Rapid determination of nematode cell and organ susceptibility to toxic treatments

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.

Cell Death and Transcriptional Responses Induced in Larvae of the Nematode Haemonchus contortus by Toxins/Toxicants with Broad Phylogenetic Efficacy

“…Results from a PI-based cell death assay and GRPs can augment insights provided by whole-worm motility inhibition assays that are commonly used to evaluate effects of drugs and drug-like compounds on parasitic nematodes. The rapid resolution of individual cells and organs affected by these treatments in live worms greatly enhances details of likely relevance to the anthelmintic effects at play, as was demonstrated in our previous studies in the nematode clade III parasite, A. suum [ 3 , 4 ]. In this investigation, we explored the utility of the overall experimental approach to another parasitic species, H. contortus , a clade V parasite.…”

“…In past research, the effects of six nematode intestinal toxins/toxicants (NITs) were reported for A. suum L3 and L4 larvae (staurosporine, ruxolitinib, leflunomide, sunitinib, alvocidib, and CID1067700) [ 3 , 4 ]. A seventh NIT not previously reported (p38 MAP kinase inhibitor IV, also known as MT4) was identified through the same de novo process as in Jasmer et al, 2020 [ 3 ] and is a prospective inhibitor of p38 MAP kinases.…”

“…Cellular proteins annotated as targets for each NIT collectively identify a diverse set of potential target proteins of significance in A. suum intestinal cells [ 4 ]. Although new approaches to kill nematode intestinal cells was a goal of this research, expression in other tissues was expected for many of the potential target proteins.…”

“…Components of early gene response patterns (GRPs) induced by NITs may reflect elements of pathways involved with initiating or mediating cell death responses. RNA-seq analysis following treatment with NITs identified early GRPs in A. suum larvae that informed on potential target proteins and pathways (e.g.,dihydroorotate dehydrogenase (uridine synthesis) and RAB GTPase(s) (vesicle transport)) that may relate to initiator events leading to cell death induced by selected NITs [ 4 ]. Information of this kind can aid in the design of experiments to manipulate cell death outcomes initiated by NITs and assessed by the PI cell death assay.…”

“…These three species were used for the initial computational prioritization of NITs [ 3 ], and they collectively represent much of the phylogenetic distance in the Nematoda, which includes parasites of many species including humans, animals, and plants (where parasitism is defined as requiring a host to complete at least one stage of its life cycle, as opposed to non-parasitic “free-living” species such as C. elegans which does not infect any hosts [ 7 ]). Although A. suum figured centrally in the development of this pan-Nematoda approach [ 3 , 4 ], we here sought to determine if NITs and the PI cell death assay have applications to rapidly detect death induced by NITs in cells and organs of H. contortus, leaving T. suis the only one of the three for which applications have yet to be investigated. H. contortus , an abomasal parasite of small ruminants, was chosen due to its veterinary importance and a number of experimental advantages it offers, including availability of large numbers of infective L3 and the ability to culture exsheathed L3 (xL3) to “parasitic” L4s.…”