Cryptosporidium mutagenesis

隐孢子虫诱变

• 批准号: 10571124
• 负责人: GIOVANNI WIDMER
• 金额: $ 20.63万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-21 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10571124
• 关键词: Acceleration; Acids; Affect; Animals; Applied Research; Area; Backcrossings; Basic Science; Biology; CRISPR/Cas technology; Cells; Chemicals; Childhood; Collection; Cryptosporidiosis; Cryptosporidium; Cryptosporidium parvum; Defect; Developing Countries; Development; Diarrhea; Dimensions; Dissection; Drug Controls; Drug resistance; Enteral; Ethyl Methanesulfonate; Etiology; Exposure to; Flushing; Frequencies; Future; Generations; Genes; Genetic; Genetic Determinism; Genome; Genomics; Goals; Human; Infantile Diarrhea; Invaded; Malaria; Maps; Mediating; Medicine; Metabolic Pathway; Methods; Multi-Drug Resistance; Mus; Mutagenesis; Mutagens; Mutation; Nitrosourea Compounds; Oocysts; Oral; Outcomes Research; Output; Parasites; Pharmaceutical Preparations; Phenotype; Piperazines; Population; Predisposition; Probability; Procedures; Proteins; Public Health; Radiation; Research; Resistance; Sporozoites; Taxonomy; Techniques; Testing; Toxoplasma; Toxoplasma gondii; Uncertainty; Virulence; Work; Zoonoses; causal variant; clinically relevant; drug action; drug development; drug-sensitive; excystation; experimental study; forward genetics; genome editing; genome sequencing; genomic locus; homologous recombination; improved; inhibitor; innovation; interest; irradiation; low and middle-income countries; model organism; mutant; pathogen; reference genome; resistance mechanism; response; tool; trait; ultraviolet irradiation; vaccine development; whole genome

ABSTRACT In low- and middle-income countries cryptosporidiosis is one of the leading causes of debilitating infant diarrhea. Effective drug option to control Cryptosporidium parasites remain limited and efforts to develop a vaccine are at an early stage. In spite of the public health impact of childhood diarrhea, the lack of research tools continues to hamper progress towards a better understanding of these parasites. The goal of this project is to develop a forward genetics method based on random mutagenesis to facilitate basic and applied research on Cryptosporidium parvum, a zoonotic species responsible for about half of human cases of cryptosporidiosis. The term Forward Genetics refers to methods used to identify genetic determinants of specific phenotypes. This approach has expanded our understanding of model organisms, including the taxonomically related species Toxoplasma gondii. The proposed project will impact primarily four areas of Cryptosporidium research. 1) It will advance drug development efforts by identifying genetic loci targeted by anti-Cryptosporidium compounds. 2) It will enable the dissection of the genetic basis of clinically relevant traits such as drug susceptibility, virulence or oocyst output, and potentially uncover genes associated with multidrug resistance. 3) It will contribute to the annotation of genes encoding proteins of unknown function, which still represent a third of the Cryptosporidium genome. 4) The identification of drug resistance mechanisms will facilitate the development of new selectable markers for advancing genome editing procedures. To reduce the frequency of unselected mutations generated by random mutagenesis, and maximize the probability of obtaining selectable C. parvum mutants, two alkylating mutagens and UV irradiation will be used. Mutagenized parasite lines will be selected by backcrossing to wildtype C. parvum in mice while applying drug selection. The genome of multiple drug-resistant C. parvum lines will be sequenced and compared to the genome of the wildtype line to identify mutations under selection. Specific Aim 1 will investigate chemical mutagenesis to generate selectable C. parvum phenotypes. We will select for resistance to two drugs: aurintricarboxylic acid, and the piperazine-based compound MMV665917 from the Medicines for Malaria Venture collection, found to be effective against C. parvum. In Specific Aim 2, UV irradiation will be used to mutagenize oocysts. For both aims, backcrossing to wildtype C. parvum will be used to flush out irrelevant mutations. The genome of multiple, independently selected drug-resistant lines will be sequenced. Mutations correlating with drug resistance in replicated selection experiments will be identified by mapping sequence reads to the C. parvum reference genome. To demonstrate causality, a mutation hypothesized based on replicated selection experiments to confer drug resistance will be introduced into wildtype C. parvum using CRISP/Cas9. A successful outcome of this research will highlight the value of forward genetics to innovate research on cryptosporidiosis.

摘要 在低收入和中等收入国家，隐孢子虫病是导致婴儿虚弱的主要原因之一。 拉肚子。控制隐孢子虫寄生虫的有效药物选择仍然有限，开发一种 疫苗目前还处于早期阶段。尽管儿童腹泻对公共健康造成了影响，但缺乏研究 工具继续阻碍着更好地了解这些寄生虫的进展。这样做的目的是 项目是开发一种基于随机突变的正向遗传学方法，以促进基本和 人畜共患微小隐孢子虫的应用研究 隐孢子虫病。术语正向遗传学指的是用来确定遗传决定因素的方法 特定的表型。这种方法扩大了我们对模型生物的理解，包括 分类上有亲缘关系的弓形虫。拟议的项目将主要影响以下四个方面 隐孢子虫研究。1)它将通过识别以下目标基因座来推进药物开发工作 抗隐孢子虫化合物。2)它将使临床相关的遗传基础的解剖成为可能。 药物敏感性、毒力或卵囊产量等特征，以及可能发现的与 多药耐药。3)有助于对编码未知功能蛋白质的基因进行注释， 仍占隐孢子虫基因组的三分之一。4)耐药性鉴定 机制将促进用于推进基因组编辑的新的可选择标记的开发 程序。减少随机诱变产生的未选定突变的频率，以及 最大限度地获得可选择的微小弧菌突变体、两种烷基化突变剂和紫外线 将使用辐射。突变的寄生虫品系将通过回交到野生型C。 小鼠，同时应用药物选择。多个抗药性细小弧菌株的基因组将是 测序并与野生型品系的基因组进行比较，以确定选择下的突变。 具体目标1将研究化学诱变以产生可选择的微小弧菌表型。 我们将选择两种药物的抗药性：金曲酸和以哌嗪为基础的化合物 MMV665917来自疟疾药物风险投资收藏，被发现对微小隐孢子虫有效。在……里面 具体目的2，将紫外线照射用于诱变卵囊。为了这两个目标，回交到野生型 微小隐孢子虫将被用来清除无关的突变。多个独立选择的基因组 耐药品系将被测序。重复选择中与耐药性相关的突变 实验将通过将序列读数映射到微小隐孢子虫的参考基因组来确定。至 证明因果关系，这是一种基于重复选择实验的突变，用于提供药物 利用CRISP/Cas9将抗性引入野生型细小球藻。这是一个成功的结果 研究将突出正向遗传学对创新隐孢子虫病研究的价值。