Elucidating the roles of transcriptional regulators during the Cryptosporidium life cycle

项目摘要 腹泻病每天造成2 195名儿童死亡，一直是儿童死亡的第五大原因。 这对低收入国家造成特别沉重的负担。在病原体中， 寄生虫隐孢子虫仍然是全球腹泻的主要原因， 年它是婴儿腹泻病的第二大原因，也是水传播疾病的主要原因。 美国的疾病。目前，硝唑尼特是治疗这种寄生虫病的唯一药物， 它对治疗最脆弱的人群，包括营养不良的儿童， 免疫功能低下的患者。隐孢子虫病的负担比以前认为的大2-5倍， 需要研究严重不足的新疗法来消除这一新兴的全球健康威胁。 寄生虫的传播通过粪口途径发生，摄入少至10 隐孢子虫卵囊导致感染。寄生虫然后通过无性生长， 在肠上皮细胞中复制和分裂，然后转变为雄性或雌性形式。性 雄性和雌性寄生虫的繁殖导致产生更具感染性的卵囊， 哺乳动物宿主虽然已经确定了一些分子标记来划分这个生命周期 进展，普遍缺乏关于信号通路和基因表达变化的知识 参与了隐孢子虫的发育在引起疟疾和弓形虫病的相关寄生虫中，DNA- 称为AP 2的结合转录因子驱动细胞周期转换，包括性承诺，宿主细胞 入侵、慢性感染和毒力蛋白的部署。初步调查结果显示， 的AP 2在无性和有性阶段的C.小，虽然更多 需要进行彻底的遗传分析和分类。我假设AP 2转录因子驱动 在隐孢子虫生命周期的关键点，如无性分裂和有性生殖， 承诺.为了进一步研究这一点，我的目标是：1）确定阶段特异性转录因子参与C。 2）决定其在发育过程中的功能作用。使用高- 通过基因组技术，我将研究转录调节因子的基因表达， 细胞周期，并优先考虑具有不同表达模式的调节因子。我将阐明它们在寄生虫中的作用 利用我们实验室开发的CRISPR/Cas9工具， 修改C.小的转基因寄生虫将用于研究蛋白质表达和定位的 候选基因以及条件敲除实验中的所得表型。破坏的能力 这些生命周期的关键调节剂将大大加速有效治疗方法的发展， 这个全球寄生虫

Project summary Diarrheal disease kills 2,195 children each day and persists as the fifth leading cause of death among children under the age of 5, with an especially high burden on low-income countries. Among pathogens, the parasite Cryptosporidium remains a leading cause of diarrhea worldwide and infects millions of people each year. It is the second leading cause of diarrheal disease in infants and is the leading cause of waterborne illness in the United States. Currently, nitazoxanide is the only drug available to treat this parasitic disease, but it is ineffective in curing the most vulnerable populations, including malnourished children and immunocompromised patients. With a burden 2-5 times greater than previously thought, cryptosporidiosis is severely understudied and novel therapeutics are needed to squander this emerging global health threat. Transmission of the parasite occurs via the fecal-oral route, with ingestion of as little as 10 Cryptosporidium oocysts leading to infection. The parasite then progresses through asexual growth, replication, and division in intestinal epithelial cells, followed by transition to a male or female form. Sexual reproduction of male and female parasites results in the production of more infectious oocysts that are shed by the mammalian host. While a few molecular markers have been identified to demarcate this life cycle progression, there is a general lack of knowledge about the signaling pathways and gene expression changes involved in Cryptosporidium development. In related parasites that cause malaria and toxoplasmosis, DNA- binding transcription factors called AP2s drive cell cycle transitions, including sexual commitment, host cell invasion, chronic infection, and deployment of virulence proteins. Preliminary findings suggest that a number of AP2s are differentially expressed between the asexual and sexual stages of C. parvum, although a more thorough genetic analysis and classification is necessary. I hypothesize that AP2 transcription factors drive cell fate decisions at key points during the Cryptosporidium life cycle, such as asexual division and sexual commitment. To investigate this further, I aim to 1) identify stage-specific transcription factors involved in C. parvum life cycle progression and 2) determine their functional roles during development. Using high- throughput genomic technologies, I will examine the gene expression of transcriptional regulators across the cell cycle and prioritize for regulators with distinct expression patterns. I will elucidate their roles in parasite development and differentiation by utilizing CRISPR/Cas9 tools developed in our laboratory to genetically modify C. parvum. Transgenic parasites will be used to study protein expression and localization of the candidate gene as well as the resultant phenotype in conditional knockout experiments. The ability to disrupt these critical regulators of the life cycle will greatly accelerate the development of effective treatments against this global parasite.