Genetic Basis of Praziquantel Resistance

 描述（由申请方提供）：吡喹酮耐药性的遗传基础使用吡喹酮（PZQ）单药治疗的大规模治疗活动对寄生虫的耐药性有很强的选择性。可以在实验室中选择耐药性，并且已经从患者中分离出耐药性寄生虫，但是使用经典的寄生虫学方法无法量化天然寄生虫种群中的当前耐药性水平。本提案旨在鉴定PZQ耐药的潜在基因，以便（a）了解该药物的作用模式，（B）确定耐药机制，以及（c）开发用于治疗方案中耐药现场监测的分子标记物。在初步工作（由R21 AI096277资助）中，我们在敏感和耐药寄生虫（通过实验室中的PZQ选择产生）之间进行了遗传杂交，并使用外显子组测序来表征PZQ治疗后存活或死亡的F2寄生虫。我们在4个支架上鉴定了与PZQ抗性相关的多个标记，跨度为4.29Mb，含有110个基因。这些支架被分配到chr 3，但目前在基因组序列中未组装。在目标1中，我们将精细定位参与PZQ抗性的基因（i）通过外显子组测序和现有遗传杂交中SNP分离的连锁分析将未组装的支架正确定位到基因组序列，（ii）通过QTL区域中优先基因的系统性RNAi敲除和成虫寄生虫的表型分析，以及（iii）通过候选基因的基于转染的操作。在目标2中，我们将研究该基因组区域与田间PZQ抗性的相关性。为了做到这一点，我们将比较全基因组等位基因频率幼虫寄生虫（毛蚴）人口收集的前和后PZQ治疗从肯尼亚和乌干达的网站的感染患者。这些分析将鉴定在PZQ治疗存活的寄生虫中显示等位基因频率系统性变化的基因组区域。这些信息对于控制程序来说是非常宝贵的。作为一种补充方法，在目标3中，我们将对先前从田间分离并在实验室中选择的冷冻PZQ抗性寄生虫的存档集合中的单个蠕虫的外显子组进行测序。这些数据将批判性地检验以下假设：与群体对照的敏感寄生虫相比，chr. 3支架中的基因在这些寄生虫中富集。我们以前已经证明了结合遗传连锁图谱研究，功能分析和分析现场收集的寄生虫，以确定奥曲喹耐药性的遗传基础的力量。平行的方法有望同样有效地确定PZQ抗性的遗传基础。

 DESCRIPTION (provided by applicant): Genetic basis of praziquantel resistance Mass treatment campaigns using praziquantel (PZQ) monotherapy impose strong selection for drug resistance in schistosome parasites. Resistance can be selected in the laboratory, and resistant parasites have been isolated from patients, but the current level of resistance in natural schistosome populations is not possible to quantify using classical parasitological methods. This proposal aims to identify the gene(s) underlying PZQ resistance in order to (a) understand the mode of action of this drug, (b) determine the mechanism of resistance, and (c) develop molecular markers for field surveillance of resistance in treatment programs. In preliminary work (funded by R21 AI096277), we conducted genetic crosses between sensitive and resistant parasites (generated by PZQ selection in the laboratory) and used exome sequencing to characterize F2 parasites that survived or died following PZQ treatment. We identified multiple markers linked to PZQ resistance on 4 scaffolds spanning 4.29Mb and containing 110 genes. These scaffolds are assigned to chr 3 but are currently unassembled in the genome sequence. In Aim 1, we will fine map the gene(s) involved in PZQ resistance (i) by correctly positioning the unassembled scaffolds to the genome sequence by exome sequencing and linkage analysis of SNP segregation in an existing genetic cross, (ii) by systematic RNAi knockdown of prioritized genes in the QTL region and phenotypic analysis of adult parasites, and (iii) by transfection-based manipulation of candidate genes. In Aim 2, we will examine the relevance of this genome region to PZQ resistance in the field. To do this we will compare genome-wide allele frequencies in larval parasite (miracidia) populations collected from schistosome-infected patients before and after PZQ treatment from sites in Kenya and Uganda. These analyses will identify genome regions showing systematic change in allele frequencies in parasites surviving PZQ treatment. This information will be invaluable for control programs. As a complementary approach, in Aim 3 we will sequence exomes of individual worms from an archived collection of frozen PZQ-resistant parasites previously isolated from the field and selected in the laboratory. These data will critically examine the hypothesis that genes in the chr. 3 scaffolds are enriched in such parasites when compared to sensitive parasites of population controls. We have previously demonstrated the power of combining genetic linkage mapping studies, functional analyses and analysis of field-collected parasites for determining the genetic basis of oxamniquine resistance. Parallel approaches promise to be equally effective for determining the genetic basis of PZQ resistance.