STUDIES ON CLOSTRIDIUM SEPTICUM LETHAL TOXIN

描述(改编自申请者摘要)：败血梭菌 导致几种危及生命的感染，如果不治疗，这些感染是致命的。 这些疾病中最具破坏性的是非创伤性气性坏疽。唯一的 败血症弧菌分泌的致死因子是一种α毒素。它是一种溶细胞剂， 一种形成毛孔的毒素，以非活性原毒素的形式产生，需要 被正常的细胞蛋白水解酶激活，如呋喃。这个 研究人员建议继续对细胞溶解机制进行详细研究 以深入了解其生物学特性并探索方法 这可能被用来改善其在体内的效果。他们建议：1)确定 阿尔法毒素前肽的关键残基促进其 与毒素主体的非共价相互作用并产生 对阿尔法毒素具有更强抑制活性的衍生物，2)鉴定 α毒素的跨膜区(S)，3)识别残基 参与受体结合的毒素，以及4)结晶更易溶的 阿尔法毒素的衍生物以及该衍生物与一种 GPI锚定的阿尔法毒素受体。为了达到第一个目标，残留物 将被甘氨酸顺序取代，分离和 测定前肽与毒素的亲和力。在《目标2》中，他们将 利用两种方法绘制出α穿膜结构域(S) 毒素。第一种方法将是用可疑的膜跨接替代 α毒素与半胱氨酸的残留物提纯这些衍生物，然后形成 每种毒素的通道都在一个平面双层中。的带电衍生工具 然后将巯基特异性试剂甲硫磺酸盐(MTS)引入到 双分子层两侧的水相。带电的MTS试剂会导致 仅当半胱氨酸被取代时通道电导的变化 作为渠道衬里残留物。相同的半胱氨酸取代残基(在 不含半胱氨酸的阿尔法毒素衍生物)也将用 环境敏感荧光探针NBD及其所检测的荧光 在毒素被允许插入细胞膜之前和之后。如果是Alpha 毒素通过一种两亲性的β-折叠或一个 他们会观察到反应周期的不同 从两种化验结果来看。受体结合域已初步定位于 在C55附近有阿尔法毒素的区域。因此，在AIM 3中，C55附近的残基 通过体外诱变将阿尔法毒素的结构模型改变为 确定哪些残基参与受体结合。在目标4中，他们提出了 使阿尔法毒素和阿尔法毒素-受体复合体结晶。这是做的 有可能通过大量获得更易溶的形式的 阿尔法毒素及其GPI锚定受体之一--人叶酸受体 (HFR)。

DESCRIPTION (Adapted from the Applicant's Abstract): Clostridium septicum causes several life-threatening infections that, without treatment, are fatal. The most devastating of these diseases is non-traumatic gas gangrene. The only lethal factor secreted by C. septicum is alpha toxin. It is a cytolytic, pore-forming toxin that is produced as an inactive protoxin which requires proteolytic activation by normal cellular proteases such as furin. The investigators propose to continue the detailed study of the cytolytic mechanism of alpha toxin in order to gain insight into its biology and to explore ways that may be used to ameliorate its effect in vivo. They propose to: 1) identify the crucial residues of the propeptide of alpha toxin which facilitate its non-covalent interactions with the main body of the toxin and generate derivatives with a greater inhibitory activity towards alpha toxin, 2) identify the transmembrane domains(s) of alpha toxin, 3) identify the residues of the toxin involved in receptor binding, and 4) crystallize a more soluble derivative of alpha toxin and the complex of this derivative with one of the GPI-anchored receptors for alpha toxin. To achieve the first aim, the residues of the propeptide will be sequentially substituted with glycine, isolated and the affinity of the propeptide for the toxin determined. In aim 2 they will utilize two approaches to map out the membrane-penetrating domains(s) of alpha toxin. The first approach will be to substitute suspected membrane-spanning residues of alpha toxin with cysteine purify these derivatives and then form channels with each toxin in a planar bilayer. Charged derivatives of the sulfhydryl-specific reagent methanethiosulfonate (MTS) are then introduced into aqueous phase on either side of the bilayer. The charged MTS reagent will cause a change in the channel conductance only if the cysteine has been substituted for a channel-lining residue. The same cysteine-substituted residues (in a cysteine-less derivative of alpha toxin) will also be modified with the environmentally sensitive fluorescent probe NBD and the fluorescence examined before and after the toxin have been allowed to insert into membranes. If alpha toxin interacts with the membrane via either an amphipathic beta sheet or an alpha helix they will observe a difference in the periodicity of the response from both assays. The receptor-binding domain has tentatively been localized to a region near C55 of alpha toxin. Thus, in aim 3, residues near C55 in the structural model of alpha toxin will be changed by in vitro mutagenesis to determine which residues participate in receptor binding. In aim 4 they propose to crystallize alpha toxin and the alpha toxin-receptor complex. This is made possible by the availability of large quantities of a more soluble form of alpha toxin and one of its GPI-anchored receptors, the human folate receptor (hFR).