Elucidating the roles of transcriptional regulators during the Cryptosporidium life cycle

项目总结 腹泻病每天导致2195名儿童死亡，仍是 5岁以下儿童，低收入国家负担特别重。在病原体中， 寄生虫隐孢子虫仍然是全球腹泻的主要原因，每个人都感染数百万人 年。它是导致婴儿腹泻的第二大原因，也是导致水传播的主要原因。 疾病在美国。目前，硝唑尼特是治疗这种寄生虫病的唯一药物，但 它在治疗最脆弱的人群方面无效，包括营养不良的儿童和 免疫功能受损的病人。隐孢子虫病的负担比之前认为的要大2-5倍 要消除这一新的全球健康威胁，需要研究严重不足的新疗法。 寄生虫的传播是通过粪便-口腔途径进行的，只需摄入10 隐孢子虫卵囊导致感染。然后寄生虫通过无性生长而发展， 在肠道上皮细胞中复制和分裂，然后过渡到雄性或雌性形式。有性 雄性和雌性寄生虫的繁殖导致产生更多传染性卵囊，这些卵囊通过 哺乳动物的宿主。虽然已经确定了一些分子标记来划分这个生命周期 进展过程中，普遍缺乏对信号通路和基因表达变化的了解 参与了隐孢子虫的发育。在导致疟疾和弓形虫病的相关寄生虫中，DNA- 称为AP2的结合转录因子驱动细胞周期转变，包括性承诺、宿主细胞 入侵、慢性感染和毒力蛋白的部署。初步调查结果表明，一些人 的AP2在无性和有性期之间有差异地表达，尽管更多 有必要进行彻底的遗传分析和分类。我假设AP2转录因子驱动 隐孢子虫生命周期关键点的细胞命运决定，如无性分裂和有性分裂 承诺。为了进一步研究这一点，我的目标是：1)鉴定与C. 微小胚胎的生命周期进程和2)决定了它们在发育过程中的功能作用。使用高- 通过基因组技术，我将研究转录调控因子的基因表达 细胞周期和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 利用本实验室开发的CRISPR/CAS9工具进行遗传学研究 对微小隐孢子虫C.parvum进行修饰。转基因寄生虫将被用于研究蛋白质的表达和定位 候选基因以及条件性基因敲除实验中的表型。颠覆一切的能力 这些生命周期的关键调节因子将极大地加速开发有效的治疗方法。 这种全球寄生虫。

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.