Early interaction between clostridium perfringens epsilon toxin and host cells

产气荚膜梭菌epsilon毒素(ETX)是一种B类CDC/USDA重叠毒素，是一种主要毒力 产产ETX的产气荚膜梭菌分离株引起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，因为在 人类是不常见的)。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 行动，并开始探索ETX疗法的发展，将追求以下具体目标 在这个MARGE项目中(方案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 肾、脑和肺中ETX受体的表达克隆方法；ii)Aim 2将免疫定位 已鉴定的受体在动物和人体组织中的分布；iii)目标3将评估致病 使用抗体阻断方法识别ETX受体的重要性，siRNA介导的减少 受体表达，以及(如果有)受体敲除小鼠；iv)Aim 4将通过质谱学分析， 在敏感宿主细胞质膜上形成的ETX复合体的蛋白质组成 对ETX作用的进一步洞察；v)因为先前的研究表明唾液酸酶可以增加ETX 在MDCK细胞中的结合/活性，Aim 5将使用同基因唾液酸酶突变来评估这一假设 唾液酸酶同样会增强ETX产生菌的毒力，而Aim 6将评估 ETX受体诱饵的治疗潜力。预计这些研究将产生新的方法 治疗抑制ETX活性，这是目前NIH生物防御活动的优先事项。

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.