Determining the molecular mechanism of anthelmintic resistance in hookworms

描述（由申请人提供）：钩虫感染仍然是全世界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，导致身体虚弱，有时甚至致命的缺铁性贫血，这是由于在肠道内喂养成虫时失血造成的。儿童、孕妇和老年人特别容易感染钩虫。控制策略仅限于使用苯并咪唑（BZ）驱虫药对受感染个体进行周期性驱虫。随着大规模给药（MDA）计划用于控制钩虫，对BZ药物的耐药性将会增加，这是相当令人担忧的。监测耐药性出现的分子测试是必要的，但在钩虫中，赋予BZ抗性的基因突变是未知的，而且似乎与赋予其他寄生线虫抗性的基因突变不同。钩虫BZ抗性菌株的缺乏阻碍了抗性突变的鉴定。为了确定赋予钩虫BZ抗性的潜在基因突变，我们提出了一种新的体外诱变和选择方案，以产生对BZ具有表型抗性的犬钩虫Ancylostoma caninum菌株。在目标1中，我们将在体外诱变成年钩虫以产生遗传变异，并将它们返回naïve宿主进行繁殖。F2后代将通过体外暴露于BZ药物来选择耐药性。幸存者将被用来感染新的宿主，而F3后代将被BZ选中。F3幸存者将与野生型亲本菌株进行回交，BZ用于选择抗性蠕虫进行抗性菌株的繁殖。表型抗性将通过标准卵孵化和幼虫发育测定来证实。在目标2中，我们将通过全基因组测序和与易感野生型亲本菌株的比较来确定与抗性相关的突变。候选抗性等位基因将通过其赋予模式线虫线虫BZ抗性的能力得到证实。产生耐药的钩虫菌株和鉴定对BZ驱虫药产生耐药性的基因突变，将允许开发基于分子的工具，以监测正在接受治疗的种群中抗性等位基因的频率。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 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.