Determining the molecular mechanism of anthelmintic resistance in hookworms

描述(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，由于肠道中的成虫失血，导致虚弱的、有时是致命的缺铁性贫血。儿童、孕妇和老年人特别容易受到钩虫感染的影响。控制策略仅限于使用苯并咪唑(BZ)驱虫剂对感染者进行定期驱虫。有相当多的人担心，随着大规模药物管理(MDA)项目中控制钩虫的使用增加，对BZ药物的抗药性将会发展。监测耐药性出现的分子测试是必要的，但钩虫对BZ产生耐药性的基因突变尚不清楚，而且似乎与其他寄生线虫的耐药性不同。缺乏对BZ耐药的钩虫株阻碍了对耐药性突变的识别。为了确定钩虫对BZ产生抗性的潜在基因突变，我们建议使用一种新的体外突变和选择方案来产生一种对BZ具有表型抗性的犬钩虫品系。在目标1中，我们将在体外诱变成虫以产生遗传变异，并将它们送回幼稚的宿主进行繁殖。F2代的后代将通过体外暴露于BZ药物来选择抗药性。幸存者将被用来感染新的宿主，F3的后代将被选为BZ。F3幸存者将与野生型亲本品系进行双向回交，BZ用于选择抗性蠕虫进行抗性品系的繁殖。表型抗性将通过标准的卵孵化和幼虫发育试验来确认。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株的比较来鉴定与耐药性相关的突变。候选抗性等位基因将通过它们在反式中赋予模式线虫BZ抗性的能力来确认。钩虫抗药性菌株的产生和引起对BZ驱虫剂抗药性的基因突变的鉴定将使基于分子的工具的开发能够监测正在接受治疗的人群中抗药性等位基因的频率。这将允许及时修改治疗方案，以防止广泛耐药的发展。

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 naïve 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.