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Transcriptional profile of Paracoccidioides induced by oenothein B, a potential antifungal agent from the Brazilian Cerrado plant Eugenia uniflora.

Zambuzzi-Carvalho PF, Tomazett PK, Santos SC, Ferri PH, Borges CL, Martins WS, de Almeida Soares CM, Pereira M - BMC Microbiol. (2013)

Bottom Line: Infection experiments in macrophages corroborated the in vitro results.Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment.The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICBII, Campus II, Universidade Federal de Goiás, C,P, 131, 74001-970 Goiânia, GO, Brazil. maristelaufg@gmail.com.

ABSTRACT

Background: The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-β-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress.

Results: We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment.

Conclusions: The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus.

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Carbohydrate content in the cell wall of Paracoccidioides yeast cells exposed to OenB. The cell walls of Paracoccidioides that were either untreated (control) or treated with OenB were isolated. The alkali-soluble (AS) and alkali-insoluble (AI) fractions were separated. (A) Total amount of carbohydrates was measured in the AS and AI fractions. (B) The amount of 1,3-β-D-glucan polymer was estimated in the AI fraction. (C) The amount of N-acetylglucosamine (GlcNAc) residue was measured in the total cell wall fraction. All data were normalized relative to the control. Three independent experiments were performed. *Significantly increased amount (p ≤ 0.05).
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Figure 5: Carbohydrate content in the cell wall of Paracoccidioides yeast cells exposed to OenB. The cell walls of Paracoccidioides that were either untreated (control) or treated with OenB were isolated. The alkali-soluble (AS) and alkali-insoluble (AI) fractions were separated. (A) Total amount of carbohydrates was measured in the AS and AI fractions. (B) The amount of 1,3-β-D-glucan polymer was estimated in the AI fraction. (C) The amount of N-acetylglucosamine (GlcNAc) residue was measured in the total cell wall fraction. All data were normalized relative to the control. Three independent experiments were performed. *Significantly increased amount (p ≤ 0.05).

Mentions: The cell wall is divided in alkali-soluble (AS) and alkali-insoluble (AI) fractions according to its solubility in alkaline substances. In the Paracoccidioides Pb01 yeast phase, the glucan polymer consists mainly of 1,3-α-glucan (95%) which is present in the AS fraction, and a small amount of the 1,3-β-D-glucan (5%), which is present in the AI fraction [34]. GlcNAc residues are also found in the AI fraction. Figure 5A shows a decrease in total carbohydrate content in the AI fraction after OenB-treatment; no alteration in carbohydrate content was observed in the AS fraction. 1,3-β-D-glucan content was estimated after the digestion of the AI cell wall fraction with the 1,3-β-glucanase enzyme. As shown in Figure 5B, there was a decrease in 1,3-β-D-glucan content. In addition there was an increase in GlcNAc residue content in this fraction (Figure 5C), suggesting an increase in the chitin polymer concentration.


Transcriptional profile of Paracoccidioides induced by oenothein B, a potential antifungal agent from the Brazilian Cerrado plant Eugenia uniflora.

Zambuzzi-Carvalho PF, Tomazett PK, Santos SC, Ferri PH, Borges CL, Martins WS, de Almeida Soares CM, Pereira M - BMC Microbiol. (2013)

Carbohydrate content in the cell wall of Paracoccidioides yeast cells exposed to OenB. The cell walls of Paracoccidioides that were either untreated (control) or treated with OenB were isolated. The alkali-soluble (AS) and alkali-insoluble (AI) fractions were separated. (A) Total amount of carbohydrates was measured in the AS and AI fractions. (B) The amount of 1,3-β-D-glucan polymer was estimated in the AI fraction. (C) The amount of N-acetylglucosamine (GlcNAc) residue was measured in the total cell wall fraction. All data were normalized relative to the control. Three independent experiments were performed. *Significantly increased amount (p ≤ 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852496&req=5

Figure 5: Carbohydrate content in the cell wall of Paracoccidioides yeast cells exposed to OenB. The cell walls of Paracoccidioides that were either untreated (control) or treated with OenB were isolated. The alkali-soluble (AS) and alkali-insoluble (AI) fractions were separated. (A) Total amount of carbohydrates was measured in the AS and AI fractions. (B) The amount of 1,3-β-D-glucan polymer was estimated in the AI fraction. (C) The amount of N-acetylglucosamine (GlcNAc) residue was measured in the total cell wall fraction. All data were normalized relative to the control. Three independent experiments were performed. *Significantly increased amount (p ≤ 0.05).
Mentions: The cell wall is divided in alkali-soluble (AS) and alkali-insoluble (AI) fractions according to its solubility in alkaline substances. In the Paracoccidioides Pb01 yeast phase, the glucan polymer consists mainly of 1,3-α-glucan (95%) which is present in the AS fraction, and a small amount of the 1,3-β-D-glucan (5%), which is present in the AI fraction [34]. GlcNAc residues are also found in the AI fraction. Figure 5A shows a decrease in total carbohydrate content in the AI fraction after OenB-treatment; no alteration in carbohydrate content was observed in the AS fraction. 1,3-β-D-glucan content was estimated after the digestion of the AI cell wall fraction with the 1,3-β-glucanase enzyme. As shown in Figure 5B, there was a decrease in 1,3-β-D-glucan content. In addition there was an increase in GlcNAc residue content in this fraction (Figure 5C), suggesting an increase in the chitin polymer concentration.

Bottom Line: Infection experiments in macrophages corroborated the in vitro results.Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment.The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, ICBII, Campus II, Universidade Federal de Goiás, C,P, 131, 74001-970 Goiânia, GO, Brazil. maristelaufg@gmail.com.

ABSTRACT

Background: The compound oenothein B (OenB), which is isolated from the leaves of Eugenia uniflora, a Brazilian Cerrado plant, interferes with Paracoccidioides yeast cell morphology and inhibits 1,3-β-D-glucan synthase (PbFKS1) transcript accumulation, which is involved in cell wall synthesis. In this work we examined the gene expression changes in Paracoccidioides yeast cells following OenB treatment in order to investigate the adaptive cellular responses to drug stress.

Results: We constructed differential gene expression libraries using Representational Difference Analysis (RDA) of Paracoccidioides yeast cells treated with OenB for 90 and 180 min. Treatment for 90 min resulted in the identification of 463 up-regulated expressed sequences tags (ESTs) and 104 down-regulated ESTs. For the 180 min treatment 301 up-regulated ESTs and 143 down-regulated were identified. Genes involved in the cell wall biosynthesis, such as GLN1, KRE6 and FKS1, were found to be regulated by OenB. Infection experiments in macrophages corroborated the in vitro results. Fluorescence microscopy showed increased levels of chitin in cells treated with OenB. The carbohydrate polymer content of the cell wall of the fungus was also evaluated, and the results corroborated with the transcriptional data. Several other genes, such as those involved in a variety of important cellular processes (i.e., membrane maintenance, stress and virulence) were found to be up-regulated in response to OenB treatment.

Conclusions: The exposure of Paracoccidioides to OenB resulted in a complex altered gene expression profile. Some of the changes may represent specific adaptive responses to this compound in this important pathogenic fungus.

Show MeSH
Related in: MedlinePlus