Early interaction between clostridium perfringens epsilon toxin and host cells

产气荚膜梭菌ε毒素与宿主细胞之间的早期相互作用

• 批准号: 7670079
• 负责人: Bruce A Mc Clane
• 金额: $ 31.14万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-03 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7670079
• 关键词: Animals; Binding; Blocking Antibodies; Brain; Cell membrane; Cells; Clostridium perfringens; Clostridium perfringens epsilon toxin; Complex; Development; Disease; Human; Infection; Kidney; Knockout Mice; Knowledge; Lung; MDCK cell; Mass Spectrum Analysis; Mediating; Neuraminidase; Proteins; Small Interfering RNA; Therapeutic; Toxin; United States National Institutes of Health; Vaccines; Virulence; Virulence Factors; biodefense; expression cloning; human tissue; improved; insight; mutant; novel therapeutic intervention; programs; receptor; receptor expression; toxin V

Clostridium perfringens epsilon toxin (ETX) is a class B CDC/USDA overlap toxin and a major virulence factor in natural veterinary infections caused by ETX-producing C. perfingens isolates. Currently there are no ETX therapeutics (NIH prefers ETX therapeutics over vaccines because natural ETX-related disease in humans is uncommon). Early steps in ETX action on host cells are poorly understood, which has negatively impacted the development of ETX therapeutics. To remedy the limited understanding of early steps in ETX action and begin exploring the development of ETX therapeutics, the following specific aims will be pursued in this MARGE project (part of Program V, interactions between toxins and host cells): i) Aim 1 will identify the ETX receptor in kidney, brain and lung by expression cloning approaches; ii) Aim 2 will immunolocalize the distribution of the identified receptor in animal and human tissues; iii) Aim 3 will evaluate the pathogenic importance of the identified ETX receptor using antibody blocking approaches, siRNA-mediated reduction of receptor expression, and (if available) receptor knock-out mice; iv) Aim 4 will analyze, by mass spectrometry, the protein composition of ETX complexes formed in the plasma membrane of sensitive host cells to gain further insights into ETX action; v) since previous studies have shown that sialidases can increase ETX binding/activity in MDCK cells, Aim 5 will use isogenic sialidase mutants to evaluate the hypothesis that sialidases similarly potentiate the virulence of ETX-producing isolates and vi) Aim 6 will evaluate the therapeutic potential of ETX receptor decoys. These studies are expected to produce new approaches for therapeutic inhibition of ETX activity, which is a current priority for NIH biodefense activities.

产气荚膜梭菌 epsilon 毒​​素 (ETX) 是 B 类 CDC/USDA 重叠毒素，是一种主要毒力 产生 ETX 的产气荚膜梭菌分离株引起的自然兽医感染的因素。目前没有 ETX 疗法（NIH 更喜欢 ETX 疗法而不是疫苗，因为自然 ETX 相关疾病在 人类并不常见）。 ETX 对宿主细胞作用的早期步骤知之甚少，这对宿主细胞产生了负面影响 影响了 ETX 疗法的发展。弥补对 ETX 早期步骤的有限理解 采取行动并开始探索 ETX 疗法的开发，将追求以下具体目标 在这个 MARGE 项目中（计划 V 的一部分，毒素与宿主细胞之间的相互作用）： i) 目标 1 将确定 通过表达克隆方法研究肾、脑和肺中的 ETX 受体； ii) 目标 2 将免疫定位 已识别受体在动物和人体组织中的分布； iii) 目标 3 将评估致病性 使用抗体阻断方法鉴定 ETX 受体的重要性，siRNA 介导的减少 受体表达和（如果有的话）受体敲除小鼠； iv) 目标 4 将通过质谱分析， 敏感宿主细胞质膜中形成的 ETX 复合物的蛋白质组成，以获得 对 ETX 行动的进一步见解； v) 因为之前的研究表明唾液酸酶可以增加 ETX MDCK 细胞中的结合/活性，目标 5 将使用同基因唾液酸酶突变体来评估以下假设： 唾液酸酶同样会增强产生 ETX 的分离株的毒力，并且 vi) 目标 6 将评估 ETX受体诱饵的治疗潜力。这些研究预计将产生新的方法 治疗性抑制 ETX 活性，这是 NIH 生物防御活动当前的首要任务。