Phospholipase gene expression during
                  <i>Paracoccidioides brasiliensis</i> morphological transition and
               infection

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

host-pathogen interaction; Paracoccidioides brasiliensis ; pathogenicity; phospholipases

Authorship

Deyze Alencar Soares

Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, BrasilUniversidade Federal de GoiásBrasilGoiânia, GO, BrasilInstituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO, Brasil

Marilia Barros Oliveira

Adriane Feijó Evangelista

Departamento de Ciências Patológicas, Universidade Estadual de Londrina, Londrina, PR, BrasilUniversidade Estadual de LondrinaBrasilLondrina, PR, BrasilDepartamento de Ciências Patológicas, Universidade Estadual de Londrina, Londrina, PR, Brasil

Emerson José Venancio

Rosângela Vieira Andrade

Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, BrasilUniversidade Católica de BrasíliaBrasilBrasília, DF, BrasilPrograma de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brasil

Maria Sueli Soares Felipe

Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, BrasilUniversidade de BrasíliaBrasilBrasília, DF, BrasilDepartamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brasil

Silvana Petrofeza ⁺+ Corresponding author: petrofez@uol.com.br

+ Corresponding author: petrofez@uol.com.br

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Real time reverse transcription-polymerase chain reaction. Analysis of phospholipase (PL) gene ( plb 1, plc , and pld ) transcript level during Paracoccidioides brasiliensis dimorphic mycelium-to-yeast (M-Y) transition. Mycelial cells grown for 48 h at 26ºC were submitted to temperature shift to 35ºC to induce their differentiation into yeast cells. Samples were collected at 1 h, 2 h, 6 h, 12 h, and 24 h and yeast cells grown at 35ºC. Results are expressed as means and standard error (SEM) of three technical replicates. RNA was isolated from pooled three independent experiments. Î"CT values were used for statistical analysis (Livak & Schmittgen 2001) using P. brasiliensis Î±-tubulin gene as the internal control {**: p < 0.01 (ANOVA, Tukey-Kramer test)} (A). P. brasiliensis yeast cells 6 h after MH-S cell interaction. In all experiments, MH-S cells and opsonised yeast cells were incubated at a yeast-to-macrophage ratio of 1:5 at 37ºC in an atmosphere of 5% CO ₂ , as described. Results are expressed as means and SEM of three technical replicates. RNA was isolated from pooled three independent experiments. The expression of each target gene is presented as fold change normalised to the reference gene Î±-tubulin and relative to the respective yeast PL genes obtain from yeast cells grown on Fava-Netto medium at 35ºC (B). P. brasiliensis yeast cells during BALB/c mice lung infection. Results are expressed as the means and SEM of three technical replicates. Five-10 mice per time point were used. The expression of each target gene is presented as fold change normalised to the reference gene Î±-tubulin and relative to the respective yeast PL genes obtain from yeast cells grown on Fava-Netto medium at 35ºC {**: p < 0.01 (ANOVA, Tukey-Kramer test)} (C).

Real time reverse transcription-polymerase chain reaction. Analysis of phospholipase (PL) gene ( plb 1, plc , and pld ) transcript level during Paracoccidioides brasiliensis dimorphic mycelium-to-yeast (M-Y) transition. Mycelial cells grown for 48 h at 26ºC were submitted to temperature shift to 35ºC to induce their differentiation into yeast cells. Samples were collected at 1 h, 2 h, 6 h, 12 h, and 24 h and yeast cells grown at 35ºC. Results are expressed as means and standard error (SEM) of three technical replicates. RNA was isolated from pooled three independent experiments. Î"CT values were used for statistical analysis (Livak & Schmittgen 2001) using P. brasiliensis Î±-tubulin gene as the internal control {: p < 0.01 (ANOVA, Tukey-Kramer test)} (A). P. brasiliensis yeast cells 6 h after MH-S cell interaction. In all experiments, MH-S cells and opsonised yeast cells were incubated at a yeast-to-macrophage ratio of 1:5 at 37ºC in an atmosphere of 5% CO ₂ , as described. Results are expressed as means and SEM of three technical replicates. RNA was isolated from pooled three independent experiments. The expression of each target gene is presented as fold change normalised to the reference gene Î±-tubulin and relative to the respective yeast PL genes obtain from yeast cells grown on Fava-Netto medium at 35ºC (B). P. brasiliensis yeast cells during BALB/c mice lung infection. Results are expressed as the means and SEM of three technical replicates. Five-10 mice per time point were used. The expression of each target gene is presented as fold change normalised to the reference gene Î±-tubulin and relative to the respective yeast PL genes obtain from yeast cells grown on Fava-Netto medium at 35ºC {: p < 0.01 (ANOVA, Tukey-Kramer test)} (C).

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Phospholipase gene expression during <i>Paracoccidioides brasiliensis</i> morphological transition and infection

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[1] + Corresponding author: petrofez@uol.com.br