Reshuffling of Aspergillus fumigatus cell wall components chitin and β-glucan under the influence of caspofungin or nikkomycin Z alone or in combination

Abstract

Chitin and β-glucan are major cell wall components of Aspergillus spp. We investigated the antifungal activity of chitin synthesis inhibitors nikkomycin Z, polyoxin D, flufenoxuron, lufenuron, and teflubenzuron, alone and combined with the β-glucan synthesis inhibitor caspofungin. Only nikkomycin Z and caspofungin were found to act synergistically. The nikkomycin Z-induced chitin decrease corresponded with a β-glucan increase, while with the caspofungin-induced β-glucan decrease, an increase in chitin was found. This could explain the synergistic activity of this combination of drugs.

Fig 1

Cells of A. fumigatus (original magnification, ×400). (a) A. fumigatus not exposed to any antifungal agent. Elongated hyphae are noted; no swollen cells are present. (b) A. fumigatus exposed to 8 μg/ml nikkomycin Z. Elongated hyphae are still seen, but some of the hyphae appear to be swollen and rounded. (c) A. fumigatus exposed to 0.125 μg/ml caspofungin. Hyphae are shortened, stubby, and broad based. (d) A. fumigatus exposed to 4 μg/ml caspofungin. Hyphae are further shortened, stubby, and broad based. This morphology is more evident than with 0.125 μg/ml caspofungin. (e) A. fumigatus exposed to 0.125 μg/ml caspofungin and 8 μg/ml nikkomycin Z. Cell morphology is disrupted, and many cells are swollen and rounded, though normal hyphae are still seen. (f) A. fumigatus exposed to 4 μg/ml caspofungin and 8 μg/ml nikkomycin Z. Cell morphology is completely disturbed, and no normal hyphae are seen.

Fig 2

Concentrations of the cell wall components β-glucan and chitin in A. fumigatus ATCC 204305 after exposure to caspofungin and nikkomycin Z at various concentrations. (A and B) Glucosamine concentrations represent chitin concentrations. Means from three independent experiments are shown. Bars represent standard deviations. β-Glucan and chitin concentrations were corrected to the number of viable cells with the XTT assay by the following formula: (amount of β-glucan or chitin measured) × (number of viable cells in the growth control/number of viable cells in the well tested). (C) Fungal response in cell wall components. Concentrations of β-glucan and chitin after exposure to 0.125 μg/ml of caspofungin combined with 2 μg/ml nikkomycin Z, each agent alone, and the growth control. This graph shows that combining caspofungin and nikkomycin Z causes a drastic decrease in β-glucan content, resulting in a compensatory increase in chitin content. (D) Shift in cell wall components β-glucan and chitin after exposure to caspofungin and nikkomycin Z at two concentrations (schematic representation). Both components are standardized to 50% in the drug-free control.