Determining the molecular mechanism of anthelmintic resistance in hookworms

 描述（由申请人提供）：钩虫感染仍然是全球最重要的公共卫生威胁之一，估计有8亿人感染。严重的钩虫感染是热带地区贫血的主要原因，由于肠道中蠕虫而导致失血，导致虚弱，有时是致命的缺铁性贫血。儿童、孕妇和老年人特别容易因钩虫感染而发病。控制策略仅限于定期用苯并咪唑（BZ）驱虫剂对感染个体进行驱虫。有相当大的关注，耐药性BZ药物将发展与使用越来越多的大规模药物管理（MDA）计划，以控制钩虫。监测耐药性出现的分子测试是必要的，但在钩虫中赋予BZ耐药性的基因突变是未知的，并且似乎与赋予其他寄生线虫耐药性的基因突变不同。缺乏BZ抗性钩虫株阻止了抗性突变的鉴定。为了确定潜在的基因突变，赋予BZ抗性钩虫，我们建议使用一种新的体外诱变和选择协议，以产生一个菌株的犬钩虫钩虫犬钩虫是表型耐BZ。在目标1中，我们将在体外诱变成年钩虫以产生遗传变异，并将其返回到幼稚宿主中繁殖。将通过体外暴露于BZ药物来选择F2后代的耐药性。将存活者用于感染新的宿主，并用BZ选择F3后代。F3幸存者将与野生型亲本菌株进行反向回交，BZ用于选择抗性蠕虫以繁殖抗性菌株。将通过标准卵孵化和幼虫发育试验确认表型抗性。在目标2中，我们将通过全基因组测序和与敏感野生型亲本菌株的比较来鉴定与耐药性相关的突变。候选人的耐药等位基因将被确认的能力，赋予BZ抗性的模式线虫秀丽隐杆线虫在反式。一代的抗钩虫菌株和识别的基因突变，导致耐药性BZ驱虫剂将允许开发基于分子的工具，以监测频率的耐药等位基因的人口正在接受治疗。这将允许及时修改治疗方案，以防止产生广泛的耐药性。

 DESCRIPTION (provided by applicant): Hookworm infection remains one of the most important public health threats worldwide, with an estimated 800 million people infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia caused by blood loss to feeding adult worms in the intestine. Children, pregnant women, and the elderly are particularly susceptible to morbidity from hookworm infection. Control strategies are restricted to periodic de-worming of infected individuals with benzimidazole (BZ) anthelmintics. There is considerable concern that resistance to BZ drugs will develop with increased use in mass drug administration (MDA) programs to control hookworms. Molecular tests to monitor the emergence of resistance are necessary, but the genetic mutations that confer resistance to BZ are unknown in hookworms, and appear to be different than those that confer resistance in other parasitic nematodes. The lack of a BZ resistant strain of hookworm prevents identification of the resistance mutations. To determine the underlying genetic mutations that confer BZ resistance in hookworms, we propose using a novel in vitro mutagenesis and selection protocol to generate a strain of the canine hookworm Ancylostoma caninum that is phenotypically resistant to BZ. In Aim 1 we will mutagenize adult hookworms in vitro to generate genetic variation, and return them to a na¿ve host to reproduce. F2 offspring will be selected for resistance by exposure to BZ drugs in vitro. Survivors will be used to infect new hosts, and the F3 offspring selected with BZ. F3 survivors will be reciprocally backcrossed to the wild type parental strain, and BZ used to select resistant worms for propagation of the resistant strains. Phenotypic resistance will be confirmed by standard egg hatch and larval development assays. In Aim 2, we will identify mutations associated with resistance by whole genome sequencing and comparison to the susceptible wild type parental strain. Candidate resistance alleles will be confirmed by their ability to confer BZ resistance on the model nematode Caenorhabditis elegans in trans. Generation of a resistant strain of hookworm and the identification of the genetic mutations that cause resistance to BZ anthelmintics will permit the development of molecular based tools to monitor the frequency of resistance alleles in populations undergoing treatment. This will allow modification of treatment regiments in time to prevent widespread resistance from developing.