Sha Jian, Erova Tatiana E, Alyea Rebecca A, Wang Shaofei, Olano Juan P, Pancholi Vijay, Chopra Ashok K
Department of Microbiology and Immunology, Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555, USA.
J Bacteriol. 2009 May;191(9):3095-107. doi: 10.1128/JB.00005-09. Epub 2009 Mar 6.
In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.
在本研究中，我们证明了嗜水气单胞菌腹泻分离株SSU表面表达的烯醇化酶与人纤溶酶原结合，并促进后者在组织型纤溶酶原激活剂介导下激活为纤溶酶。细菌表面结合的纤溶酶对其特异性生理抑制剂抗蛋白酶α2-抗纤溶酶的作用更具抗性。我们发现，用纯化的重组烯醇化酶免疫小鼠可显著保护动物免受致死剂量野生型嗜水气单胞菌的攻击。与感染且未免疫动物组中发现的严重病理变化相比，用烯醇化酶免疫然后用野生型细菌攻击的小鼠器官中的组织学变化最小。这与烯醇化酶免疫小鼠肝脏和脾脏中野生型细菌的载量低于未免疫对照组相关。我们还表明，烯醇化酶基因可能对嗜水气单胞菌SSU的生存能力很重要，因为只有当靶向基因的另一个拷贝通过反式提供时，我们才能删除烯醇化酶基因的染色体拷贝。通过定点诱变，我们改变了嗜水气单胞菌SSU烯醇化酶位于第343、394、420、427和430位的五个赖氨酸残基；烯醇化酶的突变形式在大肠杆菌中过表达，并对蛋白质进行了纯化。我们的结果表明，烯醇化酶第420和427位的赖氨酸残基在纤溶酶原结合活性中至关重要。我们还在嗜水气单胞菌SSU烯醇化酶中鉴定出一段参与纤溶酶原结合的氨基酸残基序列（(252)FYDAEKKEY(260)）。据我们所知，这是关于表面表达的烯醇化酶直接参与嗜水气单胞菌SSU感染发病机制以及一般革兰氏阴性菌发病机制的首次报道。
