Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.
Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum.
复制标题
腹泻病原体隐孢子虫的遗传修饰。
DOI：
10.1038/nature14651
复制
发表时间：
2015-07-23
期刊：
影响因子：
64.8
通讯作者：
Striepen B
中科院分区：
文献类型：
--
作者：
Vinayak S;Pawlowic MC;Sateriale A;Brooks CF;Studstill CJ;Bar-Peled Y;Cipriano MJ;Striepen B
最近对幼儿严重腹泻的全球原因的研究发现，原生动物寄生虫隐孢子虫是仅次于轮状病毒的第二大腹泻病原体。据估计，腹泻病占儿童总死亡率的 10.5%。隐孢子虫也是艾滋病毒/艾滋病和器官移植背景下的机会病原体。目前还没有疫苗，也只有一种经批准的药物，无法为处于最严重危险的人、营养不良的儿童和免疫功能低下的患者提供任何益处。隐孢子虫病药物和疫苗的开发受到寄生虫不易驯化的限制，其中包括缺乏连续培养、简便的动物模型和分子遗传工具。在这里，我们描述了一个对这种重要的人类病原体进行基因改造的实验框架。我们在组织培养中建立并优化了 C. parvum 子孢子的转染。为了分离稳定的转基因，我们开发了一种将子孢子直接输送到肠道的小鼠模型、隐孢子虫 CRISPR/Cas9 系统以及氨基糖苷类耐药性的体内选择。我们衍生出适合体外和体内药物筛选的报告寄生虫，并通过基因敲除评估药物敏感性的基础。我们预计对寄生虫进行基因改造的能力将为隐孢子虫研究带来变革。基因报告器将提供疾病、治疗和保护的定量相关性，寄生虫基因在这些过程中的作用现在正在接受严格的调查。
Recent studies into the global causes of severe diarrhea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrheal pathogen after rotavirus. Diarrheal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the context of HIV-AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger, malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes lack of continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We establish and optimize transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we develop a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium CRISPR/Cas9 system, and in vivo selection for aminoglycoside resistance. We derive reporter parasites suitable for in vitro and in vivo drug screening, and we evaluate the basis of drug susceptibility by gene knock out. We anticipate the ability to genetically engineer the parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection and the role of parasite genes in these processes is now open to rigorous investigation.