<i>Paracoccidiodes, Pb</i>01 yeast cells secreted proteins in macrophages by nanoLC-MS/MS.

1<p>NCBI database general information number (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p

Graphic summation of proteomics analysis.

<p><b>A-</b> The comparative analysis using Image master 2D Platinum software displays the analyzed spots and the preferentially expressed spots in mycelia and yeast secretomes, while the mass spectrometry analyses displays the identified differentially expressed spots. <b>B-</b> The Venn diagram shows the number of identified proteins via MS/MS. The preferentially secreted extracellular proteins include those with statistically significant alteration in the mycelia and yeast secretome, while the constitutive proteins refer to those with similar expression patterns.</p

Constitutive proteins secreted by <i>Paracoccidioides Pb</i>01 yeast and mycelia.

1<p>NCBI database general information number (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p>2<p>Number of identified isoforms of protein in <i>Paracoccidioides, Pb01</i> secretome.</p>3<p>The average of amount of values of abundances of all identified isoforms used to statistical test.</p>4<p>ANOVA – statistically significant differences are considered with <i>p</i><0.05 (*).</p>5<p>Secretion prediction according to Signal P 3.0 server, the number corresponds to signal peptide probability (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>).</p>6<p>Secretion prediction according to Secretome P 2.0 server, the number corresponds to neural network that exceeded a value of 0.5 (NN-score <b>≥</b>0.50) (<a href="http://www.cbs.dtu.dk/services/SecretomeP/" target="_blank">http://www.cbs.dtu.dk/services/SecretomeP/</a>).</p

Proteins detected in the secretome of yeast cells and mycelia of <i>Paracoccidiodes</i> via 2D-gel analysis.

<p>Protein profile generated after the separation of the secreted fraction of proteins by yeast cells (<b>A</b>) and mycelia (<b>B</b>) using 2D-eletrophoresis (first dimension: IEF pH range 3 –11 non-linear, second dimension: 12% (w/v) SDS-PAGE) and visualized using Coomassie brilliant blue staining. The 2-D gel images of three biological replications of each phase were compared to identify the differential expression levels of proteins using Image master 2D Platinum software. The protein spots that were identified via MS/MS are numbered and listed in Table S1. The pH gradient is shown above the gel, and the molecular mass protein standards (kDa) are indicated to the left of the gels.</p

Enzymatic activity analysis validates the secretome data for <i>Paracoccidioides</i> mycelia and yeast cells.

<p>Activity assay results of (<b>A</b>) formamidase (FMD), (<b>B</b>) superoxide dismutase (SOD) and (<b>C</b>) glutathione S-transferase (GST) assessed for mycelia and yeast protein extracts. FMD activity was assessed by measuring the levels of ammonia released using a standard curve. The SOD and GST Assay Kit were used to determine SOD and GST enzymatic activity, respectively. The student's t test was used for statistical comparisons, and the observed differences were statistically significants (<i>p</i>≤0.05). The erros bars represent the standard deviation of three biological replicates.</p

Blocking the conventional protein secretion pathway leads to a decrease in <i>Paracoccidioides</i> yeast cell phagocytosis.

<p><b>A</b>- The protein profile of the cell-free supernatant samples reveals the effect of blocking the protein secretion pathway on yeast cells. <i>Paracoccidioides</i> yeast cells were cultivated in Fava Netto's liquid medium in either the absence (control) (lanes 1, 3 and 5) or presence of Brefeldin A (BFA) at 6 μg/mL (lanes 2, 4 and 6) for 6, 12 and 24 hours, respectively. The cell-free supernatant samples were prepared (as described in the Materials and Methods section), reduced to equal final volumes (1 mL), and processed for one-dimensional electrophoresis (SDS-PAGE). Thirty microliters of each sample was separated via SDS-PAGE and visualized using Coomassie brilliant blue staining. The numbers on the left side correspond to the molecular mass standard. <b>B</b>- The average number of internalized/adhered <i>Paracoccidioides</i> cells by macrophages was determined. Macrophages were infected with <i>Paracoccidioides</i> yeast cells, which were pre-cultivated previously without BFA (control), in the presence of BFA or the presence of concentrated culture supernatant containing extracellular proteins (EP). The adhered/internalized cells were analyzed as described in the materials and methods section. <b>C</b>- The number of viable yeast cells after phagocytosis by macrophages was evaluated by counting the number of colony forming units (CFUs). The results are representative of triplicate biological samples. Statistical significance (* <i>p</i>≤0.05) was determined by comparing the results with the control group.</p

Validation of the extracellular protein extraction method.

<p><b>A-</b> The viability of <i>Paracoccidioides</i> yeast cells incubated in Fava Netto's liquid medium (dark gray square) and the incubation of yeast cells in Fava Netto's liquid medium containing 6 µg/mL Brefeldin A (light gray square). Viability was assessed using trypan blue staining. The error bars represent the standard deviation of three biological replicates. <b>B-</b> The growth of <i>Paracoccidioides</i> yeast cells in liquid medium in either the absence (dark line) or presence of 6 µg/mL Brefeldin A (light gray line). Culture growth was evaluated by quantifying the number of yeast cells per mL. The error bars represent the standard deviation of three biological replicates. <b>C-</b> PCR sensitivity for the formamidase gene was assessed using <i>Paracoccidiodes Pb01</i> genomic DNA (at five dilutions) as a template (50 ng to 1 pg). Lanes: 1 −50 ng; 2 −5 ng; 3 −50 pg; 4 −5 pg; 5 −1 pg; 6 - negative control (without genomic DNA). The formamidase PCR amplicons were assessed via 1% agarose gel electrophoresis and stained with ethidium bromide. <b>D-</b> The yeast and mycelia cell-free supernatant samples (2 µL) were assessed for the presence of <i>Paracoccidiodes</i> DNA via PCR using oligonucleotides specific for the formamidase gene.</p

Preferentially secreted proteins by <i>Paracoccidioides Pb</i>01 mycelia.

1<p>NCBI database general information number (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p>2<p>Number of identified isoforms of protein in <i>Paracoccidioides, Pb01</i> mycelia phase secretome.</p>3<p>The average of amount of values of abundances of all identified isoforms used to statistical test.</p>4<p>The ratio of M to Y abundance in <i>Paracoccidioides</i> secretome.</p>5<p>ANOVA – statistically significant differences are considered with <i>p</i><0.05 (*); proteins were not detected in yeast secretome (**).</p>6<p>Secretion prediction according to Signal P 3.0 server, the number corresponds to signal peptide probability (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>).</p>7<p>Secretion prediction according to Secretome P 2.0 server, the number corresponds to neural network that exceeded a value of 0.5 (NN-score <b>≥</b>0.50) (<a href="http://www.cbs.dtu.dk/services/SecretomeP/" target="_blank">http://www.cbs.dtu.dk/services/SecretomeP/</a>).</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

<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