Microsporidia: Genetic Techniques

描述（申请人提供）：微孢子虫是一种人畜共患病原生生物，可通过食物或水传播。它们被美国国立卫生研究院列为B类优先病原体，也在美国环保署的高优先生物体和污染物清单上。最常见的人致病性小孢子虫双胞虫是不可培养的；然而，其他致病性微孢子虫，如锥体脑虫和球形脑虫，可以在体外培养，此外，存在脑虫感染的小动物模型，可以在实验室中进行详细的发病机制研究。对这些神秘的原生生物的描述所缺乏的是允许对这些生物进行遗传操作的技术，为解剖这些生物的生物学提供了工具。我们对微孢子虫必需的2型蛋氨酸氨基肽酶（MetAP2）进行了研究和鉴定。MetAP2是一个合理的治疗靶点，因为微孢子虫缺乏MetAP1，使得MetAP2成为必需的酶。利用人类MetAP2的数据，我们已经能够修改微孢子虫MetAP2，使其对卵泡霉素（一种已知的MetAP2抑制剂）具有耐药性。这为这些生物的遗传研究提供了第一个可选择的标记。我们建议在此基础上，利用马氏微孢子虫和赫勒姆微孢子虫建立一个有用的转染系统。该应用程序还将利用我们的合作者在开发各种顶复合体转染系统方面的经验，以及我们对E. cucululi和E. hellem基因组rRNA位点的表征。微孢子虫转染技术的发展虽然风险较高，但将对该领域产生重大影响，并将解决这些新兴病原体研究的主要研究障碍。

DESCRIPTION (provided by applicant): The Microsporidia are zoonotic pathogenic protists which can be transmitted by food or water. They are classified as category B priorty pathogens by the NIH and are also on the EPA high priority organism and contaminant lists. The most common human pathogenic microsporidia Enterocytozoon bieneusi is non cultivatable; however other pathogenic microsporidia such as Encephalitozoon cuniculi and Encephalitozoon hellem can be grown in vitro and in addition small animal models of Encephalitozoonidae infections exist allowing detailed pathogenesis studies in the laboratory. What is lacking for the characteriziation of these enigmatic protists are techniques which would permit the genetic manipulation of these organisms providng the tools for disseciton of the biology of these organisms. We have studied and characterized microsporidian methionine aminopeptidase type 2 (MetAP2) which is an essential enzyme in these organisms. MetAP2 is a logical therapeutic target as microsporidia lack MetAP1 making MetAP2 an essential enzyme. Using human MetAP2 data we have been able to modify microsporidian MetAP2 to make it resistant to ovalicin, a known inhibitor of MetAP2. This provides the first selectable marker for genetic studies in these organisms. We propose to build on this work to develop a useful transfection system for the microsporidia using E. cunicuili and E. hellem. This application will also take advantage of the experience of our collaborators in the development of transfection systems for various Apicomplexa and our characterization of the E. cuniculi and E. hellem genomic rRNA loci. The development of microsporidian transfection, while high risk, would have a major impact on the field and would solve a major research impediment for study of these emerging pathogens. PUBLIC HEALTH RELEVANCE: Microsporidia cause opportunistic infections in patients with AIDS and are emerging infections in immune competent patients. Research on these enigmatic pathogens has been limited by the absence of genetic systems that would allow examination of gene function. We propose to develop such genetic systems based on our previous laboratory work that resulted in the identification of the first potential selectable marker for these organisms.