Abstract
In research focused on the intestine of parasitic nematodes, we recently identified small molecule inhibitors toxic to intestinal cells of larval Ascaris suum (nematode intestinal toxins/toxicants; "NITs"). Some NITs had anthelmintic activity across the phylogenetic diversity of the Nematoda. The whole-worm motility inhibition assay quantified anthelmintic activity, but worm responses to NITs in relation to pathology or affected molecular pathways was not acquired. In this study we extended this research to more comprehensively determine in whole larval A. suum the cells, organ systems, molecular targets, and potential cellular pathways involved in mechanisms of toxicity leading to cell death. The experimental system utilized fluorescent nuclear probes (bisbenzimide, propidium iodide), NITs, an A. suum larval parasite culture system and transcriptional responses (RNA-seq) to NITs. The approach provides for rapid resolution of NIT-induced cell death among organ systems (e.g. intestine, excretory, esophagus, hypodermis and seam cells, and nervous), discriminates among NITs based on cell death profiles, and identifies cells and organ systems with the greatest NIT sensitivity (e.g. intestine and apparent neuronal cells adjacent to the nerve ring). Application was extended to identify cells and organs sensitive to several existing anthelmintics. This approach also resolved intestinal cell death and irreparable damage induced in adult A. suum by two NITs, establishing a new model to elucidate relevant pathologic mechanisms in adult worms. RNA-seq analysis resolved A. suum genes responsive to treatments with three NITs, identifying dihydroorotate dehydrogenase (uridine synthesis) and RAB GTPase(s) (vesicle transport) as potential targets/pathways leading to cell death. A set of genes induced by all three NITs tested suggest common stress or survival responses activated by NITs. Beyond the presented specific lines of research, elements of the overall experimental system presented in this study have broad application toward systematic development of new anthelmintics.