Dissecting the mechanism of pyrantel resistance in hookworm

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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.