Talaromyces marneffei laccase modifies THP-1 macrophage responses.

Talaromyces (Penicillium) marneffei is an emerging opportunistic pathogen associated with HIV infection, particularly in Southeast Asia and southern China. The rapid uptake and killing of T. marneffei conidia by phagocytic cells along with the effective induction of an inflammatory response by the host is essential for disease control. T. marneffei produces a number of different laccases linked to fungal virulence. To understand the role of the various laccases in T. marneffei, laccase-encoding genes were investigated. Targeted single, double and triple gene deletions of laccases encoding lacA, lacB, and lacC showed no significant phenotypic effects suggesting redundancy of function. When a fourth laccase-encoding gene, pbrB, was deleted in the ΔlacA ΔlacB ΔlacC background, the quadruple mutant displayed delayed conidiation and the conidia were more sensitive to H2O2, sodium dodecyl sulfate (SDS), and antifungal agents than wild-type and other transformants. Conidia of the quadruple mutant showed marked differences in their interaction with the human monocyte cell line, THP-1 such that phagocytosis was significantly higher when compared with the wild-type at one and 2 hours of incubation while the phagocytic index was significantly different from 15 to 120 minutes. In addition, killing of the quadruple mutant by THP-1 cells was more efficient at 2 and 4 hours of incubation. The levels of the proinflammatory cytokines TNF-α, IL-1ß and IL-6 from THP-1 cells infected with the quadruple mutant were also significantly increased in comparison with wild-type. The results demonstrate that production of laccases by T. marneffei actually promotes the pathogen's resistance to innate host defenses.

Melanization and morphological effects on antifungal susceptibility of Penicillium marneffei.

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.