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.
Highlights
Fungal diseases are major threats to human health and food security (Strange and Scott, 2005; Fisher et al, 2012)
The in vitro effects of K20 on the growth of bacterial species E. coli TG1, M. luteus ATCC10240 and S. aureus ATCC6538 were assayed in 96-well uncoated polystyrene microtiter plates and Minimal inhibitory concentration (MIC) were determined using Clinical and Laboratory Standards Institute (CLSI) protocols with modification (NCCLS, 1993)
K20 MICs with yeasts were uniformly higher than MICs achieved with itraconanzole and fluconazole except with azole resistant strains, C. albicans strains 64124 and B-311 and C. tropicalis 95-41
Summary
Fungal diseases are major threats to human health and food security (Strange and Scott, 2005; Fisher et al, 2012). We reported on a novel aminoglycoside analog of kanamycin B, FG08, with broad-spectrum antifungal properties that did not inhibit tested bacterial and mammalian cells (Figure 1) (Chang et al, 2010). K20’s antifungal mechanism of action was determined using intact fungal cells and model lipid bilayer membranes.
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