STUDIES ON CLOSTRIDIUM SEPTICUM LETHAL TOXIN

败血梭菌致死毒素的研究

• 批准号: 6373255
• 负责人: Rodney K. Tweten
• 金额: $ 14.76万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6373255
• 关键词: Clostridium; bacterial toxins; chimeric proteins; fluorescence spectrometry; membrane permeability; pore forming protein; protein engineering; protein protein interaction; protein sequence; protein structure function; receptor; receptor binding; synthetic peptide

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).

描述（改编自申请人的摘要）：败血性梭菌 导致几种危及生命的感染，如果不治疗，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 C.败血症是α毒素它是一种溶细胞的， 作为一种非活性原毒素产生的成孔毒素， 通过正常细胞蛋白酶如弗林蛋白酶的蛋白水解激活。的 研究人员建议继续进行细胞溶解机制的详细研究 为了深入了解它的生物学， 可用于改善其在体内的作用。他们建议：1）识别 α毒素的前肽的关键残基，其促进其 与毒素主体的非共价相互作用， 对α毒素具有更大抑制活性的衍生物，2）鉴定 α毒素的跨膜结构域，3）鉴定 毒素参与受体结合，和4）结晶更可溶 α毒素的衍生物和该衍生物与α毒素之一的复合物， GPI锚定的α毒素受体。为了实现第一个目标， 的前肽将依次被甘氨酸取代，分离并 测定前肽对毒素的亲和力。在aim 2中， 利用两种方法来绘制出α的膜穿透结构域 毒素第一种方法将是用怀疑的跨膜 具有半胱氨酸的α毒素残基纯化这些衍生物， 在平面双层中具有每种毒素的通道。的带电衍生物 然后将巯基特异性试剂甲硫基磺酸盐（MTS）引入到 在双层的任一侧上的水相。带电荷的MTS试剂会导致 只有当半胱氨酸被取代时，沟道电导才发生变化 水道里的残留物相同的半胱氨酸取代的残基（在 α毒素的无半胱氨酸衍生物）也将用 环境敏感的荧光探针NBD和检测的荧光 在毒素进入细胞膜之前和之后如果alpha 毒素通过两亲性β折叠或两亲性β折叠与膜相互作用， 他们将观察到反应周期的差异 从这两个试验。受体结合域已暂时定位于 α毒素的C55附近区域因此，在目标3中，在C55附近的残基在C55附近的氨基酸残基在C55附近。 α毒素结构模型将通过体外诱变而改变， 确定哪些残基参与受体结合。在目标4中，他们提出 使α毒素和α毒素受体复合物结晶规定了这一点 可能是由于大量的更可溶形式的 α毒素及其GPI锚定受体之一，人叶酸受体 （hFR）。