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首页> 外文期刊>Infection and immunity >Role of Various Enterotoxins in Aeromonas hydrophila-Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity
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Role of Various Enterotoxins in Aeromonas hydrophila-Induced Gastroenteritis: Generation of Enterotoxin Gene-Deficient Mutants and Evaluation of Their Enterotoxic Activity

机译：各种肠毒素在嗜水气单胞菌引起的胃肠炎中的作用：肠毒素基因缺陷型突变体的产生及其肠毒性活性的评估

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Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.
机译：在我们的实验室中已经对三种来自嗜水气单胞菌腹泻分离株SSU的三种肠毒素进行了分子表征。这些肠毒素中的一种在性质上具有细胞毒性，而其他两种是细胞紧张性肠毒素，其中一种热不稳定，另一种热稳定。早期，通过开发同基因突变体，我们证明了细胞毒性肠毒素在引起小鼠全身性感染中的作用。在本研究中，我们通过通过标记交换诱变开发肠毒素基因缺陷型突变体的各种组合，评估了这三种肠毒素在小鼠模型中引起腹泻的作用。在 A的细胞毒性肠毒素基因（ act ）阳性或阴性背景菌株中共制备了六个同基因突变体。亲水。我们在热不稳定的（ alt ）或热稳定的（ ast ）胞吞性肠毒素基因中开发了两个带有截短的单敲除;三个双重敲除，其截短了编码（i） alt 和 ast ，（ii） act 和 alt 的基因的基因， （iii） act 和 ast 基因;以及在所有三个基因 act ， alt 和 ast 中均被截断的三敲除突变体。通过Southern印迹分析证实通过双交换同源重组产生的这些同基因突变体的身份。 Northern和Western印迹分析表明，突变体中不同肠毒素基因的表达相应地被消除。我们通过回肠结扎试验在饮食限制和抗生素治疗的小鼠模型中测试了这些突变体的生物活性。我们的数据表明，所有这些突变体与野生型 A相比，具有明显降低的诱发液体分泌的能力。亲水性;三重敲除突变体未能诱导任何可检测水平的体液分泌。所选 A的生物活性。互补后恢复了亲水性突变体。两者合计，我们已经建立了 A中三种肠毒素的作用。在小鼠模型中，嗜水杆菌引起的肠胃炎的作用最大，其起因于细胞毒性肠毒素法，其次是Alt和Ast胞质性肠毒素。

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