ABSTRACT
AIM: Candida glabrata is a major pathogen in humans known to be intrinsically resistant to fluconazole. However, genotypic, phenotypic, and proteomic changes associated with reduced susceptibility to fluconazole are not properly understood. The aim of this study was to observe specific phenotypic, chromosomal, and proteomic alterations in a Candida glabrata strain sequentially exposed to fluconazole. METHODS: Candida glabrata was exposed to increased concentrations of fluconazole in RPMI for 55 days. Phenotypic changes were evaluated using standard assays. Molecular/proteomic changes in C. glabrata were analyzed by contour-clamped homogeneous electric field electrophoresis, reverse transcription-polymerase chain reaction, and mass spectrometry. RESULTS: Candida glabrata demonstrated increased fluconazole resistance (>256 µg/mL), with extensive cross-resistance to ketoconazole (0.38-3.0 µg), itraconazole (8 to >32 µg), and voriconazole (0.125-1.5 µg). Morphologically dissimilar colonies on RPMI/fluconazole agar demonstrated variable chromosomal profiles compared with the control isolate. Stable chromosomal changes were associated with a significantly higher (P<0.05) mRNA level of the hemolysin gene compared with the control. Phenotypic switching on CuSO4 agar was associated with variable metallothionein mRNA transcription levels. The proteome analysis of a fluconazole-resistant offshoot demonstrated a total of 98 protein spots, 25 showing a twofold upregulation. CONCLUSION: Fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits.
Subject(s)
Antifungal Agents/pharmacology , Candida glabrata/genetics , Fluconazole/pharmacology , Fungal Proteins/drug effects , Proteome/drug effects , Antifungal Agents/administration & dosage , Candida glabrata/drug effects , Candida glabrata/pathogenicity , Chromosomes, Fungal/genetics , Copper Sulfate/pharmacology , Culture Media , Drug Resistance, Fungal/genetics , Fluconazole/administration & dosage , Fungal Proteins/genetics , Genotype , Hemolysin Proteins/drug effects , Hemolysin Proteins/genetics , Humans , Itraconazole/administration & dosage , Itraconazole/pharmacology , Ketoconazole/administration & dosage , Ketoconazole/pharmacology , Metallothionein/drug effects , Metallothionein/genetics , Phenotype , Proteome/genetics , Virulence , Voriconazole/administration & dosage , Voriconazole/pharmacologyABSTRACT
OBJECTIVE: To identify genes expressed in the fetal heart that are potentially important for myocardial development and cardiomyocyte proliferation. METHODS: mRNAs from fetal (29 weeks) and adult cardiomyocytes were use for suppression subtractive hybridization (SSH). Both forward (fetal as tester) and reverse (adult as driver) subtractions were performed. Clones confirmed by dot-blot analysis to be differentially expressed were sequenced and analyzed. RESULTS: Differential expressions were detected for 39 out of 96 (41%) clones on forward subtraction and 24 out of 80 (30%) clones on reverse. For fetal dominating genes, 28 clones matched to 10 known genes (COL1A2, COL3A1, endomucin, HBG1, HBG2, PCBP2, LOC51144, TGFBI, vinculin and PND), 9 clones to 5 cDNAs of unknown functions (accession AK021715, AF085867, AB040948, AB051460 and AB051512) and 2 clones had homology to hEST sequences. For the reverse subtraction, all clones showed homology to mitochondrial transcripts. CONCLUSIONS: We successfully applied SSH to detect those genes differentially expressed in fetal cardiac myocytes, some of which have not been shown relative to myocardial development.
