Identification and immunogenic potential of glycosylphosphatidylinositol-anchored proteins in Paracoccidioides brasiliensis

Abstract

In fungal pathogens the cell wall plays an important role in host-pathogen interactions because its molecular components (e.g., polysaccharides and proteins) may trigger immune responses during infection. GPI-anchored proteins represent the main protein class in the fungal cell wall where they can perform several functions, such as cell wall remodeling and adhesion to host tissues. Genomic analysis has identified the complement of GPI-anchored proteins in many fungal pathogens, but the function has remained unknown for most of them. Here, we conducted an RNA expression analysis of GPI-anchored proteins of Paracoccidioides brasiliensis which causes paracoccidioidomycosis (PCM), an important human systemic mycosis endemic in Latin America. The expression of the GPI-anchored proteins was analyzed by quantitative PCR in both the mycelium and yeast forms. qPCR analysis revealed that the transcript levels of 22 of them were increased in hyphae and 10 in yeasts, respectively, while 14 did not show any significant difference in either form. Furthermore, we cloned 46 open reading frames and purified their corresponding GPI-anchored proteins in the budding yeast. Immunoblot and ELISA analysis of four purified GPI-anchored proteins revealed immune reactivity of these proteins against sera obtained from PCM patients. The information obtained in this study provides valuable information about the expression of many GPI-anchored proteins of unknown function. In addition, based on our immune analysis, some GPI-anchored proteins are expressed during infection and therefore, they might serve as good candidates for the development of new diagnostic methods.

Keywords: GPI-anchored proteins; PCM; Paracoccidioides brasiliensis; Western blot; cell wall.

Figure 1

Quantitative transcriptional expression of ORFs from hyphae to yeast cell transition phase from Paracoccidioides brasiliensis. The color bar displays a heat map indicating the difference in abundance (ranging from 0.05 to 400-fold) of transcriptional expression intensities between yeast and hyphae in P. brasiliensis. The mRNA was grouped using unsupervised hierarchical clustering.

Figure 2

Quantitative transcriptional expression of ORFs from yeast to hyphae cell transition phase from Paracoccidioides brasiliensis. The color bar displays a heat map indicating the difference in abundance (ranging from 0.05 to 400-fold) of transcriptional expression intensities between yeast and hyphae in P. brasiliensis. The mRNA was grouped using unsupervised hierarchical clustering.

Figure 3

Shows the recognition of rPADG_02842 (SOD3) by sera from patients with Paracoccidioidomycosis (PCM), using SDS-PAGE and western blotting. (A) PB1-PB7: sera from 7 PCM patients recognized 6 polypeptides of 51 kDa (arrow), while GST-negative control and glutathione-S-transferase protein were not recognized. (B) Ca1 and Ca2: 2 sera from patients with candidiasis, and healthy sera from 2 persons served as negative controls (1 and 2). GP43 protein of P. brasiliensis native 43 kDa (14 mg/ml) was used as a positive control (arrow). (C) H1-H3: serum from 3 patients with histoplasmosis, and A1-A4: serum of 4 patients with aspergillosis. (D) C1-C6: 6 sera from unrelated diseases. All primary antibodies were diluted to 1:1000, and goat anti-human IgG and C+ GST and GST were used as secondary antibodies.

Figure 4

The protein rPADG_01494 was evaluated for recognition by sera from patients with paracoccidioidomycosis (PCM) using SDS-PAGE and western blot. (A) PB1-PB7: sera from 7 PCM patients were tested, with only a polypeptide of 78 kDa being recognized in PB1, PB2, and PB5 sera (arrow), and 2 polypeptides recognized in PB4 and PB6 sera. No reactivity was observed with sera from 2 patients PB3 and PB7. GST-negative control and glutathione-S-transferase protein were not recognized. (B) CA1 and CA2: 2 sera from patients with candidiasis, and healthy sera from 2 persons served as negative controls (1 and 2). GP43 protein of P. brasiliensis native 43 kDa (14 mg/ml) was used as a positive control (arrow). (C) H1-H3: serum from 3 patients with histoplasmosis, with H2 serum reacting with a polypeptide of about 50 kDa. A1-A4: serum of 4 patients with aspergillosis, with A2 serum reacting with a polypeptide of about 50 kDa, and A1 serum showing nonspecific reactivity. (D) C1-C6: 6 sera from unrelated diseases. All primary antibodies were diluted to 1:1000, and goat anti-human IgG and C+ GST and GST were used as secondary antibodies.

Figure 5

Detection of anti-rGPI-protein antibodies in sera from patients with PCM by ELISA. Recombinant GPI-proteins (1 μg/mL) were used to coat ELISA plates, and sera from different patient groups were added as primary antibodies. The results were evaluated based on optical density (OD₄₅₀ nm) readings. (A–D) Seven sera from patients with PCM were tested, and the healthy control group included two sera from individuals without clinical evidence of fungal infection. Additionally, two sera from patients with candidiasis, three from patients with histoplasmosis, four from patients with aspergillosis, and sera from patients diagnosed with unrelated diseases (tuberculosis, lymphoma, icterus, pneumonia, and asthma) were tested. (E) Seven sera from patients with PCM were evaluated against rPADG_02842 protein, rGST, and GP43. Bars depict the mean ± SEM and the values provided by serum reactivity against recombinant proteins, and the statistical significance was determined using one-way ANOVA, where *p <0.1, **p <0.05, ***p <0.01, and ****p <0.001; bars where p is not shown, are not significant.