The role of melanin pathways in extremotolerance and virulence of <em>Fonsecaea</em> revealed by <em>de novo</em> assembly transcriptomics using illumina paired-end sequencing

AbstractMelanisation has been considered to be an important virulence factor of Fonsecaea monophora. However, the biosynthetic mechanisms of melanisation remain unknown. We therefore used next generation sequencing technology to investigate the transcriptome and digital gene expression data, which are valuable resources to better understand the molecular and biological mechanisms regulating melanisation in F. monophora. We performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of parent (CBS 122845) and albino (CBS 125194) strains using the Illumina RNA-seq system. A total of 17 352 annotated unigenes were found by BLAST search of NR, Swiss-Prot, Gene Ontology, Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) (E-value <1e‒5). A total of 2 283 unigenes were judged to be the differentially expressed between the two genotypes. We identified most of the genes coding for key enzymes involved in melanin biosynthesis pathways, including polyketide synthase (pks), multicopper oxidase (mco), laccase, tyrosinase and homogentisate 1,2-dioxygenase (hmgA). DEG analysis showed extensive down-regulation of key genes in the DHN pathway, while up-regulation was noted in the DOPA pathway of the albino mutant. The transcript levels of partial genes were confirmed by real time RT-PCR, while the crucial role of key enzymes was confirmed by either inhibitor or substrate tests in vitro. Meanwhile, numbers of genes involved in light sensing, cell wall synthesis, morphology and environmental stress were identified in the transcriptome of F. monophora. In addition, 3 353 SSRs (Simple Sequence Repeats) markers were identified from 21 600 consensus sequences. Blocking of the DNH pathway is the most likely reason of melanin deficiency in the albino strain, while the production of pheomelanin and pyomelanin were probably regulated by unknown transcription factors on upstream of both pathways. Most of genes involved in environmental tolerance to oxidants, irradiation and extreme temperatures were also assembled and annotated in transcriptomes of F. monophora. In addition, thousands of identified cSSR (combined SSR) markers will favour further genetic linkage studies. In conclusion, these data will contribute to understanding the regulation of melanin biosynthesis and help to improve the studies of pathogenicity of F. monophora

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

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

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

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

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

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

Expression and characterization of a Talaromyces marneffei

Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS–polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation