Analysis of the secretomes of Paracoccidioides mycelia and yeast cells

Abstract

Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host.

Figure 1. Validation of the extracellular protein extraction method.

A- The viability of Paracoccidioides 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- The growth of Paracoccidioides 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. C- PCR sensitivity for the formamidase gene was assessed using Paracoccidiodes Pb01 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. D- The yeast and mycelia cell-free supernatant samples (2 µL) were assessed for the presence of Paracoccidiodes DNA via PCR using oligonucleotides specific for the formamidase gene.

Figure 2. Proteins detected in the secretome of yeast cells and mycelia of Paracoccidiodes via 2D-gel analysis.

Protein profile generated after the separation of the secreted fraction of proteins by yeast cells (A) and mycelia (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.

Figure 3. Graphic summation of proteomics analysis.

A- 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- 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.

Figure 4. Enzymatic activity analysis validates the secretome data for Paracoccidioides mycelia and yeast cells.

Activity assay results of (A) formamidase (FMD), (B) superoxide dismutase (SOD) and (C) 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 (p≤0.05). The erros bars represent the standard deviation of three biological replicates.

Figure 5. Blocking the conventional protein secretion pathway leads to a decrease in Paracoccidioides yeast cell phagocytosis.

A- The protein profile of the cell-free supernatant samples reveals the effect of blocking the protein secretion pathway on yeast cells. Paracoccidioides 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- The average number of internalized/adhered Paracoccidioides cells by macrophages was determined. Macrophages were infected with Paracoccidioides 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. C- 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 (* p≤0.05) was determined by comparing the results with the control group.