Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia.
Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia.
Int J Parasitol Drugs Drug Resist. 2023 Aug;22:9-19. doi: 10.1016/j.ijpddr.2023.03.004. Epub 2023 Mar 24.
Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind β-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by β-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in β-tubulin in ALB-resistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for HO degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (logFC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.
苯并咪唑-2-氨基甲酸酯（BZ，如阿苯达唑；ALB）与微管蛋白结合，扰乱微管聚合，是用于治疗贾第虫病的两种主要化合物类别之一。在大多数寄生线虫和真菌中，BZ 耐药性是由β-微管蛋白突变引起的，其分子作用模式（MOA）已得到很好的研究。相比之下，在十二指肠贾第鞭毛虫中，BZ 的作用模式或耐药性的了解较少，可能涉及特定靶点和更广泛的影响，包括细胞损伤和氧化应激，其根本原因尚不清楚。此前，我们在对阿苯达唑（ALB）耐药（ALB-R）的十二指肠贾第鞭毛虫 WB-1B 中发现了β-微管蛋白中单个核苷酸多态性 E198K 的获得，而在对阿苯达唑（ALB）敏感（ALB-S）的亲本对照中则没有发现该多态性。E198K 与真菌中的 BZ 耐药性有关，其等位基因频率与十二指肠贾第鞭毛虫 WB-1B 中的 BZ 耐药性程度相关。在这里，我们对这些 ALB-S 和 ALB-R 十二指肠贾第鞭毛虫 WB-1B 培养物进行了详细的转录组比较。在 WB-1B 中，与 ALB-R 相关的主要转录变化表明蛋白质降解和周转增加，微管和相关基因上调，与粘附盘和基底体有关。这些发现与之前的观察结果一致，即 ALB 暴露后，十二指肠贾第鞭毛虫的盘状结构和相关结构发生集中解体。我们还观察到与 ALB-R 相关的转录变化，这与先前观察到的从糖酵解到精氨酸代谢以产生 ATP 的转变以及可能对需要进一步研究的囊泡运输系统的方面发生变化一致。最后，我们在十二指肠贾第鞭毛虫 WB-1B 中观察到与 DNA 修复和氧化应激反应相关的混合转录变化。这些变化可能表明 HO 降解在 ALB-R 中的作用，就像在其他十二指肠贾第鞭毛虫细胞培养物中观察到的那样。然而，它们低于转录组分析中通常使用的转录变化折叠变化阈值（logFC>1），并且在 ALB-R 十二指肠贾第鞭毛虫 WB-1B 中似乎被下调 NAD 清除和转化途径所否定，这些途径需要支持这些应激途径，并且上调许多高度氧化敏感的铁硫（FeS）簇代谢酶。
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