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.
Highlights
Parasitic nematodes cause widespread, debilitating diseases that produce substantial mortality and morbidity among the global poor in human populations
The objective of the current study was to devise an experimental approach that can illuminate all cells in live A. suum L3 and L4 stages with fluorescent nuclear probes and provide a rapid resolution of cell death among organ systems conferred by nematode intestinal toxins/toxicants (NITs) treatments (BB in combination with vital dye propidium iodide, PI), while comparing the performance of NITs in causing cell death among cells and organ systems (PI labeling profiles)
In this investigation we integrate the use of i) fluorescent nuclear probes, ii) a set of 6 small molecule inhibitors recently demonstrated to target the nematode intestine (nematode in testinal toxins/toxicants, NITs (Jasmer et al, 2020)), iii) an Ascaris suum larval parasite culture system and iv) RNA-seq transcriptional response to NITs, to conduct rapid pathologic and molecular assessment of NIT toxicity relative to many cells and organ systems in the whole worm
Summary
Parasitic nematodes cause widespread, debilitating diseases that produce substantial mortality and morbidity among the global poor in human populations. A surge in research to increase the limited arsenal of available anthelmintic compounds has stemmed from several advances, including expanding parasite multi-omics resources, technological advances in biologic screening methods, access to expanding small molecule inhib itor libraries, and enhanced computational methods that integrate bio logic data with large knowledge bases related to both drug and inhibitor compounds (examples include (Taylor et al, 2013; Tyagi et al, 2018; Tyagi et al, 2019; Jasmer et al, 2020)) These omics-driven approaches have proven quite effective in identifying small molecule inhibitors that are toxic to parasitic nematodes and have multiple applications to anthelmintic research. They may warrant development as anthelmintics, or provide much-needed research tools to dissect mech anisms relevant to drug discovery
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