Cholinergic anthelmintics: Tachyphylaxis mechanisms and control in a parasitic nematode model,Brugia malayi - Project Summary Soil-transmitted helminth infections are a global concern for public health and affect over 1 billion people worldwide. Infections caused by Ascaris lumbricoides, Trichuris trichiura (whipworms), and Ancylostoma duodenale (hookworms) affect people in warm and moist climates often lacking in hygiene and sanitation and in temperate zones during warmer months. Infected children develop severe malnutrition and show severe physical and cognitive growth. Control of these parasites depends on the administration of anthelmintic drugs like benzimidazoles like albendazole, macrocyclic lactone, ivermectin, and nicotinic agonists like levamisole and pyrantel. The development of resistance to these anthelmintics poses a great challenge in controlling the transmission of these diseases. Parasitic nematodes are complex organisms that adopt complex mechanisms to resist exposure to anthelmintic drugs. We have identified tachyphylaxis as one mechanism the parasite utilizes to resist and recover motility in the continued exposure to the cholinergic anthelmintic levamisole. The endoplasmic reticulum retention protein, NRA-2, is one of the proteins implicated in modulating tachyphylaxis in the female Brugia malayi parasites. The transcription factor, DAF-12 is upregulated in tachyphylactic worms and could play a key role in mediating tachyphylaxis. Macrocyclic lactone, abamectin, has limited effect on its own on the motility of adult B. malayi but, when applied in combination with levamisole, prevents tachyphylaxis and recovery of motility. Here, we propose, Aim #1: To test the hypothesis that DAF-12 mediates tachyphylaxis in B. malayi. We will knockdown daf-12 in adult B. malayi to determine its role in mediating tachyphylaxis. We will measure transcript levels of nra-2 in daf-12 knockdown worms to determine the role of DAF-12 in regulating the expression of nra-2. We will also target DAF-12 using agonists to prevent tachyphylaxis. Aim #2: To test the hypothesis that muscle calcium concentrations & calcium release mechanisms produced by levamisole are changed in the presence of macrocyclic lactones. We will investigate the effects of macrocyclic lactones, abamectin, ivermectin, and moxidectin in prolonging the paralysis induced by levamisole. We will evaluate the change in calcium release mechanisms due to macrocyclic lactones by performing RNAi of the ryanodine and the IP3 receptors, unc-68 and itr-1 in B. malayi, and performing in vivo calcium imaging using C. elegans. At the end of these experiments, we will have characterized DAF-12 as a novel anthelmintic target that could help prevent cholinergic tachyphylaxis. We would have an insight into the effect of macrocyclic lactones on levamisole calcium release mechanisms in nematode muscle. These studies will provide an improved mechanistic insight into cholinergic tachyphylaxis and a rational avenue to prevent worm recovery and resistance.
