Immunopathol 2009(9): 108-114.
Fletcher, T. C. & White, A. Antibody production in the Plaice (Pleuronectes platessa L.) after oral and parenteral immunization with Vibrio anguillarum antigens. Aquaculture 1973(1): 417-428.
Francis, M. S., Lloyd, S. A. & Wolf-Watz, H. The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis. Mol Microbiol 2001(4): 1075-1093.
Francis, M. S., Wolf-Watz, H. & Forsberg, A. Regulation of type III secretion systems. Curr Opin Microbiol 2002(5): 166-172.
Frithz-Lindsten, E., Rosqvist, R., Johansson, L. & Forsberg, A. The chaperone-like protein YerA of Yersinia pseudotuberculosis stabilizes YopE in the cytoplasm but is dispensible for targeting to the secretion loci. Mol Microbiol 1995(16): 635-647.
Gauthier, A. & Finlay, B. B. Translocated intimin receptor and its chaperone interact with ATPase of the type III secretion apparatus of enteropathogenic Escherichia coli. J Bacteriol 2003(15): 6747-6755.
Ghosh, P. Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 2004(6): 771-795.
Gophna, U., Ron, E. Z. & Graur, D. Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events. Gene 2003(12): 151-163.
Grefory, V. P., James, B. D. & Franco, F. Type III export: new uses for an old pathway. Mol Microbiol 2001(4): 284-293.
Grimont, P. A. D. Edwardsiella hoshinae, a new species of Enterobacteriaceae. Curr Microbiol 1980(4): 345-351.
Gudding, R., Lillehaug, A. & Evensen, O. Recent developments in fish vaccinology. Vet Immunol Immunopathol 1999(7): 203-212.
Gutierrez, M. A. & Miyazaki, T. Responses of Japanese Eels to Oral Challenge with Edwardsiella tarda after Vaccination with Formalin-Killed Cells or Lipopolysaccharide of the Bacterium. Journal of Aquatic Animal Health 1994(6): 110-117.
Hamilton, H. L., Dominguez, N. M., Schwartz, K. J., Hackett, K. T. & Dillard, J. P. Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol Microbiol 2005(5): 1704-1721.
Heckels, J. E., Fletcher, J. N. & Virji, M. The potential protective effect of immunization with outer membrane protein from Neisseria gonorhoeae. GenMicrobiol 1989(3): 2269- 2276.
Henderson, I. R., Navarro-Garcia, F., Desvaux, M., Fernandez, R. C. & Ala'Aldeen, D. Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 2004(6): 692-744.
Hirono, I., Tange, N. & Aoki, T. Iron-regulated haemolysin gene from Edwardsiella tarda. Mol Microbiol 1997(24): 851-856.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989(7): 51-59.
Holt, J. G., Krieg, N. R., Sneath, P. H. A. & al, e. Bergey’s Manual of Determinative Bacteriology (Ninth Edition). Baltimore; Williams &Wilkins, 1994. 178, 204, 225.
Hoshina, T. On a new bacterium Paracolobactrum anguillimortiferum n.sp. Bulletin of the Japanese Society of Scientific Fisheries 1962(2): 162-164.
Hueck, C. J. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 1998(6): 379-433.
Ide, T., Laarmann, S., Greune, L., Schillers, H., Oberleithner, H. & Schmidt, M. A. Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli. Cell Microbiol 2001(3): 669–679.
Igarashi, A., Iida, T. & Crosa, J. H. Iron-acquisition ability of Edwardsiella tarda with involvement in its virulence. Fish Pathology 2002(3): 53-57.
Ishibe, K., Osatomi, K., Hara, K., Kanai, K., Yamaguchi, K. & Oda, T. Comparison of the responses of peritoneal macrophages from Japanese flounder (Paralichthys olivaceus) against high virulent and low virulent strains of Edwardsiella tarda. FishShellfish Immunol 2008(24): 243-251.
Jackson, M. W., Day, J. B. & Plano, G. V. YscB of Yersinia pestis functions as a specific chaperone for YopN. J Bacteriol 1998(1): 4912-4921. Janda, J. M. & Abbott, S. L. Pathogenic properties of Edwardsiella tarda. J Clin Microbiol 1991(2): 1998-2001.
Janda, J. M. & Abbott, S. L. Infections associated with the genus Edwardsiella: the role of Edwardsiella tarda in human disease. Clin Infect Dis 1993(7): 742-748.
Joosten, P. H. M. & Tiemersma, E. Oral vaccination of fish against Vibrio anguillarum using alginate microparticles. Molecular Immunology 1997(7): 471-294.
Kalogeraki, V. S. & Winans, S. C. Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 1997(8): 69-75.
Katinger, A., Lubitz, W., Szostak, M. P., Stadler, M., Klein, R., Indra, A., Huter, V. & Hensel, A. Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses. J Biotechnol 1999(3): 251-260.
Kim, J. & Marshall, D. L. Influence of catfish skin mucus on trisodium phosphate inactivation of attached Salmonella Typhimurium, Edwardsiella tarda, and Listeria monocytogenes. J Food Prot 2002(5): 1146-1151.
Kokubo, T., Iida, T. & Wakabayashi, H. Production of Siderophore by Edwardsiella tarda. Fish Pathology 1990(5): 237-241.
Kubori, T., Matsushima, Y., Nakamura, D., Uralil, J., Lara-Tejero, M., Sukhan, A., Galan, J. E. & Aizawa, S. I. Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 1998(2): 602-605.
Kumar, G., Rathore, G., Sengupta, U., Kapoor, D. & Lakra, W. S. Production of monoclonal antibodies specific to major outer membrane protein of Edwardsiella tarda. Comp Immunol Microbiol Infect Dis. 2008(9): 886-892.
Kwon, S. R., Nam, Y. K. & al., S. K. K. e. Generation of Edwardsiella tarda ghosts by bacteriophage PhiX174 lysis gene E. Aquaculture 2005(2): 16-21.
Kwon, S. R., Nam, Y. K., Kim, S. K. & Kim, K. H. Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts.Fish Shellfish Immunol 2006(2): 621-626.
Kwong, J. C., Stukel, T. A., McGeer, A. J. & Manuel, D. G. Appropriate measures of influenza immunization program effectiveness. Vaccine 2007(5): 967-969.
Langermann, S., Palaszynski, S., Barnhart, M. & other authors. Prevention of mucosal Escherichia coli infection by FimH-adhesin-based systemic vaccination. Science 1997(6): 607-611.
Lin, L. Oral vaccine:a new idea for vaccination. Fisheries International 2001(10): 46.
Ling, S. H., Wang, X. H., Xie, L., Lim, T. M. & Leung, K. Y. Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models. Microbiology 2000(4): 7-19.
Liu, Y., Oshima, S., Kurohara, K., Ohnishi, K. & Kawai, K. Vaccine efficacy of recombinant GAPDH of Edwardsiella tarda against Edwardsiellosis. Microbiol Immunol 2005(9): 605-612.
Lloyd, S. A., Forsberg, A., Wolf-Watz, H. & Francis, M. S. Targeting exported substrates to the Yersinia TTSS: different functions for different signals? Trends Microbiol 2001(9): 367-371.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951(13): 265-275.
Luo, Y., Bertero, M. G., Frey, E. A. & other authors. Structural and biochemical characterization of the type III secretion chaperones CesT and SigE. Nat Struct Biol 2001(8): 1031-1036.
Macnab, R. M. The bacterial flagellum: reversible rotary propellor and type III export apparatus. J Bacteriol 1999(11): 7149-7153.
Marchart, J., Dropmann, G., Lechleitner, S., Schlapp, T., Wanner, G., Szostak, M. P. & Lubitz, W. Pasteurella multocida- and Pasteurella haemolytica-ghosts: new vaccine candidates. Vaccine 2003(21): 3988-3997.
Marsden, M. J., Vaughan, L. M., Foster, T. J. & Secombes, C. J. A live (delta aroA) Aeromonas salmonicida vaccine for furunculosis preferentially stimulates T-cell responses relative to B-cell responses in rainbow trout (Oncorhynchus mykiss). Infect Immun 1996(6): 3863-3869.
Marsh, P. K. & Gorbach, S. L. Invasive enterocolitis caused by Edwardsiella tarda. Gastroenterology 1982(8): 336-338.
Mathew, J. A., Tan, Y. P., Srinivasa Rao, P. S., Lim, T. M. & Leung, K. Y. Edwardsiella tarda mutants defective in siderophore production, motility, serum resistance and catalase activity. Microbiology 2001(14): 449-457.
Mavris, M., Page, A. L., Tournebize, R., Demers, B., Sansonetti, P. & Parsot, C. Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus. Mol Microbiol 2002(4): 1543-1553.
McKenney, D., Pouliot, K. L., Wang, Y., Murthy, V., Ulrich, M., Doring, G., Lee, J. C., Goldmann, D. A. & Pier, G. B. Broadly protective vaccine for Staphylococcus aureus based on an in vivo-expressed antigen. Science 1999(8): 1523-1527.
Menard, R., Sansonetti, P., Parsot, C. & Vasselon, T. Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri. Cell 1994(7): 515-525.
Menard, R., Dehio, C. & Sansonetti, P. J. Bacterial entry into epithelial cells: the paradigm of Shigella. Trends Microbiol 1996(4): 220-226.
Meyer, F. P. & Bullock, G. L. Edwardsiella tarda, a new pathogen of channel catfish (Ictalurus punctatus). Appl Microbiol 1973(5): 155-156.
Mietzner, T. A. & Morse, S. A. The role of iron-binding proteins in the survival of pathogenic bacteria. Annu Rev Nutr 1994(14): 471-493. Miller, M. B. & Bassler, B. L. Quorum sensing in bacteria. Annual Review of Microbiology 2001(5): 165-199.
Miller, V. L. & Mekalanos, J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 1988(10): 2575-2583.
Mizunoe, S., Yamasaki, T., Tokimatsu, I., Matsunaga, N., Kushima, H., Hashinaga, K. & Kadota, J. A case of empyema caused by Edwardsiella tarda. J Infect 2006(3): 255-258.
Mo, Z. L., Xiao, P., Mao, Y. X., Zou, Y. X., Wang, B., Li, J., Xu, Y. L. & Zhang, P. J. Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.). Fish and Shellfish Immunology 2007(23): 521-530.
Mougous, J. D., Cuff, M. E., Raunser, S. & other authors A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006(12):1526-1530.
Mougous, J. D., Gifford, C. A., Ramsdell, T. L. & Mekalanos, J. J. Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat Cell Biol 2007(9): 797-803.
Muyembe, T., Vandepitte, J. & Desmyte.r, J. Natural colistin resistance in Edwardsiella tarda. Antimicrob Agents Chemother 1973(4): 521-524.
Nakatsugawa, T. Edwardsiella tarda isolated from cultured young flounder. Fish Pathology 1983(18): 99-101.
Neyt, C. & Cornelis, G. R. Role of SycD, the chaperone of the Yersinia Yop translocators YopB and YopD. Mol Microbiol 1999(1): 143-156.
Okuda, J., Arikawa, Y., Takeuchi, Y., Mahmoud, M. M., Suzaki, E., Kataoka, K., Suzuki, T., Okinaka, Y. & Nakai, T. Intracellular replication of Edwardsiella tarda in murine macrophage is dependent on the type III secretion system and induces an up-regulation of anti-apoptotic NF-kappaB target genes protecting the macrophage from staurosporine-induced apoptosis. Microb Pathog 2006(4): 226-240.
Okuda, J., Kiriyama, M., Yamanoi, E. & Nakai, T. The type III secretion system-dependent repression of NF-kappaB activation to the intracellular growth of Edwardsiella tarda in human epithelial cells. FEMS Microbiol Lett 2008(8): 9-14.
Ottemann, K. M. & Miller, J. F. Roles for motility in bacterial-host interactions. Mol Microbiol 1997(24): 1109-1117.
Padros, F., Zarza, C., Dopazo, L., Cuadrado, M. & Crespo, S. Pathology of Edwardsiella tarda infection in turbot, Scophthalmus maximus (L.). J Fish Dis 2006(9): 87-94.
Page, A. L., Fromont-Racine, M., Sansonetti, P., Legrain, P. & Parsot, C. Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri. Mol Microbiol 2001(2): 1133-1145.
Page, A. L. & Parsot, C. Chaperones of the type III secretion pathway: jacks of all trades. Mol Microbiol 2002(6): 1-11.
Page, A. L., Sansonetti, P. & Parsot, C. Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway. Mol Microbiol 2002(3): 1533-1542.
Pallen, M. J., Beatson, S. A. & Bailey, C. M. Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective. FEMS MicrobiolRev 2005(9): 201-229.
Park, L., Wakabayashi, H. & Y, W. Serotype and virulence of Edwardsiella tarda isolated from eel and their environment. Fish Pathol 1983(18): 85-89.
Parsot, C., Hamiaux, C. & Page, A. L. The various and varying roles of specific chaperones in type III secretion systems. Curr Opin Microbiol 2003(6): 7-14.
Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M. & Endo, M. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet Immunol Immunopathol 2006(11): 339-347.
Pirarat, N., Maita, M. & M. Endo, e. a. Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus. Fish & Shellfish Immunology 2007(22): 608-616.
Plano, G. V., Day, J. B. & Ferracci, F. Type III export: new uses for an old pathway. Mol Microbiol 2001(4): 284-293.
Plumb, J. A. Edwardsiella septicemias. Fish diseases and disorders 1999(3): 479-521.
Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W. C., Heidelberg, J. F. & Mekalanos, J. J. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 2006(13): 1528-1533.
Rao, P. S., Yamada, Y., Tan, Y. P. & Leung, K. Y. Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol Microbiol 2004(5): 573-586.
Reed, L. J. & Muench, H. A simple method of estimating fifty percent end points. Am J Hyg 1938(2): 493-497.
Roberts, V. & R, B. N. Bacterial diseases of fish. Cambridge: The University press. 1999. 61-79.
Rshid, M., Nakai, T. & Miroga K, e. a. Pathogenesis of experimental Edwardsiellosis in Japanese flounder Paralichthys olivaceus. Fish Science 1997(6): 384-387.
Rubires, X., Saigi, F., Pique, N., Climent, N., Merino, S., Alberti, S., Tomas, J. M. & Regue, M. A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives. J Bacteriol 1997(17): 7581-7586.
Sae-Oui, D., Muroga, K. & Nakai, T. A case of Edwardsiella tarda infection in cultured colored carp Cyprinus carpio. Fish Pathology 1984(19): 197-199.
Saier, M. H., Jr. Evolution of bacterial type III protein secretion systems. Trends Microbiol 2004(12): 113-115.
Salati, F., K, K. & R, K. Immunoresponse of eel against Edwardsiella tarda antigens. Fish Pathology 1983(18): 135-141.
Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning a Laboratory Manual, second ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1989.
Sasaki, T. & Aita, A. A study of fish diseases. Part I. Edwardsiella isolated from Sea Urchin. Jap J Microbiol 1975(3): 368.
Schell, M. A., Ulrich, R. L., Ribot, W. J. & other authors. Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol 2007(6): 1466-1485.
Schlumberger, M. C., Muller, A. J., Ehrbar, K., Winnen, B., Duss, I., Stecher, B. & Hardt, W. D. Real-time imaging of type III secretion: Salmonella SipA injection into host cells. Proc Natl Acad Sci U S A 2005(2): 12548-12553.
Simon, L. M., Nemcsok, J. & Boross, L. Studies on the effect of paraquat on glycogen mobilization in liver of common carp (Cyprinus carpio L.). Comp Biochem Physiol C 1983(7): 167-169.
Srinivasa Rao, P. S., Yamada, Y. & Leung, K. Y. A major catalase (KatB) that is required for resistance to H2O2 and phagocyte-mediated killing in Edwardsiella tarda. Microbiology 2003(1): 2635-2644.
Stathopoulos, C., Hendrixson, D. R., Thanassi, D. G., Hultgren, S. J., St Geme, J. W., 3rd & Curtiss, R., 3rd. Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story. Microbes Infect 2000(2) 1061-1072.
Stebbins, C. E. & Galan, J. E. Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 2001(4): 77-81.
Strauss, E. J., Ghori, N. & Falkow, S. An Edwardsiella tarda strain containing a mutation in a gene with homology to shlB and hpmB is defective for entry into epithelial cells in culture. Infect Immun 1997(5): 3924-3932.
Suprapto, H., Hara, T. & Nakai T, e. a. Purification of a lethal toxin of Edwardsiellatarda. Fish Pathology 1996(3): 203-207.
Szostak, M. P., Hensel, A., Eko, F. O. & other authors. Bacterial ghosts: non-living candidate vaccines. J Biotechnol 1996(4): 161-170.
Takeuchi, H., Fujita, Y., Ogawa, H., Shiomi, K., Toyokawa, Y., Yamamoto, T., Furukawa, T. & Ebisu, T. Multiple brain abscesses in neonate caused by Edwardsiella tarda: case report. Neurol Med Chir (Tokyo) 2009(1): 85-89.
Tampakaki, A. P., Fadouloglou, V. E., Gazi, A. D., Panopoulos, N. J. & Kokkinidis, M. Conserved features of type III secretion. Cell Microbiol 2004(6): 805-16.
Tamura, K., Sakazaki, R., McWhorter, A. C. & Kosako, Y. Edwardsiella tarda serotyping scheme for international use. J Clin Microbiol 1988(2): 2343-2346.
Tan, Y. P., Zheng, J., Tung, S. L., Rosenshine, I. & Leung, K. Y. Role of type III secretion in Edwardsiella tarda virulence. Microbiology 2005(5): 2301-2313.
Thomas, J., Stafford, G. P. & Hughes, C. Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. Proc Natl Acad Sci U S A 2004(10): 3945-3950.
Thomas, N. A., Deng, W., Puente, J. L., Frey, E. A., Yip, C. K., Strynadka, N. C. & Finlay, B. B. CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli. Mol Microbiol 2005(7): 1762-1779.
Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979(7): 4350-4354.
Tsuji, A., Hirasawa, K., Arakuma, T., Izumi, K., Kobori, K., Sunohara, K., Yoshizawa, N., Watanabe, H. & Izumiya, H. A 12-year-old boy with acute gastroenteritis caused by Edwardsiella tarda O4:H4. J Infect Chemother 2008(14): 433-435.
Ullah, M. A. & Arai, T. Pathological activities of the naturally ocurring strains of Edwardsiella tarda. Fish Pathology 1983(18): 65~70.
Verjan, N., Hirono, I. & Aoki, T.. Genetic loci of major antigenic protein genes of Edwardsiella tarda. Appl Environ Microbiol 2005(7): 5654-5658.
Vinogradov, S. The second annual symposium on nanomedicine and drug delivery: exploring recent developments and assessing major advances. 19-20 August 2004,Polytechnic University, Brooklyn, NY, USA. Expert Opin Drug Deliv 2004. 181-184.
Wainwright, L. A. & Kaper, J. B. EspB and EspD require a specific chaperone for proper secretion from enteropathogenic Escherichia coli. Mol Microbiol 1998(2): 1247-1260.
Wakabayashi, H. & Egusa, S. Edwardsiella tarda (Paracolobactrum anguillimortiferum) associated with pond-cultured eel disease. Bulletin of the Japanese Society of Scientific Fisheries 1973(9): 931-936.
Waltman, W. D., B, S. E. & C, H. T. Biochemical and enzymatic characterization of Edwardsiella tarda from the United States and Taiwan. Fish Pathology 1986(21): 1-8.
Wang, F., Cheng, S., Sun, K. & Sun, L. Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain. J Microbiol 2008(6): 350-355.
Wang, X. & Lu, C. Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection. Vaccine 2009(2): 1571-1578.
Watson, J. J. & White, F. H. Hemolysins of Edwardsiella tarda. Can J Comp Med 1979(3): 78-83.
Wattiau, P., Bernier, B., Deslee, P., Michiels, T. & Cornelis, G. R. Individual chaperones required for Yop secretion by Yersinia. Proc Natl Acad Sci U S A 1994(9): 10493-10497.
Witte, A., Wanner, G., Sulzner, M. & Lubitz, W. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli. Arch Microbiol 1992(7): 381-388.
Woestyn, S., Sory, M. P., Boland, A., Lequenne, O. & Cornelis, G. R. The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes. Mol Microbiol 1996(20): 1261-1271.
Wong, J. D., Miller, M. A. & Janda, J. M. Surface properties and ultrastructure of Edwardsiella species. J Clin Microbiol 1989(2): 1797-1801.
Woo, P. T. K. & Bruno, D. W. viral, bacterial and fungal infections. Fish diseases and disorders 1999(3).
Wulff-Strobel, C. R., Williams, A. W. & Straley, S. C. LcrQ and SycH function together at the Ysc type III secretion system in Yersinia pestis to impose a hierarchy of secretion. Mol Microbiol 2002(3): 411-423.
Xiao, J., Wang, Q., Liu, Q., Xu, L., Wang, X., Wu, H. & Zhang, Y. Characterization of Edwardsiella tarda rpoS: effect on serum resistance, chondroitinase activity, biofilm formation, and autoinducer synthetases expression. Appl Microbiol Biotechnol. 2009.
Zhang, M., Sun, K. & Sun, L. Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain. Microbiology 2008(5): 2060-2069.
Zheng, J. & Leung, K. Y. Dissection of a type VI secretion system in Edwardsiella tarda. Mol Microbiol 2007(12): 1192-1206.
Zheng, J., Li, N., Tan, Y. P., Sivaraman, J., Mok, Y. K., Mo, Z. L. & Leung, K. Y. EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD. Microbiology 2007(10): 1953-1962.
Zurawski, D. V. & Stein, M. A. SseA acts as the chaperone for the SseB component of the Salmonella Pathogenicity Island 2 translocon. Mol Microbiol 2003(4): 1341-1351.
Zurawski, D. V. & Stein, M. A. The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD. Microbiology 2004(10): 2055–2068.