Microsporidia: Genetic Techniques

描述(申请人提供)：微孢子虫是人畜共患病的原生动物，可通过食物或水传播。它们被美国国立卫生研究院归类为B类优先病原体，也在EPA的高度优先生物体和污染物名单上。最常见的人类致病微孢子虫比内氏肠孢子虫是不可培养的；然而，其他致病微孢子虫，如楔形脑孢子虫和赫莱姆脑孢子虫，也可以在体外生长，此外，还有脑孢子虫感染的小动物模型，使实验室能够进行详细的发病机制研究。对于这些神秘的原生生物的描述，缺乏的是允许对这些有机体进行基因操作的技术，这些技术为解剖这些有机体的生物学提供了工具。我们研究和鉴定了微孢子虫蛋氨酸氨基肽酶2(MetAP2)，它是这些生物中的一个重要酶。MetAP2是一个合理的治疗靶点，因为微孢子虫缺乏MetAP1，使MetAP2成为一种必不可少的酶。利用人类MetAP2的数据，我们已经能够修改微孢子虫MetAP2，使其对已知的MetAP2抑制剂卵胞苷具有抗药性。这为这些生物的遗传学研究提供了第一个可选择的标记。我们建议在这项工作的基础上，利用E.cunicuili和E.Hellem开发一种有用的微孢子虫转导系统。这项应用还将利用我们的合作者在开发各种顶体复合体的转基因系统方面的经验，以及我们对楔形棘球藻和海莱姆乳杆菌基因组rRNA基因座的鉴定。微孢子虫转基因的发展虽然风险很高，但将对该领域产生重大影响，并将解决这些新出现的病原体研究的主要障碍。 公共卫生相关性：微孢子虫在艾滋病患者中引起机会性感染，是免疫能力强的患者中新出现的感染。由于缺乏能够检测基因功能的遗传系统，对这些神秘病原体的研究一直受到限制。我们建议在我们之前的实验室工作的基础上开发这样的遗传系统，这些工作导致了这些生物的第一个潜在的可选择标记的识别。

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.