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Transcriptional profile of Paracoccidioides spp. in response to itraconazole.

da Silva Neto BR, Carvalho PF, Bailão AM, Martins WS, Soares CM, Pereira M - BMC Genomics (2014)

Bottom Line: Among the genes affected, we identified genes in common with other fungi, as well as genes unique to Paracoccidioides Pb01.Voltage-gated Ca2+ alpha subunit (CAV), Tetracycline resistance protein (TETA) and Hemolisyn-iii channel protein (HLYiii) were found only here and a probably involvement with resistance to itraconazole could be investigated in the future.However our findings do not permit inference to current clinical practice.

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: Itraconazole is currently used to treat paracoccidioidomycosis. The mechanism of action of azoles has been elucidated in some fungi, although little is known regarding its mechanism of action in Paracoccidioides spp. The present work focused on identification of regulated transcripts using representational difference analysis of Paracoccidioides spp. yeast cells treated with itraconazole for 1 and 2 h.

Results: Paracoccidioides Pb01 genes up-regulated by itraconazole included genes involved in cellular transport, metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6, ERG3, ERG5 and ERG25 were up-regulated at multiple time points. In vivo infection experiments in mice corroborated the in vitro results. Ergosterol levels and distribution were evaluated in Paracoccidioides Pb18 yeast cells, and the results demonstrate that both factors were changed in the fungus treated with itraconazole.

Conclusion: To our knowledge, this is the first transcriptional analysis of Paracoccidioides spp. exposed to a triazole drug. Here acetyl seems to be intensively produced from different metabolic pathways to produce ergosterol by the action of ergosterol synthesis related enzymes, which were also affected in other fungi. Among the genes affected, we identified genes in common with other fungi, as well as genes unique to Paracoccidioides Pb01. Those genes could be considered target to new drugs. Voltage-gated Ca2+ alpha subunit (CAV), Tetracycline resistance protein (TETA) and Hemolisyn-iii channel protein (HLYiii) were found only here and a probably involvement with resistance to itraconazole could be investigated in the future. However our findings do not permit inference to current clinical practice.

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Related in: MedlinePlus

Functional classification of genes responding to itraconazole in Paracoccidioides. cDNAs obtained from RNAs from yeast cells after incubation with itraconazole for 1 h (A) and 2 h (B). The numbers of ESTs are indicated with white bar segments for the up-regulated genes and black bar segments for the down-regulated genes. The annotation of genes was performed using the Blast2GO program with a cut-off for significant homology of ≤ 1e-5. Sequences were grouped into functional categories according to their classification in the MIPS functional catalog. Additionally, sequences were grouped into functional categories using the PEDANT 3 database. Each functional class is represented as a segment and expressed as a number of ESTs in each library.
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Figure 1: Functional classification of genes responding to itraconazole in Paracoccidioides. cDNAs obtained from RNAs from yeast cells after incubation with itraconazole for 1 h (A) and 2 h (B). The numbers of ESTs are indicated with white bar segments for the up-regulated genes and black bar segments for the down-regulated genes. The annotation of genes was performed using the Blast2GO program with a cut-off for significant homology of ≤ 1e-5. Sequences were grouped into functional categories according to their classification in the MIPS functional catalog. Additionally, sequences were grouped into functional categories using the PEDANT 3 database. Each functional class is represented as a segment and expressed as a number of ESTs in each library.

Mentions: A total of 86 genes were differentially expressed upon exposure to itraconazole, of which 55 were up-regulated and 31 were down-regulated. ESTs obtained from 1 h treatment with itraconazole were clustered into functional classes which were defined as metabolism/energy (26.12%); transcription (17.09%); cell rescue, defense and virulence (10.32%); protein synthesis and biogenesis (2.90%); protein fate (2.90%); cellular transport (23.87%); biogenesis of cellular components (1.61%); cellular communication (1.29%); and unclassified proteins (13.87%). ESTs from 2 h samples were clustered into functional classes which were defined as: metabolism/energy (11.61%); transcription (19.5%); cell rescue, defense and virulence (20.74%); protein synthesis and biogenesis (0.82%); protein fate (0.41%); cellular transport (41.90%); and unclassified protein (4.97%) (Figure 1).


Transcriptional profile of Paracoccidioides spp. in response to itraconazole.

da Silva Neto BR, Carvalho PF, Bailão AM, Martins WS, Soares CM, Pereira M - BMC Genomics (2014)

Functional classification of genes responding to itraconazole in Paracoccidioides. cDNAs obtained from RNAs from yeast cells after incubation with itraconazole for 1 h (A) and 2 h (B). The numbers of ESTs are indicated with white bar segments for the up-regulated genes and black bar segments for the down-regulated genes. The annotation of genes was performed using the Blast2GO program with a cut-off for significant homology of ≤ 1e-5. Sequences were grouped into functional categories according to their classification in the MIPS functional catalog. Additionally, sequences were grouped into functional categories using the PEDANT 3 database. Each functional class is represented as a segment and expressed as a number of ESTs in each library.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3975141&req=5

Figure 1: Functional classification of genes responding to itraconazole in Paracoccidioides. cDNAs obtained from RNAs from yeast cells after incubation with itraconazole for 1 h (A) and 2 h (B). The numbers of ESTs are indicated with white bar segments for the up-regulated genes and black bar segments for the down-regulated genes. The annotation of genes was performed using the Blast2GO program with a cut-off for significant homology of ≤ 1e-5. Sequences were grouped into functional categories according to their classification in the MIPS functional catalog. Additionally, sequences were grouped into functional categories using the PEDANT 3 database. Each functional class is represented as a segment and expressed as a number of ESTs in each library.
Mentions: A total of 86 genes were differentially expressed upon exposure to itraconazole, of which 55 were up-regulated and 31 were down-regulated. ESTs obtained from 1 h treatment with itraconazole were clustered into functional classes which were defined as metabolism/energy (26.12%); transcription (17.09%); cell rescue, defense and virulence (10.32%); protein synthesis and biogenesis (2.90%); protein fate (2.90%); cellular transport (23.87%); biogenesis of cellular components (1.61%); cellular communication (1.29%); and unclassified proteins (13.87%). ESTs from 2 h samples were clustered into functional classes which were defined as: metabolism/energy (11.61%); transcription (19.5%); cell rescue, defense and virulence (20.74%); protein synthesis and biogenesis (0.82%); protein fate (0.41%); cellular transport (41.90%); and unclassified protein (4.97%) (Figure 1).

Bottom Line: Among the genes affected, we identified genes in common with other fungi, as well as genes unique to Paracoccidioides Pb01.Voltage-gated Ca2+ alpha subunit (CAV), Tetracycline resistance protein (TETA) and Hemolisyn-iii channel protein (HLYiii) were found only here and a probably involvement with resistance to itraconazole could be investigated in the future.However our findings do not permit inference to current clinical practice.

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: Itraconazole is currently used to treat paracoccidioidomycosis. The mechanism of action of azoles has been elucidated in some fungi, although little is known regarding its mechanism of action in Paracoccidioides spp. The present work focused on identification of regulated transcripts using representational difference analysis of Paracoccidioides spp. yeast cells treated with itraconazole for 1 and 2 h.

Results: Paracoccidioides Pb01 genes up-regulated by itraconazole included genes involved in cellular transport, metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6, ERG3, ERG5 and ERG25 were up-regulated at multiple time points. In vivo infection experiments in mice corroborated the in vitro results. Ergosterol levels and distribution were evaluated in Paracoccidioides Pb18 yeast cells, and the results demonstrate that both factors were changed in the fungus treated with itraconazole.

Conclusion: To our knowledge, this is the first transcriptional analysis of Paracoccidioides spp. exposed to a triazole drug. Here acetyl seems to be intensively produced from different metabolic pathways to produce ergosterol by the action of ergosterol synthesis related enzymes, which were also affected in other fungi. Among the genes affected, we identified genes in common with other fungi, as well as genes unique to Paracoccidioides Pb01. Those genes could be considered target to new drugs. Voltage-gated Ca2+ alpha subunit (CAV), Tetracycline resistance protein (TETA) and Hemolisyn-iii channel protein (HLYiii) were found only here and a probably involvement with resistance to itraconazole could be investigated in the future. However our findings do not permit inference to current clinical practice.

Show MeSH
Related in: MedlinePlus