Viscosin-like lipopeptides from frog skin bacteria inhibit Aspergillus fumigatus and Batrachochytrium dendrobatidis detected by imaging mass spectrometry and molecular networking

Abstract

Amphibian populations worldwide have declined and in some cases become extinct due to chytridiomycosis, a pandemic disease caused by the fungus Batrachochytrium dendrobatidis; however, some species have survived these fungal epidemics. Previous studies have suggested that the resistance of these species is due to the presence of cutaneous bacteria producing antifungal metabolites. As our understanding of these metabolites is still limited, we assessed the potential of such compounds against human-relevant fungi such as Aspergillus. In this work we isolated 201 bacterial strains from fifteen samples belonging to seven frog species collected in the highlands of Panama and tested them against Aspergillus fumigatus. Among the 29 bacterial isolates that exhibited antifungal activity, Pseudomonas cichorii showed the greatest inhibition. To visualize the distribution of compounds and identify them in the inhibition zone produced by P. cichorii, we employed MALDI imaging mass spectrometry (MALDI IMS) and MS/MS molecular networking. We identified viscosin and massetolides A, F, G and H in the inhibition zone. Furthermore, viscosin was isolated and evaluated in vitro against A. fumigatus and B. dendrobatidis showing MIC values of 62.50 µg/mL and 31.25 µg/mL, respectively. This is the first report of cyclic depsipeptides with antifungal activity isolated from frog cutaneous bacteria.

Figure 1

Phylogenetic tree based on 16S rRNA sequences of associated-bacteria from the skin of Panamanian frogs. Color range represents bacterial phyla. Color clades show the abundance of bacterial families. The color strips relate the bacterial isolates with their host of origin. Grey color scale strips display the inhibition zones produced by bioactive bacteria against A. fumigatus ATCC 1028 after microbial interactions.

Figure 2

MALDI IMS of microbial interaction between P. cichorii and A. fumigatus ATCC 1028. Selected ions observed for the microbial interaction are presented as columns. The first column shows mass to charge ratio, and the second to fifth columns display images of the spatial distribution of the ions produced by P. cichorii, P. cichorii vs. A. fumigatus, A. fumigatus alone, and MHA as control. The average mass spectrum highlighting selected ions is shown at the bottom. These ions were present at m/z: 1,112, 1,126, 1,134, 1,140, 1,148, 1,154, 1,162, 1,176, 2,010 and 2,032 Da.

Figure 3

Molecular networking of bacterial strains that displayed biological activity against A. fumigatus ATCC 1028 after 72 hours. (a) Molecular networking of all bioactive strains against A. fumigatus ATCC 1028. Pie charts inside nodes represent the compound distribution based on the bacterial source. (b) Sub-network of cyclic lipopeptides detected inside inhibition zone through MALDI IMS. These nodes matched in GNPS spectral library. Square node comprises GNPS library hit WLIP/viscosin/massetolides molecular family and isolated viscosin from P. cichorii. massetolides A, F, G and H.

Figure 4

Spectral comparison and structural fragmentation of selected lipopeptides produced by bioactive bacteria. (a) MS/MS spectra of amino acid losses for viscosin, massetolide F, A, G and H. The color of the squares represents the amino acids in the spectrum. (b) Colored structural fragmentation of viscosin, massetolides F, A, G, H and their corresponding functional groups.

Figure 5

Antifungal susceptibility tests of viscosin against A. fumigatus ATCC 1028. Inhibition of growth of A. fumigatus by increasing concentrations of viscosin at 48 hours of culture. Viscosin displayed significant inhibition (*) when comparing with the positive control (p ≤ 0.0068) and diluent control at each concentration (p ≤ 0.0001) by Student’s t-tests. Error bars refer to the standard error of the mean. This figure is representative of two similar experiments. In both experiments, the MIC value was 62.50 μg/mL (Figs S4 and S5).

Figure 6

Antifungal susceptibility tests of viscosin against B. dendrobatidis JEL 197. Inhibition of growth of B. dendrobatidis zoospores by increasing concentrations of viscosin at day 4 of culture. Viscosin displayed significant inhibition (*) when compared with the positive control (p ≤ 0.0013) and diluent control at each concentration (p ≤ 0.05) by Student’s t-tests. Error bars refer to the standard error of the mean. This figure is representative of two similar experiments. In both experiments, the MIC was 31.25 μg/mL (Figs S6 and S7).