Elucidating the roles of transcriptional regulators during the Cryptosporidium life cycle

阐明转录调节因子在隐孢子虫生命周期中的作用

• 批准号: 10464882
• 负责人: Katelyn Ann Walzer
• 金额: $ 6.86万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10464882
• 关键词: Affect; Africa South of the Sahara; Age; Asia; CRISPR/Cas technology; Candidate Disease Gene; Cattle; Cause of Death; Cell Cycle; Cell Cycle Regulation; Cells; Cessation of life; Child; Childhood; Classification; Complex; Cryptosporidiosis; Cryptosporidium; Cryptosporidium parvum; DNA Binding; Developing Countries; Development; Diarrhea; Enterobacteria phage P1 Cre recombinase; Epithelial Cells; Epitopes; Eukaryotic Cell; Exhibits; Female; Fertilization; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genomics; Germ Cells; Growth; Human; Immunocompromised Host; Individual; Infant; Infection; Ingestion; Knowledge; Laboratories; Life Cycle Stages; Malaria; Malnutrition; Maps; Morphology; Oocysts; Oral; Organism; Parasites; Parasitic Diseases; Pathway interactions; Pattern; Pharmaceutical Preparations; Phenotype; Plasmodium; Production; Protein Kinase; Protein phosphatase; Proteins; RNA Sequences; Regulatory Pathway; Research; Role; Route; Sex Differentiation; Sexual Development; Sexual Reproduction; Signal Transduction; Signaling Protein; Staphylococcus hominis; Symptoms; Technology; Therapeutic; Time; Toxoplasma; Toxoplasmosis; Transcriptional Regulation; Transgenic Organisms; United States; Virulence; Vulnerable Populations; asexual; base; chronic infection; conditional knockout; diarrheal disease; differential expression; drug development; effective therapy; enhancer-binding protein AP-2; experimental study; genetic analysis; global health; intestinal epithelium; low income country; male; molecular marker; mortality; new therapeutic target; nitazoxanide; novel therapeutics; pathogen; pediatric patients; protein expression; single-cell RNA sequencing; therapeutically effective; tool; transcription factor; transcriptomics; transmission process; vaccine development; waterborne illness

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.

项目摘要 腹泻病每天杀死2,195名儿童，并一直是第五大死亡原因 5岁以下的儿童，低收入国家的负担特别高。在病原体中， 寄生虫隐孢子虫仍然是全球腹泻的主要原因，并感染数百万的人 年。这是婴儿腹泻病的第二大原因，是水源性的主要原因 美国的疾病。目前，奈塔唑胺是唯一可用于治疗这种寄生虫病的药物，但 它无法治愈最脆弱的人群，包括营养不良的儿童和 免疫功能低下的患者。负担比以前想象的要大2-5倍，隐孢子虫病是 需要严重研究和新颖的治疗剂来浪费这种新兴的全球健康威胁。 寄生虫的传播是通过粪便途径发生的，摄入少于10 隐孢子虫卵囊导致感染。然后，寄生虫通过无性增长而发展 复制和肠上皮细胞中的分裂，然后过渡到雄性或女性形式。性 雄性和女性寄生虫的繁殖导致产生更多的感染性卵囊 哺乳动物主人。虽然已经确定了一些分子标记来划定这个生命周期 进展，普遍缺乏有关信号通路和基因表达变化的知识 参与隐孢子虫的发展。在引起疟疾和弓形虫病的相关寄生虫中，DNA- 称为AP2S的结合转录因子驱动细胞周期过渡，包括性承诺，宿主细胞 毒力蛋白的入侵，慢性感染和部署。初步发现表明一个数字 AP2的ap2在C. parvum的无性阶段和性阶段差异表达，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 隐孢子虫生命周期期间关键点的细胞命运决定，例如无性划分和性行为 承诺。为了进一步研究，我的目标是1）确定C涉及的特定阶段的转录因子。 Parvum生命周期的进程和2）确定其在发育过程中的功能作用。使用高 吞吐量基因组技术，我将检查转录调节剂的基因表达 细胞周期并优先考虑具有不同表达模式的调节剂。我将阐明他们在寄生虫中的角色 通过利用在我们的实验室开发的CRISPR/CAS9工具来开发和差异化 修改C. parvum。转基因寄生虫将用于研究蛋白质表达和定位 有条件敲除实验中的候选基因以及最终的表型。破坏的能力 生命周期的这些关键调节者将极大地加速有效的治疗方法 这个全球寄生虫。