Characterization of a novel AB5 cytotoxin

描述（由申请人提供）：我们最近发现了一种由滋贺致突变大肠杆菌（B类优先病原体）菌株产生的新型"枯草杆菌酶细胞毒素"，该菌株导致溶血性尿毒综合征（HUS）爆发。它属于一类新的AB 5细胞毒素，因为它的A亚基具有独特的酶活性，并且它与任何其他AB 5毒素家族（霍乱、滋贺和百日咳毒素）没有序列相似性。枯草杆菌酶细胞毒素对培养的细胞具有非常强的细胞毒性，对小鼠具有致死性（组织病理学与HUS相似）。除了在严重的人类疾病中可能发挥作用外，它还是一种可能产生全球影响的潜在生物恐怖主义制剂。对PA-04 - 119的这种反应的长期目标是对新出现的毒素进行完整的结构和功能表征。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于细胞毒性至关重要，初步数据表明ER伴侣BiP是该毒素的关键细胞靶点。在这个目标中，我们将继续我们的研究细胞毒性的分子基础，通过检查下游的后果，BiP切割，包括这是否会导致ER应激和细胞凋亡。 2.研究靶细胞中的毒素运输。我们推测，结合到细胞表面后，毒素必须进行逆行转运到ER，以发挥其毒性作用。因此，细胞内毒素运输将通过使用共聚焦显微镜将标记的毒素与细胞隔室标记物共定位来检查。 3.毒素的组织嗜性和体内效应的检查。将使用荧光、共聚焦和电子显微镜检查毒素受体在小鼠组织中的分布和标记毒素在体内的运输。还将在小鼠模型中研究枯草杆菌酶细胞毒素对STEC疾病发病机制的直接贡献。 4.确定毒素的3D结构。X射线晶体学和计算机建模将用于解决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，特别是其结构和破坏细胞和组织的机制。这将为今后开发包括治疗剂和疫苗在内的对策奠定基础。

DESCRIPTION (provided by applicant): 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 labeled 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 labeled 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 3D structure of the toxin. X-ray crystallography and computer modeling 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.