Molecular signaling mechanisms controlling Cryptosporidium proliferation and development

项目摘要 隐孢子虫是婴幼儿腹泻病(隐孢子虫病)和死亡的主要原因。 生活在资源匮乏的环境中。在美国，隐孢子虫是水媒疫情的主要原因 用于娱乐用水。目前，没有完全有效的药物，也没有疫苗来治疗或预防 隐孢子虫病。唯一可用的美国FDA批准的药物，硝唑尼特在年轻患者中没有得到证实的疗效 免疫状态较弱的儿童和免疫功能低下的个体。因此，迫切需要 开发新药和疫苗以减轻隐孢子虫病的负担。抗隐孢子虫药物研究进展 由于我们对寄生虫生物学的了解有限，疫苗的开发受到了阻碍。这个 进展缓慢的根本原因是没有一种可靠的方法来持续 传播隐孢子虫，以及缺乏操纵寄生虫基因组的分子遗传学。我们有 通过开发一种强大的技术来操纵隐孢子虫基因组和 在免疫受损的小鼠模型系统中繁殖这些转基因寄生虫。钥匙 这种遗传系统的优点是隐孢子虫的整个生命周期(无性和有性阶段) 在小鼠肠道中完成，使我们能够解开寄生虫生物学(Vinayak等人2015年，《自然》523：477)。 我们对控制寄生虫阶段发展的分子信号机制缺乏了解。 为了成功地完成复杂的生命周期。信号通路组件，如类植物 钙依赖蛋白激酶(CDPKs)已成为隐孢子虫和隐孢子虫的诱人药物靶点 相关寄生虫，由于它们在人类宿主中缺乏同源物。利用我们的基因 系统中，我们已经证明了选择性蛋白激酶抑制剂对钙依赖的疗效。 蛋白激酶-1(CDPK1)的表达，表明该信号通路在微小隐孢子虫中起着关键作用。利用 我们实验室最近开发的条件蛋白质降解系统，我们已经展示了必要的 CDPK1在寄生虫无性增殖和生存中的作用此外，我们有令人信服的初步证据 这阐明了两种信号转导蛋白在性发育阶段中的作用。这个项目的目标是 阐明这些信号蛋白在调节无性和有性发育中的机制作用 寄生虫增殖和传播所需的微小隐孢子虫阶段。对这些机制的阐明将 为隐孢子虫的基本生物学提供新的见解，并开辟新的发展途径 有效的治疗方法。

Project Abstract Cryptosporidium is a leading cause of diarrheal disease (cryptosporidiosis) and death among young children living in resource-poor settings. In the US, Cryptosporidium is the major cause of waterborne outbreaks linked to recreational water use. Currently, there is no fully effective drug and no vaccine to treat or prevent cryptosporidiosis. The only available US FDA approved drug, nitazoxanide has no proven efficacy in young children with weak immune status and immunocompromised individuals. Therefore, there is an urgent need to develop new drugs and vaccine to reduce the burden of cryptosporidiosis. Progress in anti-cryptosporidial drug and vaccine development has been hampered due to our limited understanding of parasite biology. The underlying reasons for this slow progress have been the unavailability of a robust method to continuously propagate Cryptosporidium, and the absence of molecular genetics to manipulate the parasite genome. We have overcome these hurdles by developing a powerful technology to manipulate the Cryptosporidium genome and propagate these genetically modified parasites in an immunocompromised mouse model system. The key advantage of this genetic system is that the entire life cycle of Cryptosporidium (both asexual and sexual stages) is completed in the mouse intestine, allowing us to unravel parasite biology (Vinayak et al 2015, Nature 523:477). We lack an understanding of the molecular signaling mechanisms that control development of parasite stages for successful completion of the complex life cycle. Signaling pathway components such as the plant-like calcium-dependent protein kinases (CDPKs) have emerged as attractive drug targets in Cryptosporidium and related parasites, due to the absence of their homologues in human host. Taking advantage of our genetic system, we have demonstrated the efficacy of selective bumped kinase inhibitors against calcium-dependent protein kinase-1 (CDPK1), thus indicating a critical role of this signaling kinase in C. parvum. Utilizing the conditional protein degradation system recently developed in our laboratory, we have demonstrated the essential role of CDPK1 in asexual proliferation and parasite survival. Moreover, we have compelling preliminary evidence that sheds light on the role of two signaling kinases in sexual developmental stages. The goal of this project is to elucidate the mechanistic role of these signaling proteins in regulating development of asexual and sexual stages in C. parvum required for parasite proliferation and transmission. Elucidation of these mechanisms will provide novel insights into the fundamental biology of Cryptosporidium, and open new avenues for development of effective therapies.