Dissecting the mechanism of pyrantel resistance in hookworm

摘要 钩虫感染仍然是全球最重要的公共卫生威胁之一，估计有450人 数以百万计的感染者。严重的钩虫感染是热带地区贫血的主要原因，导致虚弱。 有时甚至是致命的缺铁性贫血。儿童、孕妇和老年人尤其 易受钩虫感染致病。控制策略仅限于定期驱虫 使用苯并咪唑(BZ)和其他驱虫药的感染者。有人担心，对 驱虫药的发展将随着大规模药物管理(MDA)项目的增加而发展 钩虫，需要一年或两年治疗。驱虫药抗性(AR)的速度 在感染家畜和同伴动物的寄生线虫中发展表明，增加 丙二醛对人类寄生线虫的选择性压力会迅速产生抗性蠕虫种群，因为 井。耐药性也是其他药物的主要担忧，如伊维菌素(IVM)和吡喃酮(PYR)，这些药物 通常用于治疗淋巴丝虫病和土壤传播的线虫，包括钩虫。 监测耐药性出现的分子测试是必要的，但导致耐药性的基因突变 钩虫对PYR和IVM的耐药性未知。检测钩虫中出现的AR的分子测试 可以帮助在MDA期间进行量身定制的药物治疗，使其更加有效，并将是避免最大损失的关键 防治钩虫的有效驱虫剂。此前，我们发现了一种钩虫菌株，命名为KGR，它 对BZ驱虫剂噻菌灵(TBZ)和伊维菌素(IVM)具有抗药性。最近，我们 分离出第二个独立菌株，命名为BCR，它对三种药物TBZ、IVM和吡喃酮具有抗药性 (PYR)。我们在bcr分离株中观察到一种独特的表型，我们的初步数据表明这一表型与 为了抵抗派尔。感染BCR幼虫(IL3)的激活受到抑制，以响应宿主信号 与敏感的iL3相比，这种影响在提高孵化温度时被部分挽救。 此外，我们的证据指出膜鸟苷酸环化酶(Mgc)的缺陷，它是一个关键的效应因子。 激活的结果。我们建议确定这种表型的潜在分子机制，以及它在 PYR抵抗。在目标1，我们将确定单核苷酸多态(SNPs)在功能结构域 BCR菌株所特有的mGC，并以秀丽线虫为替代品来测试其充分性 产生抗药性的突变。我们将通过在同源线虫中引入SNPs来实现这一点 通过CRISPR/Cas9和编辑后的线虫对PYR抗性进行检测。在目标2中，我们将调查 Bcr幼虫缺陷激活反应的热敏感性。我们将确定热休克的作用 在修复bcr幼虫激活缺陷中的反应，特别是伴侣HSP90。这项建议 将为钩虫对PYR抗药性的机制提供长期追求的洞察力。

SUMMARY Hookworm infection remains one of the most important public health threats worldwide, with an estimated 450 million infected. Heavy hookworm infection is the leading cause of anemia in the tropics, resulting in debilitating and sometimes fatal iron-deficiency anemia. 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) and other anthelmintics. There is concern that resistance to anthelmintics will develop with increased use in mass drug administration (MDA) programs used to control hookworms, which require annual or biannual treatment. The rapidity with which anthelmintic resistance (AR) developed in parasitic nematodes infecting livestock and companion animals suggest that increasing the selective pressure on human parasitic nematodes by MDA will rapidly generate resistant worm populations as well. Resistance is also a major concern for other drugs such as ivermectin (IVM) and pyrantel (PYR) which are commonly used to treat lymphatic filariasis and soil transmitted nematodes including hookworms. Molecular tests to monitor the emergence of resistance are necessary, but the genetic mutations that confer resistance to PYR and IVM are unknown in hookworm. Molecular tests that detect emerging AR in hookworms can help tailor drug treatments during MDA to be more effective and will be critical to avoid losing the most effective anthelmintics for hookworm control. Previously, we identified a hookworm strain, named KGR, which was resistant to the BZ anthelmintic thiabendazole (TBZ) as well as ivermectin (IVM). More recently we isolated a second independent strain, named BCR, which is resistant to three drugs, TBZ, IVM, and pyrantel (PYR). We have observed a unique phenotype in the BCR isolate that our preliminary data suggests is linked to PYR resistance. Activation of infective BCR larvae (iL3) is depressed in response to host signals when compared to susceptible iL3, and this effect is partially rescued at increased incubation temperature. Furthermore, our evidence points to a defect in the membrane guanylyl cyclase (mGC) which is a key effector of activation. We propose to determine the underlying molecular mechanism for this phenotype, and its role in PYR resistance. In Aim 1, we will identify single nucleotide polymorphisms (SNPs) in functional domains of mGCs that are unique to the BCR strain and use Caenorhabditis elegans as a surrogate to test the sufficiency of the mutations in conferring resistance. We will do this by introducing the SNPs in orthologous C. elegans genes by CRISPR/Cas9 and testing the edited nematodes for PYR resistance. In Aim 2 we will investigate the heat sensitivity of the defective activation response in BCR larvae. We will determine the role of the heat shock response and specifically the chaperone HSP90 in rescue of the activation defect in BCR larvae. This proposal will provide long sought after insight into the mechanism of PYR resistance in hookworms.