VIRULENCE FACTORS OF CORYNEBACTERIUM DIPHTHERIAE

棒状噬菌体Betatox+和一个亲缘关系密切的肾小管上皮细胞家族 携带白喉毒素的结构基因。毒素基因的一部分 已在大肠杆菌中克隆，毒素片段A已被克隆 显示从毒素启动子和输出子表达到周质 塞卡装置旁的隔间。当前的具体目标 建议继续研究白喉毒素的调节 通过(I)对毒素启动子的详细分析来表达 在白喉棒杆菌和大肠杆菌中的功能和(Ii)a 推测的毒素操纵子的详细分析及其分子克隆 白喉弯曲菌的抑制子。 进一步建议我们对白喉毒素的研究 结构/功能域继续存在。具体地说，我们将继续 真核细胞毒素的定位及氨基酸序列研究 细胞受体结合域，以及可能的加工域 片段B，它似乎是膜所必需的 片段A转位到真核细胞胞浆中。两者都有 分子生物学和分子遗传学方法，包括 重组DNA、定点突变、DNA序列分析 因为蛋白质特性和体外毒性测定将是 在调查过程中使用。 这项研究的长期目标是了解分子 铁介导的白喉毒素调控机制研究进展 并对毒素启动子活性进行了比较分析。 白喉和大肠杆菌。我们还将详细了解 白喉毒素结构/功能关系的定点研究 和缺失诱变以及突变体的后续特性 毒素相关多肽。

Corynebacteriophage Betatox+ and a family of closely related corynephage carry the structural gene for diphtheria toxin. Portions of the tox gene have been cloned in Escherichia coli, and fragment A of toxin has been shown to be expressed from the tox promoter and exporter to the periplasmic compartment by the SecA apparatus. The specific aims of the present proposal are to continue the study of diphtheria tox regulation of expression through (i) a detailed analysis of the tox promoter as it functions both in Corynebacterium diphtheriae and in E. coli and (ii) a detailed analysis of the putative tox operator and the molecular cloning of the C. diphtheriae repressor. It is further proposed that our study of diphtheria toxin structural/functional domains be continued. Specifically, we will continue the study of the location and amino acid sequence of the toxin eukaryotic cell receptor binding domain, as well as the putative processing domain in fragment B which appears to be absolutely necessary for the membrane translocation of fragment A into the eukaryotic cell cytosol. Both molecular biologic and molecular genetic methodologies, which include recombinant DNA, site-directed mutagenesis, DNA sequence analysis, as well as protein characterization and in vitro toxicity determinations will be used during the course of this investigation. The long-range goals of this investigation are to understand the molecular mechanisms involved in the iron-mediated regulation of diphtheria tox expression, and the comparative analysis of tox promoter activity in C. diphtheriae and E. coli. We will also provide a detailed understanding of diphtheria toxin structural/functional relationships by site-directed point and deletion mutagenesis and subsequent characterization of mutant toxin-related polypeptides.