Studies on ETP-like toxin gene expression and toxin detection in Batrachochytrium dendrobatidis

The continual decline of amphibians worldwide has been partially attributed to the fungus, Batrachochytrium dendrobatidis. This fungus is known to cause inflammation and thickening of the keratinized layer of amphibian skin causing a disturbance in the osmotic balance within the amphibian leading to cardiac arrest. The action by which B. dendrobatidis penetrates the skin and causes disease is unknown. Previous studies have demonstrated the ability of B. dendrobatidis to produce numerous proteases some of which may be responsible for breaching the skin and outer layers of the frog. Other studies have shown that Bd can cause death in Caenorhabditis elegans (nematodes) without observable physical interaction, and when frogs are infected with Bd, they lose their righting reflex. We therefore postulated that Bd may produce a toxin. The epipolythiodioxopiperazine (ETP) class of toxins has been shown to increase the virulence of fungi and is produced by a cluster of 13 genes. Since Aspergillus fumigatus is known to produce gliotoxin, a specific ETP toxin, through the production of core biosynthetic enzymes, we hypothesized that the presence and expression of core biosynthetic enzymes for gliotoxin in B. dendrobatidis would indicate that this fungus could produce the ETP-class toxin. By using NCBI BLAST we observed the presence of all 13 genes that make up the cluster required to produce ETP toxins in B. dendrobatidis. By using reverse transcriptase-PCR and Real Time Relative-PCR, the differential expression of seven of the genes grown in three different media types and through a time course of day 1 through day 7 of incubation were studied. High pressure liquid Texas Tech University, Amanda M. Hicks, August 2013 viii chromatography (HPLC) - mass spectrometry (MS) were used to further detect the presence of the ETP toxin in media in which B. dendrobatidis was grown. Even though B. dendrobatidis appears to express the genes required to make gliotoxin, the presence of the toxin was not detected. It is likely the in vitro conditions used in this study to induce production of the toxin have not been identified. Future studies will contribute to our understanding of the mechanism(s) by which B. dendrobatidis is able to kill amphibians and can also aid in understanding other pathogenic fungi.