CYTOLYTIC ENTEROTOXIN OF AEROMONAS HYDROPHILA

描述(改编自申请人的摘要)：长期目标 本研究确定了A。 嗜水症在疾病发病机制中的作用。调查人员已经分离出 嗜水气单胞菌腹泻分离株(SSU)的52 kDa多肽 与溶血、细胞毒性和肠道毒性活动有关，并且是 对老鼠来说是致命的。该毒素基因已被克隆、表达和测序 调查人员。最近完成的定点突变研究 建议对相关的三种生物活动进行不同的基因编码 带着毒素。关于《法案》在 传染过程是由一项行为的毒性不足所支持的 生物减量嗜水气单胞菌和转座子突变株的分离 活动。最近对SSU菌株的等基因突变体的研究 没有体外溶血和细胞毒活性也支持这样的观点 这种毒素是一个重要的致病因子。刻画人物形象 毒素的作用将在最近利用毒素阴性突变体进行 制作。探讨猪瘟病毒的抗原性和生物活性结构域 毒素将在ACT基因的高效表达后完成 多主机距离向量。毒素分子的功能结构域将 通过产生抗肽抗体和单抗来定位。 定点突变将用来提炼氨基酸的作用 残留在生物活性的毒素中。调查人员还 建议研究ACT基因的调节，要么将其与一种 报告基因或对气单胞菌染色体的诱变 赋予抗生素耐药性的转座子用于筛选毒素阴性 变种人。最后，研究人员建议对其机制进行研究。 通过探讨细胞损伤的机制和通过检测毒素的作用 细胞表面的毒素受体。

DESCRIPTION (Adapted from applicant's abstract): The long term objective of this proposal is determine the role of a cytolytic enterotoxin (Act) of A. hydrophila in the pathogenesis of disease. The investigators have isolated a 52 kDa polypeptide from a diarrheal isolate of A. hydrophila (SSU) that is associated with hemolytic, cytotoxic and enterotoxic activities and is lethal for mice. The toxin gene has been cloned, expressed and sequenced by the investigators. Site directed mutagenesis studies recently completed suggest different loci coding for the three biological activities associated with the toxin. The suggestion that Act plays a significant role in the infectious process is supported by the lack of virulence of an Act minus isolate of A. hydrophila and transposon mutants with reduced biological activity. More recent studies with an isogenic mutant of strain SSU that is without in vitro hemolytic and cytotoxic activity also supports the belief that this toxin is an important virulence factor. Characterization of the role of the toxin will proceed using the toxin negative mutant recently produced. Probing the antigenic and biologically active domains of the toxin will be accomplished following hyperexpression of the Act gene with a multi-host range vector. The functional domains of the toxin molecule will be mapped by generating anti-peptide antibodies and monoclonal antibodies. Site directed mutagenesis will be used to refine the role of amino acid residues in the biological activities of the toxin. The investigators also propose to study the regulation of the Act gene either by fusing it with a reporter gene or by mutagenesis of the chromosome of Aeromonas using a transposon which imparts antibiotic resistance to screen for toxin negative mutants. Finally, the investigators propose to study the mechanism of action of the toxin by probing the mechanism of cell damage and by examining the toxin receptor on the cell surface.