Mimicking early host-chytrid interactions using an in vitro-infection model

While the greatest loss of vertebrate biodiversity from disease is due to chytridiomycosis, knowledge of this disease’s pathogenesis is still poorly known. The early interactions of Batrachochytrium dendrobatidis (Bd) with the amphibian skin have been documented as attachment of zoospores to host skin (adhesion), zoospore germination, germ tube development, penetration into skin cells (invasion) and invasive growth in the host skin. Mostly, transmission electron microscopy and light microscopy on skin tissues are used to visualize and examine the early infection stages, but an assay that can be applied to examine the fundamentals of early host-chytrid interactions at cellular level is still lacking. The aim of this study was to elaborate a cell-based in vitro invasion test mimicking early host-Bd interactions. Therefore primary keratinocytes from Litoria caerulea were isolated and fluorescently labelled using a green celltracker. Bd spores were labelled with a red celltracker and added to the cells. Using fluorescent microscopy, clear contact between the spores and host cells was observed after an invasion period of 4 hours, mimicking adhesion. When increasing the contact time till 24 hours, host cells were now invaded by Bd spores and intracellular chytrid thalli were observed. Confocal microscopy was used to confirm the intracellular localization. In conclusion we optimized a fluorescent in vitro model that mimics early host-pathogen interactions (attachment and invasion), reflecting endobiotic chytrid development. The availability of a cell-based assay that allows rapid and efficient screening of the early host-Bd interactions opens new perspectives in chytrid research.