Molecular physiology of pumiliotoxin sequestration in a poison frog

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

箭毒蛙（Poison frogs）会从节肢动物食谱中生物富集生物碱以实现化学防御。尽管多数生物碱会以未修饰的形式被富集，但部分箭毒蛙物种可将箭毒蛙毒素（pumiliotoxin, PTX 251D）代谢为活性更强的别箭毒蛙毒素（allopumiliotoxin, aPTX 267A）。尽管已有大量研究对箭毒蛙的化学防御武器库进行了表征，但针对单一生物碱的富集与代谢相关生理机制仍不明晰。我们首先以染色箭毒蛙（Dendrobates tinctorius）为对象开展喂食实验，旨在探究该物种是否可将PTX 251D代谢为aPTX 267A，以及在肠道、肝脏与皮肤组织中，PTX 251D暴露会引发哪些基因表达变化。研究结果显示，D. tinctorius确实能够将PTX 251D代谢为aPTX 267A，且PTX 251D暴露会改变与免疫系统功能、小分子代谢及转运相关的基因表达水平。为进一步明确上述基因表达变化的功能意义，我们随后开展了一系列高通量筛选，以确定PTX 251D的分子靶点，并鉴定出可能负责将PTX 251D代谢为aPTX 267A的潜在蛋白。尽管针对PTX 251D与人类电压门控离子通道及G蛋白偶联受体的结合实验未得到明确结果，但我们在细胞色素P450（cytochrome P450）筛选中鉴定出人类CYP2D6可快速代谢PTX 251D。此外，在喂食PTX的箭毒蛙肠道中，一类类似CYP2D6的基因表达量上调，提示该蛋白可能参与PTX的代谢过程。本研究结果表明，单一生物碱可改变多个组织的基因表达，其中包括参与生物碱代谢的基因。从更广泛的视角来看，本研究暗示野生食谱中的特定生物碱类别可能诱导针对性的生理变化，以实现生物碱的靶向富集与代谢。