Article Title: Disarming Fungal Pathogens: Bacillus safensis Inhibits Virulence Factor Production and Biofilm Formation by Cryptococcus neoformans and Candida albicans
Figure Lengend Snippet: B. safensis targets the C. neoformans cell surface and bacterial chitinase activity contributes to the antimelanization effect during the dual-species interaction. (A) Individual coincubation of 154 different C. neoformans transcription factor (TF) mutants with B. safensis identified the fungal cell wall and cell membrane as possible targets. For each TF, two independent mutants were incubated in liquid l -DOPA medium with or without bacteria (308 TF strains in total) at 30°C for 5 days. Growth was determined via OD 405 measurements. Results are plotted relative to the fungus-only control. Each circle represents the mean of the results for two independent mutants of the same TF. The black dotted lines and the gray dotted lines indicate the overall means of all values ± SD, respectively. The experiment (exp.) was performed twice, and TF mutant outliers (in at least one experiment) are indicated and color-coded according to previously published roles of these TFs in cell membrane and cell wall integrity (see scheme on the right). Rel. growth, relative growth of fungal cells in coculture versus monoculture, expressed in percentages. (B) Sorbitol bypassed B. safensis -mediated suppression of fungal melanization, and this effect was not observed in a C. neoformans nrg1 Δ mutant. C. neoformans wild-type strain H99 or the nrg1 Δ mutant was incubated alone or mixed with bacteria on l -DOPA agar with or without sorbitol for 48 h. Ctrl, control; Sorb, sorbitol. Scale bar, 5 mm. The experiment was performed twice, and representative images are shown. (C) Coincubation of C. neoformans nrg1 Δ with B. safensis in YPD for 24 h resulted in fungal cell aggregation and abnormal cell wall chitin staining. White squares in the panels of the middle row indicate regions that are shown in a magnified view in the bottom row. Arrows point to abnormal, "scratch-like" CFW staining. DIC, differential interference contrast; CFW, calcofluor white. Scale bars indicate 5 µm (middle row) and 2 µm (bottom row). The experiment was performed twice, and representative images are shown. (D) B. safensis and CFW synergistically inhibit cryptococcal growth. CFU-based analysis of C. neoformans growth in YPD in presence of CFW or bacteria or a combination of the two was performed. Results are the means ± SD of two independent experiments, each performed in duplicate. (E) A chitinase inhibitor partially rescues B. safensis -mediated inhibition of fungal melanization on l -DOPA. Strains were incubated at 30°C for 48 h. Ctrl, control; BisC, bisdionine C. Scale bar, 5 mm. The experiment was performed three times, and representative images are shown.
Article Snippet: To analyze the effects of chitinase on formation of melanin, fungal cells were resuspended in 1 mg ml−1 chitinase (from Streptomyces griseus ; Sigma) solution before being spotted onto l -DOPA agar.
Techniques: Activity Assay, Incubation, Mutagenesis, Staining, Inhibition