Pore Formation by Cholesterol Dependent Cytolysins

性状（由申请方提供）：产气荚膜梭菌溶素0（PFO），一种溶细胞素 (Mr 54，000）由产气荚膜梭菌产生和分泌，属于 相关溶细胞素家族，现称为胆固醇依赖性溶细胞素 CDCs是由多种革兰氏阳性病原菌产生的 物种PFO代表CDC，具有亲水性一级结构， 最终形成溶细胞膜复合物。在与目标结合后 膜，PFO单体低聚成超分子复合物，并裂解 cell.在当前的赠款期间，我们确定了PFO的区域， 形成水膜界面，并确定插入的每个单体 两个B-发夹进入双层以形成孔的B-桶。我们还发现 PFO在插入这些结构域之前形成前孔复合物。的 本文的研究旨在通过以下方式进一步理解该机制： 这些有趣的毒素改变了它们的结构， 另一个和膜表面，因为它们从可溶性的 单体与膜结合的低聚复合物的结合。具体目标是 建议是：1）确定PFO相对于膜的形貌 并确定了不同孔隙状态下分子内的构象机会 阵2)阐明跨膜B-hairpins之间的相互作用， 低聚物。3)鉴定参与亚基-亚基的PFO残基 交互. 4)确定中间溶素受体的性质。我会瞄准的 通过使用荧光共振能量转移（FRET）来实现， 测量PFO结构中各点到膜的距离 在不同阶段的膜渗透。目标2将是 这是通过表征天然毒素诱导 PFO突变体的跨膜B-发夹（TMH）的插入， 形成寡聚前孔，但不能插入它们的TMH。在aim 3中， 膜结合低聚复合物中FF 0的单体-单体界面将 通过缺乏对水和膜限制的可及性来揭示 荧光探针的碰撞猝灭剂将被放置在不同的位置， 半胱氨酸取代的衍生物中的单体表面上的位置 PFO。界面处残留物的位置将通过以下方式确认： 位点特异性交联。最后，我们将研究有趣的 中间溶素是CDC家族的一员，它限制了其 红细胞对人红细胞的特异性，与所有其他已知的 疾病控制中心。我们将鉴定和表征中间溶素的受体， 受体印迹和亲和纯化方法的组合。

DESCRIPTION (provided by the applicant): Perfringolysin 0 (PFO), a cytolysin (Mr 54,000) produced and secreted by Clostridium perfringens, belongs to a family of related cytolysins now termed the cholesterol-dependent cytolysins (CDCs) and is produced by a variety of Gram positive pathogenic bacterial species. PFO typifies the CDCs, with a hydrophilic primary structure that ultimately forms a cytolytic membrane complex. After binding to the target membrane, PFO monomers oligomerize into supramolecular complexes and lyse the cell. During the current grant period, we identified the regions of PFO that form the aqueous-membrane interface and determined that each monomer inserted two B-hairpins into the bilayer to form the B-barrel of the pore. We also found that PFO forms a prepore complex prior to the insertion of these domains. The studies herein are designed to further our understanding of the mechanism by which these intriguing toxins alter their structure and interact with one another and the membrane surface as they make the transition from a soluble monomer to a membrane-bound oligomeric complex. The specific aims of this proposal are to: 1) Determine the topography of PFO relative to the membrane and identify intramolecular conformational chances at different sta2es of pore formation. 2) Elucidate the interactions between transmembrane B-hairpins in the oligomer. 3) Identify the PFO residues involved in subunit-subunit interactions. 4)Identify the nature of the intermedilysin receptor. Aim I will be accomplished by the use of fluorescence resonance energy transfer (FRET) to measure distances from various points in the PFO structure to the membrane surface at different stages of its membrane penetration. Aim 2 will be accomplished by characterizing the ability of native toxin to induce the insertion of the transmembrane B-hairpins (TMHs) of PFO mutants that alone can form an oligomeric prepore, but cannot insert their TMHs. In aim 3 the monomer-monomer interfaces of FF0 in the membrane-bound oligomeric complex will be revealed by the lack of accessibility to aqueous and membrane-restricted collisional quenchers of a fluorescent probe that will be placed at various locations on the surface of the monomer in cysteine-substituted derivatives of PFO. The location of the residue at an interface will be confirmed by site-specific crosslinking. Finally, we will investigate the intriguing property of intermedilysin, a member of the CDC family, which restricts its erythrocyte specificity to human erythrocytes, in contrast to all other known CDCs. We will identify and characterize the receptor for intermedilysin by a combination of receptor blots and affinity purification methods.