Xu X J, Ferguson M R, Popov V L, Houston C W, Peterson J W, Chopra A K
Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1070, USA.
Infect Immun. 1998 Aug;66(8):3501-9. doi: 10.1128/IAI.66.8.3501-3509.1998.
Transposon and marker exchange mutagenesis were used to evaluate the role of Aeromonas cytotoxic enterotoxin (Act) in the pathogenesis of diarrheal diseases and deep wound infections. The transposon mutants were generated by random insertion of Tn5-751 in the chromosomal DNA of a diarrheal isolate SSU of Aeromonas hydrophila. Some of the transposon mutants had dramatically reduced hemolytic and cytotoxic activities, and such mutants exhibited reduced virulence in mice compared to wild-type Aeromonas when injected intraperitoneally (i.p.). Southern blot data indicated that transposition in these mutants did not occur within the cytotoxic enterotoxin gene (act). The transcription of the act gene was affected drastically in the transposon mutants, as revealed by Northern blot analysis. The altered virulence of these transposon mutants was confirmed by developing isogenic mutants of the wild-type Aeromonas by using a suicide vector. In these mutants, the truncated act gene was integrated in place of a functionally active act gene. The culture filtrates from isogenic mutants were devoid of hemolytic, cytotoxic, and enterotoxic activities associated with Act. These filtrates caused no damage to mouse small intestinal epithelium, as determined by electron microscopy, whereas culture filtrates from wild-type Aeromonas caused complete destruction of the microvilli. The 50% lethal dose of these mutants in mice was 1.0 x 10(8) when injected i. p., compared to 3.0 x 10(5) for the wild-type Aeromonas. Reintegration of the native act gene in place of the truncated toxin gene in isogenic mutants resulted in complete restoration of Act's biological activity and virulence in mice. The animals injected with a sublethal dose of wild-type Aeromonas or the revertant, but not the isogenic mutant, had circulating toxin-specific neutralizing antibodies. Taken together, these studies clearly established a role for Act in the pathogenesis of Aeromonas-mediated infections.
转座子诱变和标记交换诱变被用于评估嗜水气单胞菌细胞毒素肠毒素（Act）在腹泻性疾病和深部伤口感染发病机制中的作用。通过将Tn5 - 751随机插入嗜水气单胞菌腹泻分离株SSU的染色体DNA中来产生转座子突变体。一些转座子突变体的溶血和细胞毒性活性显著降低，与野生型嗜水气单胞菌相比，经腹腔注射（i.p.）后，这些突变体在小鼠中的毒力降低。Southern印迹数据表明，这些突变体中的转座并非发生在细胞毒素肠毒素基因（act）内。Northern印迹分析显示，转座子突变体中act基因的转录受到严重影响。通过使用自杀载体构建野生型嗜水气单胞菌的同基因突变体，证实了这些转座子突变体毒力的改变。在这些突变体中，截短的act基因取代了功能活性的act基因。同基因突变体的培养滤液缺乏与Act相关的溶血、细胞毒性和肠毒性活性。通过电子显微镜观察发现，这些滤液对小鼠小肠上皮没有造成损伤，而野生型嗜水气单胞菌的培养滤液则导致微绒毛完全破坏。经腹腔注射时，这些突变体在小鼠中的半数致死剂量为1.0×10⁸，而野生型嗜水气单胞菌为3.0×10⁵。将同基因突变体中截短的毒素基因替换为天然act基因后，Act的生物学活性和在小鼠中的毒力完全恢复。注射亚致死剂量野生型嗜水气单胞菌或回复突变体（而非同基因突变体）的动物体内有循环的毒素特异性中和抗体。综上所述，这些研究明确证实了Act在嗜水气单胞菌介导的感染发病机制中的作用。
