Functional genomics in <i>Brugia malayi</i> reveal diverse muscle nAChRs and differences between cholinergic anthelmintics.

Abstract

Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to <i>Caenorhabditis elegans</i> because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite <i>Brugia malayi,</i> which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (<i>M-, L-, P-,</i> and <i>N-</i>). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the <i>unc-38</i> and/or <i>unc-29</i> genes. Derquantel behaved as a competitive antagonist and distinguished <i>M-</i>nAChRs activated by morantel (<i>K</i>_b 13.9 nM), <i>P-</i>nAChRs activated by pyrantel (<i>K</i>_b 126 nM), and <i>L-</i>nAChRs activated by levamisole (<i>K</i>_b 0.96 µM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing <i>N-</i>type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the <i>C. elegans</i> model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of <i>unc-38+unc-29</i> (<i>L-</i> and/or <i>P-</i>receptors) inhibited motility but knockdown of <i>acr-16+acr-26</i> (<i>M-</i> and/or <i>N-</i>receptors) did not.