Antifungal amphiphilic aminoglycoside K20: bioactivities and mechanism of action

Abstract

K20 is a novel amphiphilic antifungal aminoglycoside that is synthetically derived from the antibiotic kanamycin A. Reported here are investigations of K20's antimicrobial activities, cytotoxicity, and fungicidal mechanism of action. In vitro growth inhibitory activities against a variety of human and plant pathogenic yeasts, filamentous fungi, and bacteria were determined using microbroth dilution assays and time-kill curve analyses, and hemolytic and animal cell cytotoxic activities were determined. Effects on Cryptococcus neoformans H-99 infectivity were determined with a preventive murine lung infection model. The antifungal mechanism of action was studied using intact fungal cells, yeast lipid mutants, and small unilamellar lipid vesicles. K20 exhibited broad-spectrum in vitro antifungal activities but not antibacterial activities. Pulmonary, single dose-administration of K20 reduced C. neoformans lung infection rates 4-fold compared to controls. Hemolysis and half-maximal cytotoxicities of mammalian cells occurred at concentrations that were 10 to 32-fold higher than fungicidal MICs. With fluorescein isothiocyanate (FITC), 20-25 mg/L K20 caused staining of >95% of C. neoformans and Fusarium graminearum cells and at 31.3 mg/L caused rapid leakage (30-80% in 15 min) of calcein from preloaded small unilamellar lipid vesicles. K20 appears to be a broad-spectrum fungicide, capable of reducing the infectivity of C. neoformans, and exhibits low hemolytic activity and mammalian cell toxicity. It perturbs the plasma membrane by mechanisms that are lipid modulated. K20 is a novel amphiphilic aminoglycoside amenable to scalable production and a potential lead antifungal for therapeutic and crop protection applications.

Keywords: Cryptococcus neoformans; K20; amphiphilic aminoglycoside; antifungal; kanamycin.

Figure 1

Structures of aminoglycosides FG08, kanamycin A, and K20.

Figure 2

Antimicrobial activities of K20, FG08, and kanamycin A. Disk agar diffusion assays show that K20 and FG08 are antifungal, but not antibacterial. Kanamycin A is antibacterial, but not antifungal. Ten μL aliquots of K20, FG08, and kanamycin A solutions were applied to paper disks (0.6 cm diameter) on surfaces of PDA and LB agar at concentrations of 10 and 5 mg/mL, respectively, and with spread-plated fungal (S. cerevisiae strain W303C and F. graminearum B4-5A) and bacterial (E. coli TG1and S. aureus ATCC 6538) cultures, respectively.

Figure 3

Time kill curves for C. neoformans H99 exposed to K20. Cultures were exposed to K20 at 2 mg/L (open triangles), 4 mg/L (filled circles), and 8 mg/L (open circles) or to no K20 (filled triangles).

Figure 4

Hemolysis of sheep erythrocytes with various concentrations of K20 after 1 h exposure at 37°C (white bars). Controls were exposure to Triton X-100^® (1%, vol/vol) giving 100% hemolysis (black bar) and no exposure to K20 (gray bar). The HC50 value is >500 mg/L. Standard deviation was used as the statistical parameter.

Figure 5

Toxicities of NIH3T3 mouse fibroblast cells (white bars) and C8161.9 melanoma cells (gray bars) with 24 h exposure to K20 at various concentrations. Positive control (0% cell survival) was provided by treatment with Triton X100^® (1%, vol/vol) (black bar).

Figure 6

Dose-dependent membrane perturbation effects of K20 on C. neoformans H99. FITC dye uptake without (A1,A2) and with K20 (4 mg/L) (B1,B2), (8 mg/L) (C1,C2), and (25 mg/L) (D1,D2) exposure for 10 min. Bright-field images (A1,B1,C1,D1) are compared with fluorescence images (A2,B2,C2,D2). Bar length is 10 μm. (E1) Shows dose-dependent effects of K20 on FITC dye uptake and effects of kanamycin A and no treatment Triton X-100^® (1%,vol/vol) gave 100% dye influx (data not shown). The error bars show SD from analyses of 10 separate microscopic image fields randomly selected from at least two separate experiments. Numbers above the range bars indicate the number of cells analyzed.

Figure 7

Dose-dependent membrane perturbation effects of K20 on F. graminearum. FITC dye uptake without (A1,A2) and with K20 (7.8 mg/L) (B1,B2), (15.6 mg/L) (C1,C2), and (32 mg/L) (D1,D2) exposure for 10 min. Bright-field images (A1,B1,C1,D1) and fluorescence images (A2,B2,C2,D2). Image a2 (no antimicrobial agent added) shows no fluorescent cells against a fluorescent background. Bar length is 10 μm. Triton X-100^® (1%, vol/vol) was assumed to give 100% dye influx (data not shown).

Figure 8

Effect of K20 on calcein release from SUVs that mimic fungal plasma membranes. Calcein released from SUVs made with PC/PE/PI/ergosterol (5:4:1:2) (A) and PC/ergosterol (7:3) (B) were exposed to K20 at 31.3 mg/L (open circles), 62.5 mg/L (open triangles), and 125 mg/L (open squares), kanamycin A at 62.5 mg/L (filled triangles), and Triton X-100^® (1% vol/vol) (filled circles). Data were compiled from three separate experiments. Standard deviation was used as the statistical parameter.

Figure 9

Mean percent body weight change in groups of mice receiving treatments of K20. Treatments with K20 were one dose/animal of 100 μL of 200 mg/L K20 (filled circles), infected with one dose/animal of 100 μL of C. neoformans H99 (5 × 10³ cells/mL) mixed with K20 (200 mg/L) (open triangles), and infected with 1 dose/animal of 100 μL C. neoformans H99 (5 × 10³ cells/mL) only (filled squares). Data were statistically analyzed and P-values determined by One-Way ANOVA methods.

Figure 10

Effect of K20 on C. neoformans H99 infectivity in a RAG^−/− mouse model. (A) Mouse lung fungal burden at day 15 after infection with C. neoformans H99 cells mixed (black bar) or not mixed (white bar) with K20. Fungal burdens were assessed by plating lung homogenate suspensions for CFU determinations. Control treatment with K20 and no cells (gray bar) showed no fungal burden. ^*** Indicates a p-value < 0.001 determined by One-Way ANOVA. Light microscopic images of lung homogenates were prepared from C. neoformans H99 infected mice with no K20 (B) and with K20 (C) exposure. Methanol-fixed homogenates on glass slides were stained with Diff-Quik™. Yeast cells were visible as large purple colored cells surrounded by an opaque halo.