Melanization and morphological effects on antifungal susceptibility of Penicillium marneffei

Abstract

The biosynthesis of melanin has been linked with virulence in diverse pathogenic fungi. Penicillium marneffei, a dimorphic fungus, is capable of melanization in both mycelial and yeast phases, and the pigment may be produced during infection to protect the fungus from the host immune system. To investigate the impact of yeast morphological transformation on antifungal susceptibility, P. marneffei was cultured on various media including minimal medium, 1 % tryptone, brain heart infusion broth, and malt extract broth by using the standardized susceptibility protocol (the M27-A protocol, RPMI medium) for yeasts. We also investigated whether P. marneffei melanization affected its susceptibility to antifungal drugs by adding L-DOPA into culture broths. There were no differences in the minimum inhibitory concentrations of P. marneffei yeast cells previously grown in various culture broths with or without L-DOPA using the M27A protocol (into which no melanin substrate can be added due to a rapid colour change of the RPMI medium to black) for testing amphotericin B, clotrimazole, fluconazole, itraconazole and ketoconazole. However, both melanized and non-melanized P. marneffei displayed increased resistance to antifungal drugs when L-DOPA was added into a selected assay medium, 0.17 % yeast nitrogen base, 2 % glucose, and 1.5 % agar. Hence, active melanin formation appears to protect P. marneffei by enhancing its resistance to antifungal drugs.

Fig. 1

Corresponding immunofluorescence (A,C) and bright field (B,D) microscopy images demonstrating the labeling of mycelial phase (A,B) and yeast cells of P. marneffei by the melanin-binding MAb 8D6. No reactivity was observed when FITC labeled goat-anti mouse IgM alone was used (not shown). Bar, 5 μm.

Fig. 2

Morphology of P. marneffei yeast cells grown in various culture media: A, minimal medium; B, 1% tryptone; C, BHI broth; D, ME broth. Bar, 5 μm.

Fig. 3

The effect of melanization of P. marneffei yeast cells on susceptibility to AMB. Ten fold dilutions of melanized and non-melanized cells, at a concentration 5 × 10⁶ cells/ml, were prepared and then 10 μl of each dilution were inoculated onto YNB agar containing AMB with or without L-DOPA (A, and B, respectively).

Fig. 4

The effect of melanization of P. marneffei yeast cells on susceptibility to CLT. Ten fold dilutions of melanized and non-melanized cells, at a concentration 5 × 10⁶ cells/ml, were prepared and then 10 μl of each dilution were inoculated onto YNB agar containing CLT with and without L-DOPA (A, and B, respectively).

Fig. 5

The effect of melanization of P. marneffei yeast cells on susceptibility to FLC. Ten fold dilutions of melanized and non-melanized cells, at a concentration 5 × 10⁶ cells/ml, were prepared and then 10 μl of each dilution were inoculated onto YNB agar containing FLC with and without L-DOPA (A, and B, respectively).

Fig. 6

The effect of melanization of P. marneffei yeast cells on susceptibility to ITC. Ten fold dilutions of melanized and non-melanized cells, at a concentration 5 × 10⁶ cells/ml, were prepared and then 10 μl of each dilution were inoculated onto YNB agar containing ITC with and without L-DOPA (A, and B, respectively).

Fig. 7

The effect of melanization of P. marneffei yeast cells on susceptibility to KTC. Ten fold dilutions of melanized and non-melanized cells, at a concentration 5 × 10⁶ cells/ml, were prepared and then 10 μl of each dilution were inoculated onto YNB agar containing KTC with and without L-DOPA (A and B, respectively).