Molecular responses of Batrachochytrium dendrobatidis following exposure to thyroid hormone
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Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has been implicated in worldwide amphibian decline. This fungus causes a deadly disease, known as chytridiomycosis which is characterized by hyperkeratosis symptoms whereby susceptible animals exhibit thickening of the epidermal layer and the sloughing of the skin. Severely infected animals lose their righting reflex and eventually die from cardiac arrest. The fungal life cycle consists of motile zoospores and substrate- dependent sporangia. In aquatic environments, zoospores are capable of moving towards their hosts and infecting the keratinized mouth parts of tadpoles and the epidermal layer of adult amphibians. Although Bd infects tadpoles, these infections are non-lethal. In amphibian biology, metamorphosis is the transition from young tadpoles to adult amphibians. Thyroid hormone (TH) plays a central role in this transition process. In an effort to understand if the fungus responds to the host-derived TH, a motility assay was performed. This study demonstrated the positive movement of Bd zoospores toward the chemical T3, a form of TH at a physiologically relevant concentration of 50 nM as compared to the solvent control or intermediates in T3 biosynthesis. A significant (19-fold) increase in the expression of subtilisin-like serine protease gene was observed in Bd within 3 hours following exposure to T3. The gene encoding this protease was cloned from Bd and expressed in Escherichia coli. The recombinant protein was partially purified and its enzymatic properties were determined. The partially purified enzyme was capable of degrading casein. Additionally, this enzyme was found to be optimally active at low millimolar concentrations of calcium and sodium. The optimum pH for the enzyme activity was 8.5 and was strongly inhibited by 10 mM PMSF, a serine protease inhibitor. Similarity at the amino acid level between subtilases of human fungal dermatophytes and the Bd subtilisin-like serine protease suggests the importance of this enzyme in Bd pathogenicity. To further understand the Bd- T3 interaction, a liquid chromatography-mass spectrophotometry (LC-MS) based proteomics approach was utilized. The total cellular protein and membrane protein profile expression in the fungus following exposure to T3 was assessed. Proteins that may play a role in Bd life cycle, pathogenicity, metabolism, and in other cellular functions of the fungus were identified. Furthermore, a transcriptome study also revealed the activity of genes that may be involved in several cellular pathways and Bd pathogenicity. A hypothetical model of the mechanism of T3 action was generated based on the integrated transcriptomics and proteomics studies. Taken together, these studies may shed light on our understanding of the early events that may occur in Bd- amphibian interactions.