Cryptosporidium mutagenesis

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 腹泻控制隐孢子虫寄生虫的有效药物选择仍然有限， 疫苗处于早期阶段。尽管儿童腹泻对公共卫生有影响，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。这个目标 项目是开发一种基于随机诱变的正向遗传学方法， 关于隐孢子虫的应用研究，这是一种人畜共患物种，约占人类病例的一半 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。这种方法扩大了我们对模式生物的理解，包括 分类学上相关的物种弓形虫。拟议项目将主要影响四个领域， 隐孢子虫研究。1)它将通过识别靶向的遗传位点来推进药物开发工作， 抗隐孢子虫化合物。2)它将使解剖的遗传基础，临床相关的 性状，如药物敏感性，毒力或卵囊输出，并可能发现与 多药耐药3)它将有助于编码未知功能蛋白质的基因的注释， 仍占隐孢子虫基因组的三分之一4)耐药性鉴定 机制将促进新的选择性标记的开发，以推进基因组编辑 程序.降低随机诱变产生的突变频率， 最大化获得可选C的概率。parvum突变体，两种烷基化诱变剂和紫外线 将使用辐射。将通过与野生型C回交来选择诱变的寄生虫系。微小的 小鼠，同时应用药物选择。多重耐药念珠菌的基因组。parvum线将是 测序并与野生型品系的基因组比较以鉴定选择下的突变。 具体目标1将研究化学诱变以产生可选择的C。parvum表型 我们将选择对两种药物有耐药性的：金精三羧酸和哌嗪类化合物 MMV 665917来自Medicines for Malaria Venture collection，发现对C.小的在 具体目标2，UV辐照将用于诱变卵囊。为了这两个目的，回交到野生型 C.将使用parvum来清除不相关的突变。多个独立选择的基因组 将对耐药株系进行测序。重复选择中与抗药性相关的突变 实验将通过将序列读数映射到C.参考基因组。到 证明因果关系，一种基于重复选择实验假设的突变， 将抗性引入野生型C。使用CRISP/Cas9对parvum进行鉴定。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。

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.