title: Genotypic, phenotypic, and proteomic characterization of Candida glabrata during sequential fluconazole exposure.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  Fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits.
authors.affiliation: Department of Oral Biosciences, Faculty of Dentistry, University of Hong Kong, Hong Kong, China.
authors.forename: Yuthika Hemamala
authors.initials: YH
authors.lastname: Samaranayake
authors.forename: Becky Pik Ki
authors.initials: BP
authors.lastname: Cheung
authors.forename: Joyce Yick Yee
authors.initials: JY
authors.lastname: Yau
authors.forename: Kim Wai
authors.initials: KW
authors.lastname: Yeung
authors.forename: Lakshman Perera
authors.initials: LP
authors.lastname: Samaranayake
mesh_terms: D000935:Antifungal Agents / Q000008:administration & dosage / Q000494:pharmacology*; D041221:Candida glabrata / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity; D015825:Chromosomes, Fungal / Q000235:genetics; D019327:Copper Sulfate / Q000494:pharmacology; D003470:Culture Media; D025141:Drug Resistance, Fungal / Q000235:genetics; D015725:Fluconazole / Q000008:administration & dosage / Q000494:pharmacology*; D005656:Fungal Proteins / Q000187:drug effects* / Q000235:genetics; D005838:Genotype; D006460:Hemolysin Proteins / Q000187:drug effects / Q000235:genetics; D006801:Humans; D017964:Itraconazole / Q000008:administration & dosage / Q000494:pharmacology; D007654:Ketoconazole / Q000008:administration & dosage / Q000494:pharmacology; D008668:Metallothionein / Q000187:drug effects / Q000235:genetics; D010641:Phenotype; D020543:Proteome / Q000187:drug effects* / Q000235:genetics; D014774:Virulence; D065819:Voriconazole / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D003470:Culture Media; D005656:Fungal Proteins; D006460:Hemolysin Proteins; D020543:Proteome; D017964:Itraconazole; D015725:Fluconazole; D008668:Metallothionein; D065819:Voriconazole; D019327:Copper Sulfate; D007654:Ketoconazole
keywords: Candida; fluconazole resistance; hemolysin; metallothionein; virulence