MOLECULAR MECHANISMS OF BACTERIAL PATHOGENESIS

这笔赠款的长期目标是了解 ADP-核糖化外毒素的详细信息；并在可行的情况下，应用我们的 了解这类毒素，以开发更好的疾病控制方法。 所描述的工作主要集中在白喉毒素(DT)和外毒素A 铜绿假单胞菌(PT)，两种相关的毒素，阻止蛋白质合成在 哺乳动物细胞通过催化延伸因子2的ADP-核糖化 (EF-2)。关于DT，我们建议：(I)提供生化 组件以确定协作项目的3维 毒素的结构(可能还有片段和突变形式)； 探测催化中心的结构(特别是NAD结合 站点)，目的是阐明其催化机理。 EF-2的ADP-核糖化；(Iii)研究B插入 部分进入膜，以期了解其作用机制 B促进A链的跨膜转移；(Iv)表征 并检测其与毒素受体结合部位的关系。 聚磷酸盐结合部位(P-位)；(V)为了研究结构， 高亲和力结合位点的特异性及其生物学意义 内源性二核苷酸的DT。关于PT，我们建议：(1) 为三维检测提供生化支持 结构；(Ii)克隆 毒素的结构基因；以及(Iii)寻求详细的 类似于DT概述的结构-活性研究。各种各样的 适用于这些问题的生化和生物物理方法将是 辅以使用单抗、分子克隆和 诱变。结果将与以下方面有关：(1)详细了解 在分子/亚分子水平上的细菌致病；(Ii)细胞 表面受体-配基相互作用；(Iii)插入和 蛋白质对细胞膜的穿透；以及(Iv)可能的机制 将有毒蛋白质或其部分导向特定的细胞。

The long-range goals of this grant are to understand the actions of ADP-ribosylating exotoxins in detail; and, where feasible, to apply our knowledge of such toxins to develop better methods of disease control. Work described is focused primarily on diphtheria toxin (DT) and exotoxin A of P. aeruginosa (PT), two related toxins that block protein synthesis in mammalian cells by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). With respect to DT, we propose: (i) to provide the biochemical component of a collaborative project to determine the 3-dimensional structure of the toxin (and perhaps fragments and mutant forms); (ii) to probe the structure of the catalytic center (particularly the NAD binding site) with the goal of elucidating the mechanism of catalysis of ADP-ribosylation of EF-2; (iii) to investigate the insertion of the B moiety into membranes, with a view to understanding the mechanism by which B promotes transmembrane transfer of the A chain; (iv) to characterize the receptor binding site on the toxin and examine its relationship with the polyphosphate binding site (P-site); (v) to study the structure, specificity, and biological significance of the hig-affinity binding site of DT for endogenous dinucleotides. With respect to PT, we propose: (i) to provide the biochemical support for determination of the 3-dimensional structure through an existing collaboration; (ii) to clone and sequence the structural gene for the toxin; and (iii) to pursue detailed structure-activity studies similar to those outlined for DT. A variety of biochemical and biophysical methods applicable to these problems will be supplemented by the use of monoclonal antibodies, molecular cloning and mutagenesis. The results will be relevant to: (i) detailed understanding of bacterial pathogenesis at the molecular/sub-molecular level; (ii) cell surface receptor-ligand interactions; (iii) mechanisms of insertion and traversal of membranes by proteins; and (iv) potential mechanisms of directing toxic proteins or moieties thereof to specific cells.