A Novel Cholesterol-Dependent Cytolysin Receptor

说明(申请人提供)：胆固醇依赖细胞溶血素(CDCs)是由20多种革兰氏阳性病原菌产生的一大类相关细菌毒素。一些疾控中心已被证明对这些细菌的致病机制做出了贡献。CDC是以可溶性蛋白质的形式产生的，这些蛋白质在含胆固醇的膜表面齐聚成大的成孔复合体。膜胆固醇的存在是CDC成孔所必需的，也是CDC的一个标志。三十多年来，CDCs的受体被普遍认为是胆固醇，但最近的研究表明，CDCs的前孔到孔的转换是必要的，这是膜结合的下游事件。因此，疾控中心使用胆固醇作为受体的长期教条似乎不太可信。我们现在已经确定，CDC家族中至少有一个成员，来自中间链球菌的中间溶素(ILY)，利用GPI锚定的人CD59(HCD59)作为其受体。根据初步研究，我们假设：1)ILY结构域4含有与hCD59结合和特异性所需的残基，2)人补体蛋白C8和C9识别的hCD59区域和残基也包括ILY的hCD59结合部位，3)ILY在形成孔道之前与其受体脱离。这项建议旨在研究ILY与hCD59的相互作用，并绘制参与这种配体-受体相互作用的ILY和hCD59的结构域。ILY与hCD59的相互作用也将在细胞溶解机制的不同阶段进行检测，以确定毒素在其毛孔复合体组装过程中是否与受体脱离。这些假说将在三个特定的目标中得到解决，这三个目标将确定hCD59和ILY上的结合位点的位置，并将确定ILY在组装成孔复合体的过程中是否与受体脱离。这些研究的结果将为研究疾病预防控制中心的一个新的和重要的方面提供基础，以前这些毒素的研究是无法获得的。CDC受体的发现和研究将对CDC的研究产生重大影响，并将改变我们对CDC在由多种革兰氏阳性病原体引起的大量疾病中所起作用的看法。

DESCRIPTION (provided by applicant): The cholesterol-dependent cytolysins (CDCs) are a large family of related bacterial toxins produced by over 20 species of Gram-positive pathogenic bacteria. Several CDCs have been shown to contribute to the pathogenic mechanisms of these bacterial species. The CDCs are produced as soluble proteins that oligomerize into large pore-forming complexes on the surface of cholesterol-containing membranes. The presence of membrane cholesterol is required for the CDC pore-formation and is a hallmark of the CDCs. For over three decades the receptor for the CDCs was generally accepted as cholesterol but recent studies show that it is necessary for the prepore to pore conversion of the CDCs, an event that is downstream of membrane binding. Therefore, the longstanding dogma that the CDCs use cholesterol as their receptor appears less plausible. We have now determined that at least one member of the CDC family, intermedilysin (ILY) from Streptococcus intermedius, utilizes the GPI-anchored human CD59 (hCD59) as its receptor. Based on preliminary studies we hypothesize that 1) ILY domain 4 contains the residues required for binding to and specificity for hCD59, 2) the region and residues of hCD59 recognized by human complement proteins C8 and C9 also comprises the hCD59 binding site for ILY, and 3) ILY disengages from its receptor prior to pore formation. This proposal is designed to examine the interaction of ILY with hCD59 and to map out the structural domains of both ILY and hCD59 that are involved in this ligand-receptor interaction. The interaction of ILY with hCD59 will also be examined at various stages of the cytolytic mechanism to determine if the toxin disengages from receptor during the assembly of its pore complex. These hypotheses will be addressed in three specific aims that will establish the location of the binding sites on both hCD59 and ILY and will determine whether ILY undocks from receptor during the assembly of the pore-forming complex. The results of these studies will provide a basis for investigations of a new and significant aspect of the CDCs previously unavailable to the study of these toxins. The discovery and study of a CDC receptor will have a significant impact on the study of CDCs and will alter our perception of their role in a large number of diseases caused by a wide variety of Gram-positive pathogens.