Characterization of a novel AB5 cytotoxin

我们最近发现了一种新的由志贺产毒菌株产生的“枯草杆菌细胞毒素” 导致溶血性尿毒症暴发的大肠杆菌(B类优先病原体) 综合征(HUS)。它属于一类新的AB5细胞毒素，因为它的A亚基具有独特的酶 活性，与任何其他AB5毒素家族(霍乱、志贺氏菌和 百日咳毒素)。枯草杆菌细胞毒素对培养细胞有极强的细胞毒性，对小鼠(含 组织病理学与HUS相似)。除了它在严重的人类疾病中可能起的作用外，它还是一个潜在的 可能对全球产生影响的生物恐怖分子。对PA-04-119做出回应的长期目标 是新出现的毒素的完整结构和功能特征。其具体目标是： 1.阐明细胞毒性的分子基础。 亚基枯草杆菌酶样丝氨酸蛋白酶活性对细胞毒性是必不可少的，初步数据表明 内质网伴侣蛋白Bip是这种毒素的关键细胞靶点。为此，我们将继续调查 通过研究BiP裂解的下游后果，细胞毒性的分子基础包括 这是否会导致内质网应激和细胞凋亡。 2.靶细胞内毒素转运的研究。 我们推测，毒素在与细胞表面结合后，一定会逆行转运到内质网 以发挥其毒性作用。因此，将通过共同定位来检查细胞内毒素的运输 共聚焦显微镜下用细胞隔室标记的毒素。 3.毒素的组织趋向性和体内效应的检测。 本课程将研究毒素受体在小鼠组织中的分布和标记毒素在体内的转运。 使用荧光显微镜、共聚焦显微镜和电子显微镜。枯草杆菌细胞毒素对细胞毒性的直接作用 STEC疾病的发病机制也将在小鼠模型中进行研究。 4.确定毒素的三维结构。 将使用X射线结晶学和计算机建模来解决3D结构。 相关性：该项目将提供关于这种新出现的毒素的基本生物学的基本信息， 特别是它的结构和破坏细胞和组织的机制。这将提供一个 为包括治疗剂和疫苗在内的对策的未来发展奠定基础。

We have recently discovered a novel "Subtilase cytotoxin" produced by a strain of Shiga toxigenic Escherichia coli (a category B Priority Pathogen) that was responsible for an outbreak of hemolytic uremic syndrome (HUS). It belongs to a new class of AB5 cytotoxins, because its A subunit has distinct enzymic activity and it has no sequence similarity with any of the other AB5 toxin families (cholera, Shiga and pertussis toxins). Subtilase cytotoxin is extraordinarily cytotoxic for cultured cells and is lethal for mice (with histopathology similar to that of HUS). Apart from its possible role in severe human disease, it is a potential bioterrorism agent which could have global impact. The long-term objective of this response to PA-04-119 is complete structural and functional characterization of the emerging toxin. Its Specific Aims are: 1. Elucidation of the molecular basis for cytotoxicity. A subunit Subtilase-like serine protease activity is essential for cytotoxicity, and preliminary data indicate that the ER chaperone BiP is a key cellular target for the toxin. In this Aim, we will continue our investigation of the molecular basis for cytotoxicity by examining the downstream consequences of BiP cleavage, including whether this results in ER stress and apoptosis. 2. Investigation of toxin trafficking in target cells. We hypothesize that after binding to the cell surface, the toxin must undergo retrograde transport to the ER in order to exert its toxic effects. Intracellular toxin trafficking will therefore be examined by co-localization of labelled toxin with cellular compartmental markers using confocal microscopy. 3. Examination of tissue tropism and in vivo effects of the toxin. The distribution of toxin receptors in mouse tissues and trafficking of labelled toxin in vivo will be examined using fluorescence, confocal and electron microscopy. The direct contribution of Subtilase cytotoxin to the pathogenesis of STEC disease will also be investigated in a mouse model. 4. Determination of the 3Dstructure of the toxin. X-ray crystallography and computer modelling will be used to solve the 3D structure. Relevance: This project will provide essential information on the basic biology of this emerging toxin, particularly its structure and the mechanism whereby it damages cells and tissues. This will provide a foundation for the future development of countermeasures including therapeutic agents and vaccines.