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. 2021 Mar 26:116:e200592.
doi: 10.1590/0074-02760200592. eCollection 2021.

Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts

Jaqueline Derissi Braz  1 , Janaina de Cássia Orlandi Sardi  1   2 , Nayla de Souza Pitangui  1   3 , Aline Raquel Voltan  1 , Ana Marisa Fusco Almeida  1 , Maria José Soares Mendes-Giannini  1
Affiliations

Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts

Jaqueline Derissi Braz et al. Mem Inst Oswaldo Cruz. .

Abstract

Background: Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus.

Objectives: In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line).

Methods: The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease.

Findings: In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form.

Main conclusions: In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

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Figures

Fig. 1:
Fig. 1:. evaluation of gp43 gene expression. Quantitative polymerase chain reaction (qPCR) for (A) Paracoccidioides brasiliensis (Pb18) and (B) P. lutzii (Pb01). Y form (Y), infected macrophages (Y + AMJ2-C11), infected lung fibroblasts (Y + MRC-5); M form (M), infected macrophages (M + AMJ2-C11), infected lung fibroblasts (M + MRC-5). Analysis of variance (ANOVA) with Tukey’s multiple comparison tests and significance at p < 0.05. *: different letters denote statistical significant.
Fig. 2:
Fig. 2:. evaluation of ENO gene expression. Quantitative polymerase chain reaction (qPCR) for (A) Paracoccidioides brasiliensis (Pb18) and (B) P. lutzii (Pb01) Y form (Y), infected macrophages (Y + AMJ2-C11), infected lung fibroblasts (Y + MRC-5); M form (M), infected macrophages (M + AMJ2-C11), infected lung fibroblasts (M + MRC-5). Analysis of variance (ANOVA) with Tukey’s multiple comparison tests and significance at p < 0.05. *: different letters denote statistical significant.
Fig. 3:
Fig. 3:. evaluation of phospholipase gene expression. Quantitative polymerase chain reaction (qPCR) for (A) Paracoccidioides brasiliensis (Pb18) and (B) P. lutzii (Pb01). Y form (Y), infected macrophages (Y + AMJ2-C11), infected lung fibroblasts (Y + MRC-5); M form (M), infected macrophages (M + AMJ2-C11), infected lung fibroblasts (M + MRC-5). Analysis of variance (ANOVA) with Tukey’s multiple comparison tests and significance at p < 0.05. *: different letters denote statistical significant.
Fig. 4:
Fig. 4:. evaluation of aspartyl protease gene expression. Quantitative polymerase chain reaction (qPCR) for (A) Paracoccidioides brasiliensis (Pb18) and (B) P. lutzii (Pb01). Y form (Y), infected macrophages (Y + AMJ2-C11), infected lung fibroblasts (Y + MRC-5); M form (M), infected macrophages (M + AMJ2-C11), infected lung fibroblasts (M + MRC-5). Analysis of variance (ANOVA) with Tukey’s multiple comparison tests and significance at p < 0.05. *: different letters denote statistical significant.
Fig. 5:
Fig. 5:. evaluation of GAPDH gene expression. Quantitative polymerase chain reaction (qPCR) for (A) Paracoccidioides brasiliensis (Pb18) and (B) P. lutzii (Pb01). Y form (Y), infected macrophages (Y + AMJ2-C11), infected lung fibroblasts (Y + MRC-5); M form (M), infected macrophages (M + AMJ2-C11), infected lung fibroblasts (M + MRC-5). Analysis of variance (ANOVA) with Tukey’s multiple comparison tests and significance at p < 0.05. *: different letters denote statistical significant.
Fig. 6:
Fig. 6:. evaluation of 14-3-3 gene expression. Quantitative polymerase chain reaction (qPCR) for (A) Paracoccidioides brasiliensis (Pb18) and (B) P. lutzii (Pb01). Y form (Y), infected macrophages (Y + AMJ2-C11), infected lung fibroblasts (Y + MRC-5); M form (M), infected macrophages (M + AMJ2-C11), infected lung fibroblasts (M + MRC-5). Analysis of variance (ANOVA) with Tukey’s multiple comparison tests and significance at p < 0.05. *: different letters denote statistical significant.
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