CYTOLYTIC ENTEROTOXIN OF AEROMONAS HYDROPHILA

描述（改编自申请人的摘要）：长期目标 本建议是确定一种具有溶细胞作用的肠毒素（Act）的A。 在疾病的发病机制中起重要作用。 调查人员已经分离出 来自A.苏（SSU）， 与溶血、细胞毒性和肠毒性活性相关， 对老鼠是致命的。 该毒素基因已被克隆，表达和测序， 调查人员 最近完成的定点诱变研究 表明不同的基因座编码的三个生物活性相关 带着毒素 建议法案发挥重要作用， 一个传染过程是由缺乏毒力的行为减去支持 分离物A.具有降低的生物学活性的转座子和转座子突变体 活动 最近对SSU菌株的同基因突变体的研究表明， 没有体外溶血和细胞毒活性也支持了以下信念 这种毒素是一种重要的毒力因子 表征 毒素的作用将继续使用毒素阴性突变体最近 制作。 探测免疫球蛋白的抗原和生物活性结构域， 毒素将在Act基因的超表达后完成， 多宿主范围向量。 毒素分子的功能结构域将 通过产生抗肽抗体和单克隆抗体来定位。 定点突变将用于改进氨基酸的作用， 毒素生物活性的残留物。 调查人员还 我建议研究Act基因的调控，要么将其与一种 报告基因或通过使用 转座子赋予抗生素抗性以筛选毒素阴性 变种人 最后，研究人员建议研究 通过探测细胞损伤的机制和通过检查毒素的作用， 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.