- Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata
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Jae Il Yoo, Hwa Su Kim, Chi Won Choi, Jung Sik Yoo, Jae Yon Yu, Yeong Seon Lee
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Osong Public Health Res Perspect. 2013;4(6):293-300. Published online December 31, 2013
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DOI: https://doi.org/10.1016/j.phrp.2013.10.001
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 Abstract
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- Objectives
The proteomic analysis of voriconazole resistant Candida glabrata strain has not yet been investigated. In this study, differentially expressed proteins of intracellular and membrane fraction from voriconazole-susceptible, susceptible dose-dependent (S-DD), resistant C. glabrata strains were compared with each other and several proteins were identified.
Methods
The proteins of intracellular and membrane were isolated by disrupting cells with glass bead and centrifugation from voriconazole susceptible, S-DD, and resistant C. glabrata strains. The abundance of expressed proteins was compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteins showing continuous twofold or more increase or reduction of expression in resistant strains compared to susceptible and S-DD strain were analyzed by liquid chromatography/mass spectrometry-mass spectrometry method.
Results
Of 34 intracellular proteins, 15 proteins showed expression increase or reduction (twofold or more). The identified proteins included regulation, energy production, carbohydrate transport, amino acid transport, and various metabolism related proteins. The increase of expression of heat shock protein 70 was found. Among membrane proteins, 12, 31 proteins showed expression increase or decrease in the order of susceptible, S-DD, and resistant strains. This expression included carbohydrate metabolism, amino acid synthesis, and response to stress-related proteins. In membrane fractions, the change of expression of 10 heat shock proteins was observed, and 9 heat shock protein 70 (Hsp70) showed the reduction of expression.
Conclusion
The expression of Hsp70 protein in membrane fraction is related to voriconazole resistant C. glabrata strains.
- Proteomic Analysis of Cellular and Membrane Proteins in Fluconazole-Resistant Candida glabrata
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Jae Il Yoo, Chi Won Choi, Hwa Su Kim, Jung Sik Yoo, Young Hee Jeong, Yeong Seon Lee
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Osong Public Health Res Perspect. 2012;3(2):74-78. Published online June 30, 2012
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DOI: https://doi.org/10.1016/j.phrp.2012.04.001
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1,408
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Abstract
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- Objectives
Candida glabrata is one of the most common causes of Candida bloodstream infections worldwide. Some isolates of C glabrata may be intermediately resistant to azoles, with some strains developing resistance during therapy or prophylaxis with fluconazole. In this study, we used a proteomic approach to identify differentially expressed proteins between fluconazoleresistant and -susceptible strains.
Methods
Membrane and cellular proteins were extracted from fluconazolesusceptible and fluconazole-resistant C glabrata strains. Differentially expressed proteins were compared using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with >1.5-fold difference in expression were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS).
Results
A total of 65 proteins were differentially expressed in the cellular and membrane fractions. Among the 39 cellular proteins, 11 were upregulated and 28 were downregulated in fluconazole-resistant strains in comparison with fluconazole-susceptible strains. In the membrane fraction, a total of 26 proteins were found, of which 19 were upregulated and seven were downregulated. A total of 31 proteins were identified by LC-MS/MS that are involved in glycolysis, carbohydrate transport, energy transfer, and other metabolic pathways. Heat shock proteins were identified in various spots.
Conclusion
Heat shock and stress response proteins were upregulated in the membrane fraction of the fluconazole-resistant C glabrata strain. Compared with susceptible strains, fluconazole-resistant strains showed increased expression of membrane proteins and decreased expression of cellular proteins.
- Gene Expression and Identification Related to Fluconazole Resistance of Candida glabrata Strains
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Jae Il Yoo, Chi Won Choi, Kyeong Min Lee, Yeong Seon Lee
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Osong Public Health Res Perspect. 2010;1(1):36-41. Published online December 31, 2010
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DOI: https://doi.org/10.1016/j.phrp.2010.12.009
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1,342
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Abstract
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- Objectives
Candida glabrata has become one of the most common causes of Candida bloodstream infections worldwide. Some strains of C. glabrata may be intermediately resistant to all azoles. The several possible mechanisms of azole resistance have been reported previously, but the exact resistant mechanism is not clear. In this study, we identified differentially expressed genes (DEGs) of fluconazole-resistant C. glabrata and compared the gene expression of fluconazole-resistant strains with that of fluconazole-susceptible strains to identify gene corresponding to fluconazole resistance.
Methods
Using antifungal susceptibility test, several C. glabrata strains were selected and used for further study. The expression of CgCDR1 and CgCDR2 genes was investigated by slot hybridization against fluconazole-susceptible, -resistant, and resistant-induced strains. In addition, ERG3 and ERG11 genes were sequenced to analyze DNA base substitution. DEGs were identified by reverse transcription-polymerase chain reaction using DEG kit composed of 120 random primers.
Results
In slot hybridization, CgCDR1 gene was expressed more than CgCDR2 gene in resistant strains. Though base substitution of ERG11 and ERG3 genes was observed in several base sequences, just one amino acid change was identified in resistant strain. In the results of reverse transcription-polymerase chain reaction, 44 genes were upregulated and 34 genes were downregulated. Among them, adenosine triphosphate-binding cassette transporter-related genes, fatty acid desaturase, lyase, and hypothetical protein genes were upregulated and aldehyde dehydrogenase, oxidoreductase, and prohibitin-like protein genes were downregulated. Other DEGs were also identified.
Conclusion
This study showed that CgCDR1 gene was more closely related to fluconazole resistance of C. glabrata than CgCDR2 gene. In addition, several other genes related with fluconazole resistance of C. glabrata were identified.
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