The pbrB gene encodes a laccase required for DHN-melanin synthesis in conidia of Talaromyces (Penicillium) marneffei

Abstract

Talaromyces marneffei (Basionym: Penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. T. marneffei cells have been shown to become melanized in vivo. Melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. The synthesis of the two most commonly found melanins in fungi, the eumelanin DOPA-melanin and the allomelanin DHN-melanin, requires the action of laccase enzymes. The T. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrB, during growth and development. A strain carrying a PbrB-GFP fusion shows that pbrB is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The pbrB gene is required for the synthesis of DHN-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type.

Fig 1. The effect of tricyclazole on melanization during asexual development.

(A) The generic DHN melanin biosynthetic pathway [15, 24, 43]. Enzymes acting at each step include polyketide synthase (PKS), tetrahydroxynaphthalene reductase (4HNR), trihydroxynaphthalene reductase (3HNR), dehydratase (D), and laccase. Intermediate metabolites including 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), scytalone, 1,3,8-trihydroxynaphthalene (1,3,8-THN), vermelone, and 1,8-dihydroxynaphthalene (1,8-DHN). (B) At 28°C, T. marneffei F4 colonies appear green as a result of the pigmented conidia. The addition of 30 μg/ml tricyclazole, which inhibits two reductases (4HNR and 3HNR) functioning during DHN melanin synthesis, results in yellow conidiation.

Fig 2. Phylogenetic analysis and partial sequence alignment.

(A) Putative fungal MCO sequences from a number of species, including those predicted in T. marneffei, were obtained from Genbank (http://www.ncbi.nlm.nih.gov/protein/) and used to build sequence alignments in CLUSTALW. The alignment was then used to construct a relatedness tree using MEGA 6 software. Phylogenetic relationships of the 55 MCOs were inferred using the Neighbor-Joining method and bootstrap tested (1000 replicates). Branch lengths of the tree are drawn to scale and bootstrap support indicating at the branch sites. Fungal MCOs that share a common ancestry with more than 60% bootstrap value are shaded in gray. Functions are defined at each clade based on characterized function of MCO members. (B) A partial alignment representing the conserved motifs of copper binding sites and L1–L4 signature sequences found in T. marneffei PbrB (PMAA_082060) and its orthologs A. fumigatus Abr2 (AFUA_2G17530) and A. nidulans YA (AN6635). The numbers in front of each sequence indicate the amino acid position of a particular protein. Cu binding motifs are identified above the sequences, numbers including 1, 2, and 3 are type 1 Cu, type 2 Cu, and type 3 Cu. The asterisk indicates the potential proton donor for the reaction intermediates.

Fig 3. Expression and localization of PbrB in T. marneffei.

Epifluorescence microscopy of the T. marneffei strain expressing the PbrB::GFP fusion in vegetative cells grown at 37°C (A) or 28°C (B) for 3 days. No GFP fluorescence was noted in either cell type. Epifluorescence microscopy of the T. marneffei conidiophores from the wild type (G681) strain (C) and the strain expressing the PbrB::GFP fusion (D) under bright field and fluorescence optics. The PbrB::GFP fusion is observed as distinct spots (white arrows) in metulae and phialides. Strains were grown on slides at 28°C for 10 days. Microscopic images were captured at 1000X magnification.

Fig 4. Localization of PbrB and melanin during conidiation in T. marneffei.

Epifluorescence microscopy after immunostaining using an anti-melanin antibody staining for the wild type control (G681) and the PbrB::GFP fusion strain after growth on slides at 28°C for 10 days. To assess non-specific staining by the rhodamine-labeled secondary antibody (goat anti-mouse IgM antibody), samples were processed without the primary anti-melanin antibody step (labeled as—anti-melanin Ab.). Red fluorescent signals of melanin or melanin-like particles are observed in both wild type and PbrB::GFP fusion strain (+ anti-melanin Ab.). Green fluorescent signals indicate sites of PbrB::GFP proteins. The merged image shows co-localization of PbrB::GFP and melanin-labeled fluorescence. White arrows identify sites of fluorescence signals which melanin-labeled particles co-localize with PbrB::GFP proteins. Microscopic images were captured at 1000X magnification.

Fig 5. Macroscopic morphologies of the wild type and mutant T. marneffei strains.

Colonies of the wild type T. marneffei control strain G681, ΔpbrB 1&2 strains, and ΔpbrB pbrB ⁺ 1&2 complemented strains grow on ANM agar at 28°C for 14 days. Conidiation of the ΔpbrB strains is brown compared to the green conidiation of the parental and complemented strains.

Fig 6. Germination and growth of the ΔpbrB strain.

Microscopic imaging of the T. marneffei ΔpbrB and ΔpbrB pbrB ⁺ strains grown at 28°C for 24 h to assess germination (A, B) and 7 days to observe conidiophores (C, D). Microscopic images were captured at 200X (A, B) and 400X (C, D) magnification.

Fig 7. Vegetative growth rate effects in the ΔpbrB mutant.

Radial growth rates were assessed by measuring colony diameters for the ΔpbrB and ΔpbrB pbrB ⁺ strains growing at 28°C on ANM medium. The line plots the growth rates of ΔpbrB (solid line) and ΔpbrB pbrB ⁺ (dashed line). Error bars present standard error of the mean (SEM). Asterisks indicate significantly difference between strains determined by p-values (p = 0.002 and 0.015). X axis is time and Y axis is the mean colony diameter.

Fig 8. Predicted DHN melanin synthesis pathway in T. marneffei.

A consensus biochemical pathway for DHN melanin synthesis based on what is known from other fungal systems is shown [15, 39, 50]. Processing steps of the well-known DHN pathway are presented with dark arrows. PKS, polyketide synthase; 4HNR, tetrahydroxynaphthalene reductase; SD, scytalone dehydratase; 3HNR, trihydroxynaphthalene reductase; VD, vermelone dehydratase; 1,3,6,8-THN, tetrahydroxynaphthalene; 1,3,8-THN, trihydroxynapthalene; 1,8-DHN, dihydroxynaphthalene. Tc, tricyclazole, can inhibit both reductases (4HNR and 3HNR) presented in a model pathway. Distinctive steps described in A. nidulans (An), A. fumigatus (Af), T. marneffei (Tm) are shown with dashed arrows. Asterisks, * and **, refer to data from previous study [22] and this study, respectively.