Genetic Basis of Praziquantel Resistance

 描述(申请人提供)：吡喹酮抗药性的遗传基础使用吡喹酮(PZQ)单一疗法的大规模治疗活动对血吸虫寄生虫的抗药性进行了强烈的选择。可以在实验室中选择抗药性，并从患者身上分离出抗药性寄生虫，但目前自然血吸虫种群中的抗药性水平不可能使用经典寄生虫学方法进行量化。这项建议旨在确定导致PZQ耐药性的基因(S)，以便(A)了解该药物的作用模式，(B)确定耐药机制，以及(C)开发用于治疗计划中现场耐药监测的分子标记。在前期工作中(由R21 AI096277资助)，我们进行了敏感和抗性寄生虫(由实验室PZQ选择产生)之间的遗传杂交，并使用外显子组测序来表征在PZQ处理后存活或死亡的F2寄生虫。我们在跨度为4.29Mb的4个支架上发现了多个与PZQ抗性相关的标记，包含110个基因。这些支架被指定为CHR 3，但目前在基因组序列中是未组装的。在目标1中，我们将通过以下方法对与PZQ抗性有关的基因(S)进行精细定位：(I)通过对现有遗传杂交中的SNP分离进行外显子测序和连锁分析，(Ii)通过系统的RNAi敲除QTL区域的优先基因和成虫的表型分析，以及(Iii)通过基于转基因的候选基因的操作，将未组装的支架正确地定位到基因组序列上。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。为此，我们将比较肯尼亚和乌干达的PZQ治疗前后从血吸虫感染患者那里收集的幼虫(毛虫)种群的全基因组等位基因频率。这些分析将确定在PZQ治疗中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将是无价的。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选择的冷冻抗PZQ寄生虫的存档集合中的单个蠕虫的外显子进行测序。这些数据将批判性地检验这一假设，即CHR中的基因。3与种群控制的敏感寄生虫相比，支架中富含这种寄生虫。我们以前已经证明了结合遗传连锁图谱研究、功能分析和现场采集的寄生虫分析来确定奥沙米喹耐药性的遗传基础的力量。平行方法有望同样有效地确定PZQ耐药的遗传基础。

 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.