Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen

Abstract

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides-the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin-from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.

Biological activities of leaf extracts from selected Kalanchoe species and their relationship with bufadienolides content

“…The authors indicated that this compound modulated human innate immune responses and triggered antibacterial activity. Arenobufagin and gamabufotalin also exhibited antimicrobial properties (Barnhart et al 2017).…”

Relationships Between Chemical Defenses of Common Toad (Bufo bufo) Tadpoles and Bacterial Community Structure of their Natural Aquatic Habitat

“…Both NBC and TBQ were correlated with one or two NMDS axes describing the bacterial community of larval habitats. This may be a cause-effect relationship, because bufadienolides can have antipathogenic and antiparasitic effects (Barnhart et al 2017;Cunha Filho et al 2005;Tempone et al 2008), and Bufonid toads lack AMP-s (Conlon 2011;König et al 2015), therefore the involvement of bufadienolides in skin-based immune-defense is probable, and their synthesis may be adjusted to the presence or absence of particular pathogens or specific members of bacterial communities. The presence of certain bacteria and changes in the natural microbiota can induce responses in chemical defenses (up-or down-regulation of AMP synthesis) in adult frogs (Mangoni et al 2001;Miele et al 1998;Simmaco et al 1998).…”

“…Several toad species in the family Bufonidae are known to synthesize bufadienolides de novo in the serous glands of their skin already from early larval stages on (Hayes et al 2009a;Ujszegi et al 2017;Üveges et al 2017). These skin-toxins may be effective against natural enemies including predators (Denton and Beebee 1991;Hantak et al 2016;Kruse and Stone 1984;Peterson and Blaustein 1991;Shine 2010), competitors (Bókony et al 2018) and potential pathogens (Barnhart et al 2017;Cunha Filho et al 2005;De Medeiros et al 2019;Tempone et al 2008). However, little is known about what evolutionary processes and environmental factors influence bufadienolide synthesis.…”

“…As demonstrated by Cunha Filho et al (2005), bufadienolide compounds synthesized in the skin of Rhinella (=Bufo) rubescens have potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. Also, bufadienolides can inhibit the growth of Batrachochytrium dendrobatidis, a fungal pathogen associated with global amphibian declines (Barnhart et al 2017). Although bufadienolides are present, defensive antimicrobial peptides (AMPs) are lacking from the skin of bufonids (Conlon 2011;König et al 2015).…”

Ocular Hypotonia and Transient Decrease of Vision as a Consequence of Exposure to a Common Toad Poison

“…Bufadienolides are produced in parotid glands and the skin of a toad [2]. These toxic compounds are potent inhibitors of Na + /K + -ATPase activity, and toads use these as a natural repellent against predators and also as an immune defence against pathogens [3,4]. Bufadienolides are divided into bufagenins, the smaller, hydrolysed molecules which have stronger cardiotoxic effects, and bufotoxins, the larger bufadienolide molecules with an amino-acid side chain [5].…”