Identification of novel secreted compounds from the amphibian pathogen Batrachochytrium dendrobatidis



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Global amphibian decline is caused in large measure by the fungal pathogen, Batrachochytrium dendrobatidis. The fungus produces a motile zoospore that is attracted to the keratin rich layer of epidermal skin, where it produces a suite of proteases to help degrade intercellular junctions, elastin, and antimicrobial peptides. The zoospore resorbs its flagellum, produces a germ tube to infiltrate deeper into epidermal layers, and transitions into a thallus. The thallus then differentiates into a zoosporangium and produces secreted metabolites that alter the immunological response of the amphibian. Infected animals have thickened skin which is sloughed off. Because the skin of amphibians plays a major role in air and moisture exchange, the resulting osmotic imbalance is believed to lead to eventual death of infected animals by cardiac arrest. An unexplained symptom of the B. dendrobatidis infection on amphibians is the loss of their righting reflex. Previous research has suggested that B. dendrobatidis possesses a non-ribosomal peptide synthetase (NRPS). This dissertation research has shown that B. dendrobatidis possesses NRPS-encoding genes. We hypothesized that B. dendrobatidis produces secondary metabolites that may play a role in its pathogenicity. Growth of the fungus in rich media supplemented with lactose and heat-killed bacteria was most effective for induction of the NRPS gene cluster and other genes responsible for the synthesis of secondary metabolites. Cell culture filtrates of B. dendrobatidis grown in various modified media were subjected to high performance liquid chromatography-mass spectrometry and data analyzed by Compound Discoverer 3.0. Among the products observed, three were identified that have not been previously reported to be produced by this pathogen. The use of wax moth larvae (Galleria mellonella), as a whole organism bioassay system, to study pathogenicity of B. dendrobatidis in this work, establishes it as a novel entity for investigation as we preserve our precious resource of amphibians.



Chytrid, Virulence