Tapping into an anthelmintic Bacillus thuringiensis crystal protein arsenal for human strongyloidiasis

项目摘要/摘要 人类弓形虫病被认为是最被忽视的主要由热带疾病引起的疾病。 由肠道寄生线虫粪类圆线虫感染，估计有3亿人感染。 慢性感染斯特氏链球菌会引起腹痛、食欲不振、腹泻、便秘、荨麻疹和 瘙痒，并与儿童生长发育迟缓有关。它的幼虫也可以自我感染人类宿主， 导致了几代人的自体感染。在免疫抑制的患者中，自身感染可能成为 不受监管，导致高度感染和严重的症状加剧。超级感染可能 扩散到器官和组织，导致严重出血、小肠梗阻和结肠炎，严重肺部 疾病和败血症，病死率高达80%。目前我们只有一种主要药物可以治疗急性淋巴细胞白血病 绿僵菌、伊维菌素的寄生期。然而，伊维菌素在圆线虫和其他 肠道寄生线虫已经存在，在某些情况下还很猖獗，特别是在兽医领域。 迫切需要具有新作用模式的药物来对抗这种致命的寄生虫。土壤细菌--芽孢杆菌 苏云金杆菌(Bt)是世界上最大的生物杀虫剂。具有昆虫活性的成分是 杀死昆虫但对脊椎动物无害的晶体(Cry)蛋白(无作用；1000 mg/kg)。杀虫 Bt Cry蛋白在转基因粮食作物中表达(例如，美国80%的玉米)，并被FDA批准用于 摄取。线虫活性Cry蛋白，Cry5B(与杀虫Cry蛋白有关)，治疗钩虫和 蛔虫在大型动物中感染肠道线虫，并已被广泛研究。最近，其他 目前已鉴定出寄生线虫活性Cry蛋白，包括Cry14A和Cry21A。这些都是高度 在体外低剂量下对自由生活的斯特珊瑚成虫有效，增加了它们可能 代表了一种治疗这种寄生虫的新方法。这项提议旨在利用22种已知的天然氨基酸 寻找针对这种寄生虫的最佳Cry14A和Cry21A序列，筛选这两种蛋白的变体 针对自由生活的成虫和寄生的雌性阶段。一旦确定，最好的Cry14A和Cry21A蛋白 变种将在沙土鼠体内测试其治愈粪链霉菌感染的能力。它们之间的相互作用 伊维菌素与抗伊维菌素线虫的蛋白以及伊维菌素与Cry14A的相互作用 将对抗药性线虫进行调查。未来的计划包括剂量范围研究，最佳配方， 药效和蛋白质产量的详细工程和序列优化，作用机理研究 和耐药性，在更高的模型中进行测试，包括在非人类灵长类动物中自然感染S。 与伊维菌素和其他Cry蛋白的组合研究。最终，我们预计将确定第一个 1988年批准伊维菌素为新的铅驱虫剂。

Project Summary/Abstract Human strongyloidiasis is considered the most neglected of the neglected tropical diseases caused primarily by the intestinal parasitic nematode Strongyloides stercoralis, infecting an estimated 300 million people. Chronic infections with S. stercoralis cause abdominal pain, anorexia, diarrhea constipation, urticaria, and itching and are associated with growth stunting in children. Its larvae can also autoinfect the human host, resulting in many generations of autoinfection. In immunosuppressed patients autoinfection can become unregulated, resulting hyperinfection and in severe intensification of symptoms. Hyperinfections may disseminate to organs and tissues causing severe bleeding, small bowel obstruction and colitis, severe pulmonary disease and sepsis, with fatality rates are as high as 80%. Currently we have only one main drug to treat all parasitic stages of S. stercoralis, ivermectin. However, ivermectin resistance in Strongyloides and other intestinal parasitic nematodes is already present, and in some cases rampant, especially in veterinary medicine. Drugs with new modes of action against this deadly parasite are urgently needed. The soil-bacterium Bacillus thuringiensis (Bt) is the number one biological insecticide agent in the world. The insect-active components are crystal (Cry) proteins that kill insects but that are harmless to vertebrates (no effect >1000 mg/kg). Insecticidal Bt Cry proteins are expressed in transgenic food crops (e.g., >80% of USA corn) and are FDA approved for ingestion. The nematode-active Cry protein, Cry5B (related to insecticidal Cry proteins), cures hookworm and Ascaris intestinal nematode infections in large animals and has been studied extensively. More recently, other parasitic nematode-active Cry proteins have been identified, including Cry14A and Cry21A. These are highly effective in vitro against free-living S. stercoralis adults at low doses, raising the possibility that they may represent a new cure for this parasite. This proposal aims to take advantage of 22 known natural amino acid variants of both proteins to find the optimal Cry14A and Cry21A sequences that target this parasite, screening against both free-living adult and parasitic female stages. Once identified, the best Cry14A and Cry21A protein variants will be tested for their ability to cure S. stercoralis infections in vivo in gerbils. The interaction of these proteins with ivermectin-resistant nematodes and, conversely, the interaction of ivermectin with Cry14A resistant nematodes will be investigated. Future plans include dose ranging studies, optimum formulation, detailed engineering and sequence optimization for efficacy and protein yield, studies of mechanism of action and resistance, testing in higher models including natural S. stercoralis infections in non-human primates, and combinatorial studies with ivermectin and other Cry proteins. At the end, we expect to have identified the first new lead anthelmintic against S. stercoralis approval of ivermectin in 1988.