Erova Tatiana E, Pillai Lakshmi, Fadl Amin A, Sha Jian, Wang Shaofei, Galindo Cristi L, Chopra Ashok K
Department of Microbiology and Immunology, University of Texas Medical Branch, 3.142D Medical Research Building, 301 University Boulevard, Galveston, TX 77555-1070, USA.
Infect Immun. 2006 Jan;74(1):410-24. doi: 10.1128/IAI.74.1.410-424.2006.
Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam(AhSSU)) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N(6)-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P(BAD) promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.
在嗜水气单胞菌产生的各种毒力因子中，II型分泌系统（T2SS）分泌的细胞毒性肠毒素（Act）和III型分泌系统（T3SS）在嗜水气单胞菌相关感染的发病机制中起着关键作用。我们实验室对来自腹泻分离株嗜水气单胞菌SSU的Act和T3SS进行了分子特征分析，并确定了一些调控基因在调节Act生物学效应中的作用。在本研究中，我们使用大肠杆菌ER2566作为宿主菌株，在基于T7启动子的载体系统中克隆、测序并表达了嗜水气单胞菌SSU的DNA腺嘌呤甲基转移酶基因（dam(AhSSU)），该基因可改变嗜水气单胞菌的毒力潜力。重组Dam命名为M.AhySSUDam，作为带组氨酸标签的融合蛋白产生，并使用镍亲和层析从大肠杆菌细胞裂解物中纯化。纯化的Dam具有甲基转移酶活性，基于其将甲基从S-腺苷-L-甲硫氨酸转移到无N(6)-甲基腺嘌呤的λDNA上，并保护甲基化的λDNA不被DpnII消化但不被DpnI限制酶消化的能力。dam基因对细菌的生存能力至关重要，在嗜水气单胞菌SSU中使用基于阿拉伯糖诱导型P(BAD)启动子的系统过量表达Dam，降低了该病原体的毒力。具体而言，M.AhySSUDam的过量表达使细菌的运动性降低了58%。同样，与单独携带pBAD载体的对照嗜水气单胞菌SSU菌株相比，在感染过量表达Dam菌株的小鼠巨噬细胞中，通过乳酸脱氢酶释放量测量的T3SS相关细胞毒性降低了55%。相反，但与对照嗜水气单胞菌SSU菌株相比，过量表达Dam菌株的培养上清液中与Act相关的细胞毒性和溶血活性以及蛋白酶活性分别增加了10倍、3倍和2.4倍。当以腹腔注射途径给予过量表达Dam菌株时，其剂量为50%致死剂量的两倍，该菌株对小鼠无致死性（100%存活），而接种嗜水气单胞菌对照菌株的动物在2至3天内100%死亡。综上所述，我们的数据表明过量表达Dam会改变嗜水气单胞菌的毒力。
