Characteristics of monolayer formation in vitro by the chytrid Batrachochytrium dendrobatidis

Abstract

Batrachochytrium dendrobatidis is a globally distributed generalist pathogen that has driven many amphibian populations to extinction. The life cycle of B. dendrobatidis has two main cell types, motile zoospores, and sessile reproductive sporangia. When grown in a nutrient-rich liquid medium, B. dendrobatidis forms aggregates of sporangia that transition into monolayers on surfaces and at the air-liquid interface. Pathogenic microorganisms use biofilms as mechanisms of group interactions to survive under harsh conditions in the absence of a suitable host. We used fluorescent and electron microscopy, crystal violet, transcriptomic, and gas chromatographic analyses to understand the characteristics of B. dendrobatidis monolayers. The cell-free monolayer fraction showed the presence of extracellular ribose, mannose, xylose, galactose, and glucose. Transcriptome analysis showed that 27%, 26%, and 4% of the genes were differentially expressed between sporangia/zoospores, monolayer/zoospores, and sporangia/monolayer pairs respectively. In pond water studies, zoospores developed into sporangia and formed floating aggregates at the air-water interface and attached film on the bottom of growth flasks. We propose that B. dendrobatidis can form surface-attached monolayers in nutrient-rich environments and aggregates of sporangia in nutrient-poor aquatic systems. These monolayers and aggregates may facilitate dispersal and survival of the fungus in the absence of a host. We provide evidence for using a combination of plant-based chemicals, allicin, gingerol, and curcumin as potential anti-chytrid drugs to mitigate chytridiomycosis.

Keywords: Anti-chytrid drugs; Biofilm; Gene expression; Phytochemicals; Transcriptomics.

Graphical abstract

Fig. 1

Scanning electron microscopic images showing the time course of the B. dendrobatidis monolayer development with (a) low power and (b) high power. Monolayer development was studied every day up to 6-days under the SEM. Arrows (white) indicate microcolonies formed after 48 h of incubation. Images indicate the transition of zoospores to sporangia in 24 h then to a mature monolayer in 6-days.

Fig. 2

Scanning electron microscopic image of a 6-day old mature monolayer film. The monolayer was grown on tissue-culture treated glass coverslips in H-broth media. Mature monolayers mainly consisted of sporangia at different development stages. Thread-like rhizoids and extracellular matrix materials hold sporangia together, making the monolayer. Mature sporangia with discharge papilla and plug are clearly visible. Arrows indicate the extracellular matrix-like material.

Fig. 3

Epifluorescence images of sporangia and mature B. dendrobatidis monolayers. (a) sporangia (b) mature monolayer. Mature monolayers and sporangia were stained with Texas Red conjugated concanavalin A which binds to glucose and mannose residues and DAPI which intercalated into DNA. Blue indicates DAPI stained nucleic acids while the red color indicates concanavalin A stained thin extracellular matrix-like materials. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Transcriptomic multivariate analysis for B. dendrobatidis at different life stages. Principal component analysis (PCA) score plot showing that the transcriptome of B. dendrobatidis in different life stages. Colored circles represent life cycle stages; red – monolayer-associated cells, blue – sporangia, and green – zoospores. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Heatmap of the differentially expressed genes among three life stages of B. dendrobatidis. Based on the scaled and centered expression values of the genes. The hierarchical clustering of the samples is in columns while genes are clustered in rows. Different colors indicate differences in the gene expression levels. Purple indicates the lower relative abundance of mRNA while orange indicates the higher relative abundance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Effect of temperature on cell viability. After exposure to different temperatures for 24 ​h, cell viability was measured by XTT reduction. Bars represent the percentage mean ​± ​SEM of three independent experiments analyzed using two-way ANOVA with Dunnett’s multiple comparison test. Statistical significance of differences is indicated by asterisks. (**** p < 0.0001, F = 49.85).

Fig. 7

Effect of pH on B. dendrobatidis cell types. Bars represent the percentage mean ​± ​SEM of three independent experiments analyzed using two-way ANOVA with Dunnett’s multiple comparison tests. Statistical significance of differences is indicated by asterisks. **** p ​< ​0.0001, * p < 0.05, F = 43.76.

Fig. 8

Cytotoxicity of amphotericin B and sodium chloride to zoospores, sporangia, and monolayer-associated cells in vitro. (a) the effects of different concentrations of sodium chloride and (b) amphotericin B on B. dendrobatidis survival were tested against all three cell types by the XTT reduction. Cells were grown in H-broth alone or in H-broth with different concentrations of the chemicals. The percentage means of three independent experiments were analyzed using two-way ANOVA with Dunnett’s multiple comparison tests comparing the viabilities to H-broth media. Asterisks on bars indicate the statistical significance. **** p ​< ​0.0001, ** p < 0.01, F(a) = 22.49, F(b) = 112.4.

Fig. 9

Dose-dependent effects of plant-based chemicals on the growth of zoospores in vitro. The effects of different concentrations of (a) curcumin (b) allicin (c) 6-gingerol were tested against zoospores for 6-days at 23 ​°C by measuring the absorbance (492 ​nm) at 24 ​h intervals. Zoospores were grown in H-broth alone or in H-broth with various concentrations of plant-based chemicals. Each data point represents the mean ​± ​SEM of three independent experiments. Data were analyzed using repeated measure two-way ANOVA with Dunnett’s multiple comparison tests. **** p ​< ​0.0001, ** p ​< ​0.01, * p < 0.05, F(a) = 24.58, F(b) = 26.03, F(c) = 17.27.

Fig. 10

Cytotoxicity of plant-based chemicals on B. dendrobatidis cell types. The MICs of curcumin (6 ​μg/ml), allicin (3.375 ​μg/ml), and 6-gingerol (200 ​μg/ml) for zoospores were tested for survivability of sporangia and monolayer-associated cells by XTT reduction. Cells were grown in H-broth only or in H-broth with chemicals for 24 ​h ​at 23 ​°C. Bars represent the percentage mean ​± ​SEM of three independent experiments analyzed by two-way ANOVA with Dunnett’s multiple comparison test. Statistically significant differences from H-broth are indicated by asterisks. *** p ​< ​0.001, ** p < 0.01, F = 14.89.

Fig. 11

Combinatorial treatment of plant-based chemicals. Mixtures of the chemicals were tested in full, half and quarter strengths of MICs. Cells were grown in H-broth or in H-broth with chemicals for 24 ​h ​at 23 ​°C. The viability of the cells was measured by XTT reduction. Bars represent the percentage mean ​± ​SEM of three independent experiments analyzed by two-way ANOVA with Dunnett’s multiple comparison test. Asterisks on bars indicate the statistical significance of cell viability compared to H-broth. **** p < 0.0001, F = 100.1.