<i>Paracoccidioides</i> can internalize protoporphyrin rings.

<p>Iron deprived <i>Pb</i>01 and <i>Pb</i>18 yeast cells were incubated in MMcM medium supplemented or not (0) with different zinc protoporphyrin IX (Zn-PPIX) concentrations (20–100 µM) for 2 h. After this period, the cells were washed twice, and observed by bright field microscopy (BF) and by live fluorescence microscopy (F).</p

<i>Paracoccidioides</i> Rbt5 shows virulent and antigenic properties.

<p><b>A</b>. To test the ability to infect macrophages, <i>Pb</i>Wt, <i>Pbrbt5</i>-aRNA or <i>Pb</i>Wt+EV strains were co-cultivated with macrophages for 24 h. After this period, infected macrophages were lysed, and lysates were plated on BHI medium to recover the fungi. The data are presented as a bar graph of the means ± SEM from triplicates. *: statistically significant data as determined by Student's t-test (p<0.05) in comparison with the data that were obtained from the <i>Pb</i>Wt+EV strain. <b>B</b>. A murine model of infection was also used. Mice were infected intraperitoneally with <i>Pb</i>Wt, <i>Pbrbt5</i>-aRNA or <i>Pb</i>Wt+EV strains. After 2 weeks of infection, mice were sacrificed, the spleens were removed and samples of the homogenate were plated on BHI medium. After 15 days, the CFUs were counted to determine the fungal burden for each strain. The data are presented as a bar graph of the means ± SEM from quadruplicates. *: statistically significant data as determined by Student's t-test (p<0.05) in comparison with the data that were obtained from the <i>Pb</i>Wt+EV strain. <b>C</b>. Reaction of the recombinant <i>Pb</i>01 Rbt5 with sera of five PCM patients (lanes 1–5) or with control sera (lanes 6–10). After reacting with the anti-human IgG peroxidase coupled antibody, the reaction was developed using hydrogen peroxide and diaminobenzidine.</p

Effect of different iron sources on the growth of <i>Paracoccidioides</i> yeast cells.

<p><i>Pb</i>01 and <i>Pb</i>18 cell cultures were collected after 36 h of iron scarcity, washed and ten-fold serial dilutions of cell suspensions (10⁴ to 10 cells) were spotted on MMcM medium plates, which were supplemented with 50 µM BPS, an iron chelator. As indicated, different iron sources were added or not (no iron condition): 30 µM inorganic iron, 30 µM hemoglobin, 120 µM hemin, 30 µg/µl ferritin, 30 µM transferrin or 3 µM lactoferrin.</p

<i>Paracoccidioides</i> Rbt5 knock down via an antisense-RNA (aRNA) strategy.

<p><b>A</b>. Schematic representation of the T-DNA cassette that was used in this work to perform the <i>Agrobacterium tumefaciens</i>-mediated transformation (ATMT) of <i>Pb</i>339 (<i>Pb</i>Wt). <i>Pbrbt5</i>-aRNA was cloned in the pUR5750 binary vector under the control of the <i>Histoplasma capsulatum cbp-1</i> gene promoter region (P-<i>cbp-1</i>) and the <i>Aspergillus fumigatus cat-B</i> gene termination region (T-<i>cat-B</i>). The selection marker that was used in this work was the <i>Escherichia coli</i> hygromycin-resistance gene <i>hph</i>. In the cassette, this gene is flanked by the glyceraldehyde-3-phosphate dehydrogenase promoter region (P-<i>gapdh</i>) and by the <i>trpC</i> termination region (T-<i>trpC</i>) from <i>Aspergillus nidulans</i>. <b>B</b>. After the selection of mitotic stable isolates, a qRT-PCR was performed to analyze the silencing level of the gene in isolates that were transformed with <i>Pbrbt5</i>-aRNA. As controls, <i>rbt5</i> transcript level from <i>Pb</i>Wt and <i>Pb</i>Wt transformed with the empty vector (<i>Pb</i>Wt+EV) were also quantified. Alpha tubulin was used as the endogenous control. The data are represented as the means ± SD from triplicate determinations. *: statistically significant data as determined by Student's t-test (p<0.05) in comparison with the data that were obtained from <i>Pb</i>Wt+EV strain. <b>C</b>. Effect of <i>Pb</i>rbt5 deletion on the interaction of <i>Paracoccidioides</i> with heme-containing molecules. Hemoglobin prevents <i>Pb</i>Wt and <i>Pb</i>Wt+EV cells to be recognized by the anti-Rbt5 antibodies. However, <i>Pb</i>rbt5-aRNA cells are poorly recognized by the antibody that was raised against Rbt5, which is a process that was not affected by the previous or subsequent exposure of yeast cells to hemoglobin.</p

<i>Paracoccidioides</i> Rbt5 binds heme-containing molecules.

<p><b>A</b>. Recombinant protein Rbt5 was pre-incubated with (+) or without (−) hemoglobin (Hb) for 1 h. Subsequently, the samples were incubated with hemin-agarose resin for 1 h. After, the samples were centrifuged, the supernatants (S) were collected and the resin (R) was washed twice. After adding the buffer, the samples were boiled for 5 min and submitted to SDS-PAGE and Western blot analysis. A single 22 kDa immunoreactive species, which corresponds to the Rbt5 recombinant protein, was detected bound to the resin in the absence of hemoglobin or in the supernatant in the presence of hemoglobin. <b>B</b>. Upper and lower panels represent systems where protoporphyrin or hemoglobin Rbt5 recognition was assessed, respectively. The sequential steps of incubation in each system are indicated on the bottom of each panel. <i>Pb</i>01 denotes the background fluorescence of fungal cells alone; <i>Pb</i>01 + anti-Rbt5 represents systems where fungal cells were sequentially incubated with primary and secondary antibodies; <i>Pb</i>01 + protoporphyrin/hemoglobin + anti-Rbt5 is representative of systems that included the blocking of yeast cells with heme-containing molecules before exposure to antibodies; and <i>Pb</i>01 + anti-Rbt5 + protoporphyrin/hemoglobin represents systems that included the incubation of yeast cells with heme-containing molecules after exposure to antibodies.</p

Relevant <i>Paracoccidioides Pb</i>01 proteins that were repressed in the presence of hemoglobin as detected by nanoUPLC-MS^E.

a<p>Information that was obtained from the <i>Paracoccidioides</i> Database (<a href="http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html</a>).</p>b<p>filter 1 – proteins that were derived from PepFrag2; filter 2 – proteins that were derived from PepFrag1, as determined by PLGS and cited by Murad and Rech (2012).</p><p>***: proteins that were identified only in the presence of inorganic iron;</p><p>N.A.: not applicable.</p

<i>Paracoccidioides</i> Rbt5 is a GPI-anchored protein localized in the yeast cell wall.

<p><b>A</b>. Cell wall fraction of <i>Pb</i>01 yeast cells was obtained and analyzed by Western blot using polyclonal antibodies raised against the recombinant protein Rbt5. Proteins that were obtained from the cell wall (lane 1) were extracted by HF-pyridine digestion and analyzed (lane 2). Molecular weight markers are indicated at the right side of the panel. <b>B</b>. Immunoelectron microscopic detection of Rbt5 in <i>Pb</i>01 yeast cells by post embedding methods. (1) Negative control exposed to the rabbit preimmune serum. (2 and 3) Gold particles are observed at the fungus cell wall (arrow) and in the cytoplasm (double arrowheads). Bars: 1 µm (1 and 2) and 0.5 µm (3). v: vacuoles. m: mitochondria. w: cell wall.</p

Hemolysis of sheep erythrocytes in the presence of <i>Paracoccidioides</i> yeast cells.

<p><i>Pb</i>01 and <i>Pb</i>18 10⁷ yeast cell suspensions were incubated with 10⁸ sheep erythrocytes for 2 h at 36°C in 5% CO₂. As a negative or positive control, respectively, erythrocytes were incubated with phosphate buffered saline solution (PBS) or sterile water. The optical densities of the supernatants were determined with an ELISA plate reader at 405 nm. The experiment was performed in triplicate, and the average optical density of each condition was used to calculate the relative hemolysis of the experimental conditions or the negative control against the positive control. The data are plotted as the mean ± standard deviation. *: statistically significant difference in comparison with PBS values according to Student's t-test.</p

Relevant <i>Paracoccidioides Pb</i>01 proteins that were induced in the presence of hemoglobin as detected by nanoUPLC-MS^E.

a<p>Information that was obtained from the <i>Paracoccidioides</i> Database (<a href="http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html</a>).</p>b<p>filter 1 – proteins that were derived from PepFrag2; filter 2 – proteins that were derived from PepFrag1, as determined by PLGS and cited by Murad and Rech (2012).</p><p>***: proteins that were identified only in the presence of hemoglobin;</p><p>N.A.: not applicable.</p