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.

在专注于寄生线虫肠道的研究中，我们最近发现了对猪蛔虫幼虫肠道细胞有毒的小分子抑制剂（线虫肠道毒素/毒物；“NIT”）。一些 NIT 对线虫的系统发育多样性具有驱虫活性。全蠕虫运动抑制试验量化了驱虫活性，但未获得蠕虫对 NIT 与病理或受影响的分子途径相关的反应。在这项研究中，我们扩展了这项研究，以更全面地确定整个幼虫A. suum涉及导致细胞死亡的毒性机制的细胞、器官系统、分子靶标和潜在的细胞通路。实验系统利用荧光核探针（双苯甲酰亚胺、碘化丙啶）、NIT、A. suum幼虫寄生虫培养系统和对 NIT 的转录反应 (RNA-seq)。该方法提供了器官系统（例如肠、排泄、食道、皮下组织和缝细胞以及神经）中 NIT 诱导的细胞死亡的快速解决方案，根据细胞死亡情况区分 NIT，并识别具有最大NIT 敏感性（例如肠和神经环附近的明显神经元细胞）。应用程序扩展到识别对几种现有驱虫药敏感的细胞和器官。这种方法还解决了由两种 NIT在成虫A. suum 中诱导的肠道细胞死亡和不可修复的损伤，建立了一个新的模型来阐明成虫的相关病理机制。RNA-seq 分析解决了A. suum对三种 NIT 治疗有反应的基因，将二氢乳清酸脱氢酶（尿苷合成）和 RAB GTPase（s）（囊泡转运）鉴定为导致细胞死亡的潜在靶标/途径。由测试的所有三个 NIT 诱导的一组基因表明由 NIT 激活的常见压力或生存反应。除了所提出的具体研究方向之外，本研究中提出的整个实验系统的元素在新驱虫药的系统开发方面具有广泛的应用。