Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China.
Fish Shellfish Immunol. 2011 Aug;31(2):182-8. doi: 10.1016/j.fsi.2011.05.001. Epub 2011 May 14.
Members of the DnaJ/Hsp40 family play an important role in protein homeostasis by regulating the activity of DnaK/Hsp70. In this study, we examined the activity and function of the DnaJ from Edwardsiella tarda, a serious fish pathogen that can also infect humans and birds. In silico analysis indicated that E. tarda DnaJ contains structural features, i.e. the J domain, the glycine/phenylalanine-rich region, and the zinc-finger domain, that are conserved among Type I Hsp40. Purified recombinant DnaJ was able to stimulate the ATPase activity of DnaK. Pull-down assay indicated that DnaJ could interact specifically with DnaK. Mutation of the conserved HPD site in the J domain completely abolished the DnaK-stimulating effect of DnaJ. To examine the functional importance of DnaJ, a dnaJ-defective mutant was constructed. Compared to the wild type, the dnaJ mutant (i) was retarded in growth and more sensitive to H₂O₂-induced oxidative damage, (ii) dramatically reduced in general bacterial virulence and in blood dissemination capacity, and (iii) significantly weakened in the ability to block macrophage activation and to survive within macrophages. Furthermore, when used as a subunit vaccine, purified recombinant DnaJ induced protective immunity in Japanese flounder (Paralichthys olivaceus). Taken together, these results indicate that DnaJ plays an important role in the pathogenesis of E. tarda probably by functioning as a DnaK partner and that DnaJ, with its immunoprotective property, may be useful in the control of E. tarda infection in aquaculture.
DnaJ/Hsp40 家族成员通过调节 DnaK/Hsp70 的活性在蛋白质稳态中发挥重要作用。在这项研究中，我们研究了爱德华氏菌的 DnaJ，爱德华氏菌是一种严重的鱼类病原体，也可以感染人类和鸟类。计算机分析表明，爱德华氏菌 DnaJ 含有结构特征，即 J 结构域、甘氨酸/苯丙氨酸丰富区和锌指结构域，这些结构特征在 I 型 Hsp40 中是保守的。纯化的重组 DnaJ 能够刺激 DnaK 的 ATP 酶活性。下拉实验表明 DnaJ 可以与 DnaK 特异性相互作用。J 结构域中保守的 HPD 位点的突变完全消除了 DnaJ 对 DnaK 的刺激作用。为了研究 DnaJ 的功能重要性，构建了 dnaJ 缺陷突变体。与野生型相比，dnaJ 突变体 (i) 生长迟缓，对 H₂O₂诱导的氧化损伤更敏感，(ii) 一般细菌毒力和血液传播能力显著降低，(iii) 阻断巨噬细胞激活和在巨噬细胞内存活的能力显著减弱。此外，当用作亚单位疫苗时，纯化的重组 DnaJ 诱导日本牙鲆（Paralichthys olivaceus）产生保护性免疫。综上所述，这些结果表明 DnaJ 可能通过作为 DnaK 伴侣发挥作用而在爱德华氏菌的发病机制中发挥重要作用，并且具有免疫保护特性的 DnaJ 可能有助于控制水产养殖中的爱德华氏菌感染。
