The effect of tricyclazole on melanization during asexual development.

<p>(A) The generic DHN melanin biosynthetic pathway [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref015" target="_blank">15</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref024" target="_blank">24</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref043" target="_blank">43</a>]. 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, <i>T</i>. <i>marneffei</i> 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.</p

Predicted DHN melanin synthesis pathway in <i>T</i>. <i>marneffei</i>.

<p>A consensus biochemical pathway for DHN melanin synthesis based on what is known from other fungal systems is shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref015" target="_blank">15</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref050" target="_blank">50</a>]. 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 <i>A</i>. <i>nidulans</i> (<i>An</i>), <i>A</i>. <i>fumigatus</i> (<i>Af</i>), <i>T</i>. <i>marneffei</i> (<i>Tm</i>) are shown with dashed arrows. Asterisks, * and **, refer to data from previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122728#pone.0122728.ref022" target="_blank">22</a>] and this study, respectively.</p

Localization of PbrB and melanin during conidiation in <i>T</i>. <i>marneffei</i>.

<p>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.</p

Macroscopic morphologies of the wild type and mutant <i>T</i>. <i>marneffei</i> strains.

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

Basic measurements of radiation at station Minamitorishima (2017-02)

<div><p><i>Talaromyces marneffei</i> (Basionym: <i>Penicillium marneffei</i>) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in Southeast Asia. <i>T</i>. <i>marneffei</i> cells have been shown to become melanized <i>in vivo</i>. 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 <i>T</i>. <i>marneffei</i> genome encodes a number of laccases and this study describes the characterization of one of these, <i>pbrB</i>, during growth and development. A strain carrying a PbrB-GFP fusion shows that <i>pbrB</i> is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. The <i>pbrB</i> 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.</p></div

List of primers used in this study.

<p>List of primers used in this study.</p

Germination and growth of the Δ<i>pbrB</i> strain.

<p>Microscopic imaging of the <i>T</i>. <i>marneffei</i> Δ<i>pbrB</i> and Δ<i>pbrB pbrB</i>⁺ 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.</p

Expression and localization of PbrB in <i>T</i>. <i>marneffei</i>.

<p>Epifluorescence microscopy of the <i>T</i>. <i>marneffei</i> 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 <i>T</i>. <i>marneffei</i> 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.</p