In vitro modeling of Batrachochytrium dendrobatidis infection of the amphibian skin

Abstract

The largest current disease-induced loss of vertebrate biodiversity is due to chytridiomycosis and despite the increasing understanding of the pathogenesis, knowledge unravelling the early host-pathogen interactions remains limited. Batrachochytrium dendrobatidis (Bd) zoospores attach to and invade the amphibian epidermis, with subsequent invasive growth in the host skin. Availability of an in vitro assay would facilitate in depth study of this interaction while reducing the number of experimental animals needed. We describe a fluorescent cell-based in vitro infection model that reproduces host-Bd interactions. Using primary keratinocytes from Litoria caerulea and the epithelial cell line A6 from Xenopus laevis, we reproduced different stages of host cell infection and intracellular growth of Bd, resulting in host cell death, a key event in chytridiomycosis. The presented in vitro models may facilitate future mechanistic studies of host susceptibility and pathogen virulence.

Virulence and Pathogenicity of Chytrid Fungi Causing Amphibian Extinctions

“…surface, where it releases infectious zoospores that continue the cycle of infection. Invasion of the keratinocytes results in apoptosis of the host cell (66,140), and epidermal cell death is positively associated with infection loads and morbidity (19).…”

“…Further, enzymatic and gene expression profiles of chytrid cultures grown under different conditions yield information on potential virulence factors, the relevance of which can be confirmed using recombinantly produced or purified chytrid compounds (85,96,112,130). More-complex models such as host cell or tissue cultures allow simultaneous mapping of host and pathogen responses (22,134,140,145). Adding a live host tissue to the system increases biological relevance and has provided insights into gene regulation in the presence of host skin compounds (154), the potential of discrete host defenses to control chytrid infection [for example the skin microbiome and various compounds of skin secretions, such as antimicrobial peptides (AMPs)], and key mechanisms of pathogen adhesion and invasion (135).…”

Presence of low virulence chytrid fungi could protect European amphibians from more deadly strains

“…The Xenopus laevis kidney epithelial cell line A6 (ATCC-CCL 102) was grown in 75 cm 2 cell culture flasks and maintained in complete growth medium (74% NCTC 109 medium, 15% distilled water, 10% fetal bovine serum (FBS) and 1% of a 10,000 U ml −1 penicillin-streptomycin solution (P/S)) and the cells were incubated at 26°C and 5% CO 2 until they reached confluence. Using trypsin, the cells were detached, washed with 70% Hanks' Balanced Salt Solution without Ca 2+ , Mg 2+ (HBSS−) by centrifugation for 5 min at 1500 rpm and resuspended in the appropriate cell culture medium for invasion assays, which were performed as described in Verbrugghe et al 61 . To assess the germ tube formation, A6 cells were stained with 3 µM CellTracker TM Green CMFDA, seeded (10 5 cells per well) in 24well tissue culture plates containing collagen-coated glass coverslips and they were allowed to attach for 2 h at 20°C and 5% CO 2 .…”

Identification of a Novel Secreted Metabolite Cyclo(phenylalanyl-prolyl) from Batrachochytrium Dendrobatidis and its Effect on Galleria Mellonella.

“…The tryptophan biosynthetic pathway is already known to be important for production of immunomodulatory compounds from B. dendrobatidis. These compounds include tryptophan, kynurenine, methylthioadenosine, and tryptophol [9,71].…”