Fig 2.

Combinatorial effects of gladiolin with AmpB against drug-resistant fungal biofilms, and during immune cell and animal infections. (A) C. albicans biofilms were grown in synthetic ventilator airway mucus (SVAM) medium on endotracheal tubes cuttings for 48 h (37°C and 5% CO₂). Biofilms were treated for 24 h as indicated before being photographed. (B) Experiment as in panel A. Biofilms were dislodged after drug treatment and plated for CFUs. Data were generated from three biological repeats with three technical repeats each. Synergistic interactions were evaluated by using the Response-Additivity model as described in the Materials and Methods (“indif” is indifferent and “syn” is synergistic). (C) Larvae of Galleria mellonella were infected with C. albicans and treated with 50 µg/mL AmpB, 32 µg/mL gladiolin, or the combination of both. Larvae were monitored for survival over 7 days. Data were derived from four independent experiments with four larvae per condition and statistically analyzed using the log-rank Mantel-Cox test with P-values at days 3 and 7 shown underneath. The numbers in parenthesis represent the median survival in days. (D) The filamentation of C. albicans during the challenge of mouse bone marrow-derived macrophages (BMDMs) at 6 h post infection. In control conditions, abundant escaped hyphae are visible in the medium surrounding macrophages. There is a small reduction in visible escaped hyphae in the presence of gladiolin and an additional reduction in the gladiolin/AmpB combination. Images are stills taken from live cell imaging movies used to generate data in panel E. Shown is the overlay of bright field and Draq7-stained nuclei of macrophages that were lysed by the growing fungal hyphae. Images were cropped and adjusted for brightness. The scale bar shows 100 µm. (E) Macrophage death of C. albicans-infected BMDMs (MOI 3:1) with combinations of gladiolin and AmpB treatment was determined by Draq7 staining. A minimum of 900 macrophages were counted per experiment for each of the conditions. The half-maximal macrophage death of two independent experiments with two technical repeats each was determined using 4-parameter analysis and statistically evaluated using a one-way ANOVA with Dunnett’s multiple comparison test (**P < 0.01, ****P < 0.0001). (F) Metabolic activity of uninfected BMDMs treated for 24 h with single or drug combinations was measured using resazurin metabolic assay, and the error bars indicate SEM with n = 3.