Deletion of <i>atfA</i> does not affect chitin deposition and cell wall integrity.

<p>(A) <i>P. marneffei</i> wild type (F4), <i>atfA</i> mutant (SC) and <i>atfA</i> complemented (AC1) strains were grown for seven day at 25°C on PDA and stained with CFW day four and day seven to visualize cell wall and septa. Scale bar represents five micrometers. (B to M) five microliters of cell dilutions (5×10⁴ to 5 cells) of wild type, <i>atfA</i> mutant and <i>atfA</i> complemented strains were inoculated on media supplemented with 5 µg/ml CFW(C and I), 0.004% SDS (D and J), 10 µg/ml and 4 µg/ml amphotericin B (F and L) and 8 µg/ml and 0.8 µg/ml itraconazole (G and M). (B and E) and (H and K) represent MM control plates at 25°C and 37°C, respectively.</p

Susceptibility to oxidative stresses of <i>P. marneffei</i>.

<p>Growth of <i>P. marneffei</i> wild type (F4), the <i>atfA</i> mutant (SC) and <i>atfA</i> complemented strain (AC1) at 25°C and 37°C on MM agar supplemented with 2 and 0.5 mM <i>t</i>-BOOH (B and F), 2 and 1 mM H₂O₂ (C and G), and 0.25 mM and 25 µM menadione (D and H). Five microliters of cell dilutions (5×10⁴ to 5 cells) were inoculated on MM agar containing each compound. (A) and (E) represent MM control plates at 25°C and 37°C, respectively.</p

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

Susceptibility of conidia from <i>P. marneffei</i> wild type, <i>sakA</i> mutant (Δ<i>sakA</i>) and <i>atfA</i> mutant (Δ<i>atfA</i>) to UV light.

<p>Conidia of each strain were plated in duplicate on SDA and exposed to different UV light radiation at 0, 2000, 4000, 6000 and 8000 microjoules/cm². Data are from three independent experiments and standard error bars of the mean bars are shown (p<0.05).</p

Gene expressions and survival of <i>P. marneffei</i> under heat stress at 42°C.

<p>(A) Relative RNA expression of <i>sakA</i> and <i>atfA</i> genes of conidia from <i>P. marneffei</i> wild type determined by real-time PCR. Conidia were incubated at 42°C for 10, 20, 30 or 40 minutes. Total RNA was extracted from conidia and subjected to real-time PCR. Expression level of heat stress cells is presented as relative value to the expression level from no stress cells which is given a value of 1. GAPDH gene expression level was used to normalize amounts of input RNA. (B) Survival of conidia from <i>P. marneffei</i> wild type (WT), <i>atfA</i> mutant (Δ<i>atfA</i>) and complemented strains (Δ<i>atfA</i> + <i>atfA</i>) after incubating in BHI at 42°C for one hour. (C) Relative RNA expression level of <i>atfA</i> gene of conidia from <i>P. marneffei</i> wild type (WT), <i>sakA</i> mutant (Δ<i>sakA</i>). Conidia were incubated at 42°C for 20 minutes and total RNA was extracted and subject to real-time PCR. Results were obtained from three independent experiments and standard error bars of the mean bars are shown (p<0.05).</p

<i>atfA</i> gene is not involved in osmotic and heat stress responses in <i>P. marneffei</i>.

<p>Five microliters of cell dilutions (5×10⁴ to 5 cells) of wild type (F4), <i>atfA</i> mutant (SC) and <i>atfA</i> complemented (AC1) strains were inoculated on MM agar supplemented with 0.5 and 0.25 M NaCl (B and E) and 1 M sorbitol (C and F). (G) MM agar was incubated at 39°C. (A) and (D) represent MM control plates at 25°C and 37°C, 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

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

Strategy for construction of the <i>atfA</i> complemented strain.

<p>(A) The restriction map demonstrates recognition sites of <i>Eco</i>RV (EV) and <i>Bss</i>SI (BsI) used in Southern blot analysis to detect the presence of <i>atfA</i> gene in the <i>atfA</i> complemented strain (AC1) comparing to the wild type strain (WT). The grey bar indicates the position of probe that is specific to the <i>atfA</i> gene. (B) Southern blot hybridization of genomic DNA from the wild type (F4) and the <i>atfA</i>-complemented (AC1) strains using probe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111200#pone-0111200-g002" target="_blank">Figure 2A</a>. Probe (a 0.9 kb fragment of <i>atfA</i>) hybridized with 5.6 kb fragment and unpredicted fragment (10.0 kb fragment) of <i>Eco</i>RV- and BssSI-digested DNA from the wild type strain, respectively. For the <i>atfA</i> complemented strain, probe hybridized with unpredicted fragment (3.5 kb fragment) and 7.5 kb fragment of <i>Eco</i>RV- and BssSI-digested DNA from the <i>atfA</i> complemented strain, respectively.</p

Survival of <i>P. marneffei</i> inside macrophage.

<p>Mouse (A) and human (B) macrophages were infected with conidia of <i>P. marneffei</i> wild type (F4), <i>atfA</i> mutant (Δ<i>atfA</i>) and <i>atfA</i> complemented (Δ<i>atfA</i> + <i>atfA</i>) strains. Percent recovery was calculated from number of colonies recovered after two hours and 24 hours post-infection. Data are from three independent experiments and standard error bars of the mean bars are shown (p<0.05).</p