Molecular, Biochemical and Functional Analysis of Shiga Toxin and Subtilase Cytotoxin Subunits of Enterohemorrhagic Escherichia coli

观察到E.大肠杆菌枯草杆菌酶和滋贺毒素在没有各自B亚基的情况下也能引起细胞毒性，这代表了基本毒理学范式的变化。根据这些最近的发现，拟议项目的主要目标是详细描述两种AB 5毒素的这种意外行为。因此，我们将进一步表征SubA亚基的细胞结合和细胞内转运，并分析Stx 2 A亚基（StxA 2）在人细胞中不存在结合/转运亚基的情况下细胞毒性作用的分子机制。详细地说，我们将研究结合，摄取和细胞内运输的StxA 2和直接比较这些过程与典型的全毒素Stx 2和Stx 1，分别由1个A-和5个B-亚基。此外，将发现和表征药理学抑制剂，包括中和Stx 2和Stx 1的细胞毒性作用的人肽，如之前对SubAB所做的那样。此外，成熟毒素从细菌细胞的表达、组装和分泌的模式留下了未解决的问题。因此，将表征stx亚基基因转录和亚基组装的控制，以了解细菌培养物的上清液中是否存在游离A亚基。这项工作将有助于更深入地了解EHEC介导的疾病发病机制的分子和细胞机制，并为新的治疗方案提供一个起点。

The observation, that the A-subunits of E. coli subtilase and Shiga toxin can cause cytotoxicity without their respective B-subunits, represents a change in a basic toxicological paradigm. Prompted by these recent findings, the main objective of the proposed project is to characterize this unexpected behavior of both AB5-toxins in detail. Therefore, we will further characterize the cell binding and intracellular transport of the SubA-subunit and analyze the molecular mechanisms underlying the cytotoxic effects of the Stx2 A-subunit (StxA2) in the absence of a binding/transport subunit in human cells. In detail, we will investigate binding, uptake and intracellular transport of StxA2 and directly compare these processes with the canonical holotoxins Stx2, and Stx1, consisting of 1 A- and 5 B-subunits, respectively. Furthermore, pharmacological inhibitors including human peptides that neutralize the cytotoxic effects of Stx2 and Stx1 will be discovered and characterized, as done before for SubAB. Moreover, the mode of expression, assembly and secretion of the mature toxin from bacterial cells leaves unresolved questions. Therefore, control of stx subunit gene transcription and subunit assembly will be characterized to understand whether free A-subunits are present in the supernatant of bacterial cultures. This work will contribute to a deeper understanding of the molecular and cellular mechanisms underlying the pathogenesis of EHEC-mediated diseases and should provide a starting point for novel therapeutic options.