Early interaction between clostridium perfringens epsilon toxin and host cells

产气荚膜梭菌毒素（Clostridium perfringens ETX）是CDC/USDA的B类重叠毒素，是一种主要的致病性毒素 产生ETX的C.产气荚膜梭菌分离株。目前没有 ETX疗法（NIH更喜欢ETX疗法而不是疫苗，因为在美国， 人类是不寻常的）。ETX对宿主细胞作用的早期步骤知之甚少，这对宿主细胞具有负面影响。 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疗法的发展，将追求以下具体目标 在这个MARGE项目中（项目V的一部分，毒素和宿主细胞之间的相互作用）：i）目标1将确定 通过表达克隆方法在肾、脑和肺中的ETX受体; ii）Aim 2将免疫定位 所鉴定的受体在动物和人体组织中的分布; iii）目标3将评估病原性 使用抗体阻断方法鉴定的ETX受体的重要性，siRNA介导的减少 受体表达和（如果可用）受体敲除小鼠; iv）Aim 4将通过质谱分析， 在敏感宿主细胞的质膜中形成的ETX复合物的蛋白质组成， 对ETX作用的进一步了解; v）由于先前的研究表明唾液酸酶可以增加ETX， 结合/活性，Aim 5将使用同基因唾液酸酶突变体来评估以下假设： 唾液酸酶类似地增强产生ETX的分离株的毒力，并且vi）目的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， ETX活性的治疗性抑制，这是NIH生物防御活动的当前优先事项。

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.