Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.
Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets.
Department of Biological Sciences, The University of Toledo, Toledo, Ohio, United States of America.
Wenjing
W
Law
Department of Biological Sciences, The University of Toledo, Toledo, Ohio, United States of America.
Leah M
LM
Wuescher
Department of Biological Sciences, The University of Toledo, Toledo, Ohio, United States of America.
Amanda
A
Ortega
Department of Biological Sciences, The University of Toledo, Toledo, Ohio, United States of America.
Vera M
VM
Hapiak
Department of Biological Sciences, The University of Toledo, Toledo, Ohio, United States of America.
Patricia R
PR
Komuniecki
Department of Biological Sciences, The University of Toledo, Toledo, Ohio, United States of America.
Richard
R
Komuniecki
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D000871:Anthelmintics / Q000494:pharmacology*; D001522:Behavior, Animal / Q000187:drug effects*; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D065101:Chloride Channel Agonists / Q000494:pharmacology*; D029721:Drosophila Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D055808:Drug Discovery / Q000379:methods*; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006189:Haemonchus; D015801:Helminth Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007038:Hypotonic Solutions / Q000633:toxicity; D007395:Interneurons / Q000187:drug effects / Q000378:metabolism; D009043:Motor Activity / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D017982:Receptors, Biogenic Amine / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D058825:Serotonin 5-HT1 Receptor Agonists / Q000494:pharmacology*
D000871:Anthelmintics; D029742:Caenorhabditis elegans Proteins; D065101:Chloride Channel Agonists; D029721:Drosophila Proteins; D044385:GTP-Binding Protein alpha Subunits; D015801:Helminth Proteins; D007038:Hypotonic Solutions; D009419:Nerve Tissue Proteins; D017982:Receptors, Biogenic Amine; D011994:Recombinant Proteins; D058825:Serotonin 5-HT1 Receptor Agonists