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.

抽象的 在低收入和中等收入国家，隐孢子虫病是导致婴儿衰弱的主要原因之一 腹泻。控制隐孢子虫寄生虫的有效药物选择仍然有限，并且努力开发一种 疫苗尚处于早期阶段。尽管儿童腹泻对公共健康产生影响，但缺乏研究 工具继续阻碍更好地了解这些寄生虫的进展。此举的目标 该项目是开发一种基于随机诱变的正向遗传学方法，以促进基本和 小隐孢子虫的应用研究，一种人畜共患的物种，约一半的人类病例是由隐孢子虫引起的 隐孢子虫病。术语“正向遗传学”是指用于识别遗传决定因素的方法。 特定的表型。这种方法扩展了我们对模式生物的理解，包括 分类学相关物种弓形虫。拟议项目将主要影响四个领域 隐孢子虫研究。 1) 它将通过识别靶向基因位点来推进药物开发工作 抗隐孢子虫化合物。 2）它将能够剖析临床相关的遗传基础 药物敏感性、毒力或卵囊输出等特征，并可能发现与 多重耐药性。 3）它将有助于注释编码未知功能的蛋白质的基因， 仍然代表隐孢子虫基因组的三分之一。 4）耐药性鉴定 机制将促进新的选择性标记的开发，以推进基因组编辑 程序。减少随机诱变产生的未选择突变的频率，以及 最大化获得可选择的 C. parvum 突变体、两种烷化诱变剂和 UV 的概率 将使用辐照。诱变的寄生虫系将通过与野生型 C. parvum 回交来选择 小鼠在应用药物选择时。多个耐药 C. parvum 品系的基因组将被 测序并与野生型品系的基因组进行比较，以识别选择下的突变。 具体目标 1 将研究化学诱变以产生可选择的 C. parvum 表型。 我们将选择两种药物的耐药性：金三甲酸和哌嗪类化合物 MMV665917 来自 Medicines for Malaria Venture 系列，被发现可有效对抗微小衣原体。在 具体目标 2，将使用紫外线照射来诱变卵囊。为了实现这两个目标，与野生型回交 小隐孢子虫将用于清除不相关的突变。多个、独立选择的基因组 耐药株系将被测序。重复选择中与耐药性相关的突变 实验将通过将序列读取映射到 C. parvum 参考基因组来识别。到 证明因果关系，一种基于重复选择实验假设的突变，以赋予药物 使用 CRISP/Cas9 将抗性引入野生型 C. parvum。此次活动取得圆满成功 研究将凸显正向遗传学对隐孢子虫病创新研究的价值。