Methylxanthine inhibit fungal chitinases and exhibit antifungal activity

Abstract

Chitinases are necessary for fungal cell wall remodeling and cell replication. Methylxanthines have been shown to competitively inhibit family 18 chitinases in vitro. We sought to determine the effects of methylxanthines on fungal chitinases. Fungi demonstrated variable chitinase activity and incubation with methylxanthines (0.5-10 mM) resulted in a dose-dependent decrease in this activity. All fungi tested, except for Candida spp., demonstrated growth inhibition in the presence of methylxanthines at a concentration of 10 mM. India ink staining demonstrated impaired budding and decreased cell size for methylxanthine-treated Cryptococcus neoformans. C. neoformans and Aspergillus fumigatus treated with pentoxifylline also exhibited abnormal cell morphology. In addition, pentoxifylline-treated C. neoformans exhibited increased susceptibility to calcofluor and a leaky melanin phenotype consistent with defective cell wall function. Our data suggest that a variety of fungi express chitinases and that methylxanthines have antifungal properties related to their inhibition of fungal chitinases. Our results highlight the potential utility of targeting chitinases in the development of novel antifungal therapies.

Fig. 1

Effects of PTX on fungal chitinase activity. a Average chitinase activity in untreated fungal extracts (Control) and fungal extracts treated with (PTX). b Average chitinase activity of CN 24067 treated with aminophylline and caffeine compared to untreated organisms (Control). Activity is expressed milli Units/mg of protein relative to a Streptomyces griseus chitinase standard (Sigma). Bars represent one standard deviation. *P < 0.05 and **P %lt; 0.001 for comparison with untreated fungi (Control)

Fig. 2

Effects of PTX on CN 24067 growth. Growth curves were generated from automated turbidity measurements of CN 24067 treated with different concentrations of PTX. Growth was measured in plates incubated with continuous shaking in an Easy Bioscreen Reader (Oy Growth Curves, Piscataway, NJ). Fungi were grown 37°C in defined media (a) at 37°C and Sabouraud’s dextrose broth (b)

Fig. 3

Effects of PTX on CN 24067 growth as reflected in colony formation. CN 24067 was cultured in media with or without PTX (Control). At different times an aliquot was removed and incubated on Sabouraud’s agar and resultant colonies counted. a represents growth in defined media, while b represents growth in Sabouraud’s dextrose broth. Colony numbers represent an average of 3 plates, and bars represent one standard deviation

Fig. 4

Effects of PTX on CN and C. albicans viability. a Fungal metabolic activity was measured by XTT assay (absorbance 498_nm) for untreated fungi (Control) and fungi treated with (PTX). b XTT readings following treatment of A. fumigatus with varying concentrations of PTX or aminophyl line. Average absorbance measurements of 2 different wells are shown. Bars represent one standard deviation, **P < 0.01, ***P < 0.001 for comparison with controls

Fig. 3

Effects of PTX on CN budding. India ink staining of untreated (Controls, a) and PTX treated CN 24067 (b). PTX treated CN demonstrates increased number of budding cells and increased number of buds per cell. Original magnification, (a and b) ×200

Fig. 6

Effects of PTX on CN and A. fumigatus morphology. Top panels: Calcofluor staining (blue) demonstrates irregular cell wall shape for PTX (10 mM) treated CN 24067 (a) com pared with untreated cells (b). Center panels: WGA staining for chitosan demonstrates decreased and irregular reactivity at the surface of the cell wall for PTX (10 mM) treated CN 24067 (c) compared with controls (d) (original magnification 9365). Bottom panels Calcofluor staining of A. fumigatus reveals thinner hyphae with less extensive branching for PTX (10 mM treatment) organisms (e) compared with controls (f) (original magnification ×200)

Fig. 7

Effects of PTX on melanization of C. neoformans. Pellets and supernatants from CN24067 cultures grown in varying concentrations of PTX for 10 days are shown. Organisms treated with 10 mM of PTX produced a significantly smaller pellet with dark supernatant consistent with release of melanin into the supernatant