Pore Formation by Cholesterol Dependent Cytolysins

描述(申请人提供)：Perfringolysin 0(PFO)，一种细胞溶血素 (Mr 54,000)由产气荚膜梭菌产生和分泌，属于 相关细胞溶血素家族现在称为胆固醇依赖细胞溶血素 (CDCS)，由多种革兰氏阳性病原菌产生 物种。PFO是CDC的典型代表，具有亲水性的一级结构， 最终形成溶细胞膜复合体。在绑定到目标之后 膜，PFO单体齐聚成超分子络合物，裂解 手机。在目前的赠款期间，我们确定了PFO的区域 形成水-膜界面，并确定每个单体插入 两个B-发夹进入双层以形成毛孔的B-桶。我们还发现 该PFO在插入这些结构域之前形成了前孔复合体。这个 这里的研究旨在通过以下方式加深我们对这一机制的理解 这些耐人寻味的毒素改变了它们的结构并与 另一种是与膜表面的相互作用，使它们从可溶的 单体转变为膜结合的低聚物复合体。这样做的具体目的是 建议：1)确定PFO相对于膜的形貌 并确定不同孔道的分子内构象几率 队形。2)阐明了跨膜B-发夹之间的相互作用 低聚物。3)确定亚基-亚基中涉及的PFO残留量 互动。4)鉴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结构中不同点到膜的距离 在其膜穿透的不同阶段。目标2将是 通过表征天然毒素诱导 插入PfO突变体的跨膜B-发夹(TMH)，这种突变体单独可以 形成低聚前孔，但不能插入它们的TMH。在目标3中， 膜结合低聚物中FF0的单体-单体界面 由于无法接触到水和膜而显露出来 荧光探头的碰撞猝灭器将放置在不同的 半胱氨酸取代衍生物中单体的表面位置 PFO。残留物在界面上的位置将通过以下方式确认 特定部位的交联剂。最后，我们将调查耐人寻味的 CDC家族成员中间溶素的属性，该属性限制其 红细胞对人类红细胞的特异性，与所有其他已知的 疾控中心。我们将通过一种识别和鉴定中间溶素受体的方法 受体印迹和亲和纯化相结合的方法。

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.