Virulence associated with fluconazole (FL) resistance in Candida glabrata is a global problem and has not been well characterized at the proteome level. In this study, a stable FL-resistant (MIC >256 µg ml−1) strain of C. glabrata was generated on agar containing FL. Eight phenotypic mutants were characterized by contour-clamped homogeneous electrophoretic field analysis and two-dimensional PAGE. The secondary derivatives of C. glabrata yielded four distinct genotypes with varying chromosomal profiles. Proteomic analysis performed by tandem mass spectrometry for two of the mutants, CGL2 and CGS3, demonstrated a total of 25 differentially regulated proteins of which 13 were upregulated and 12 were downregulated or were similar compared with the parental isolate. The mRNA transcript levels of significantly (P<0.001) upregulated genes were determined by quantitative RT-PCR analysis, and their physiological relevance in terms of phenotypic expression of virulence attributes was verified by conventional laboratory methodologies. The data showed that the FL resistance (MIC >256 µg ml−1) of CGS3 was associated with significantly upregulated (P<0.001) mRNA transcript levels of several genes – ERG11, CDR1, CDR2, MFS, MTI, TPR, VPS and EFT2 – in addition to a number of other potentially virulent genes expressed differentially at a lower level. The results demonstrated accentuated phenotypic expression of bud formation of yeast and metallothionein production associated with FL resistance in C. glabrata, which may help the fungus to colonize the host.
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
CasanovaM.,
ChaffinW. L.1991; Cell wall glycoproteins of Candida albicans as released by different methods. J Gen Microbiol 137:1045–1051 [View Article][PubMed]
DograS.,
KrishnamurthyS.,
GuptaV.,
DixitB. L.,
GuptaC. M.,
SanglardD.,
PrasadR.1999; Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily. Yeast 15:111–121 [View Article][PubMed]
FidelP. L.Jr,
VazquezJ. A.,
SobelJ. D.1999; Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans
. Clin Microbiol Rev 12:80–96[PubMed]
GeberA.,
HitchcockC. A.,
SwartzJ. E.,
PullenF. S.,
MarsdenK. E.,
Kwon-ChungK. J.,
BennettJ. E.1995; Deletion of the Candida glabrataERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility. Antimicrob Agents Chemother 39:2708–2717 [View Article][PubMed]
GygiS. P.,
CorthalsG. L.,
ZhangY.,
RochonY.,
AebersoldR.2000; Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci U S A 97:9390–9395 [View Article][PubMed]
HachemR.,
HannaH.,
KontoyiannisD.,
JiangY.,
RaadI.2008; The changing epidemiology of invasive candidiasis: Candidaglabrata and Candida krusei as the leading causes of candidemia in hematologic malignancy. Cancer 112:2493–2499 [View Article][PubMed]
HajjehR. A.,
SofairA. N.,
HarrisonL. H.,
LyonG. M.,
Arthington-SkaggsB. A.,
MirzaS. A.,
PhelanM.,
MorganJ.,
Lee-YangW.other authors2004; Incidence of bloodstream infections due to Candida species and in vitro susceptibilities of isolates collected from 1998 to 2000 in a population-based active surveillance program. J Clin Microbiol 42:1519–1527 [View Article][PubMed]
HenryK. W.,
NickelsJ. T.,
EdlindT. D.2000; Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors. Antimicrob Agents Chemother 44:2693–2700 [View Article][PubMed]
HilmiogluS.,
IlkitM.,
BadakZ.2007; Comparison of 12 liquid media for germ tube production of Candida albicans and C. tropicalis
. Mycoses 50:282–285 [View Article][PubMed]
JinY.,
YipH. K.,
SamaranayakeY. H.,
YauJ. Y.,
SamaranayakeL. P.2003; Biofilm-forming ability of Candida albicans is unlikely to contribute to high levels of oral yeast carriage in cases of human immunodeficiency virus infection. J Clin Microbiol 41:2961–2967 [View Article][PubMed]
KatiyarS. K.,
EdlindT. D.2001; Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei
. Med Mycol 39:109–116[PubMed][CrossRef]
KuschH.,
EngelmannS.,
BodeR.,
AlbrechtD.,
MorschhäuserJ.,
HeckerM.2008; A proteomic view of Candida albicans yeast cell metabolism in exponential and stationary growth phases. Int J Med Microbiol 298:291–318 [View Article][PubMed]
LachkeS. A.,
SrikanthaT.,
TsaiL. K.,
DanielsK.,
SollD. R.2000; Phenotypic switching in Candida glabrata involves phase-specific regulation of the metallothionein gene MT-II and the newly discovered hemolysin gene HLP
. Infect Immun 68:884–895 [View Article][PubMed]
LawI. K. M.,
LiuL.,
XuA.,
LamK. S.,
VanhoutteP. M.,
CheC.-M.,
LeungP. T.,
WangY.2009; Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti-aging and metabolic effects of sirtuins. Proteomics 9:2444–2456 [View Article][PubMed]
Lopez-RibotJ. L.,
McAteeR. K.,
LeeL. N.,
KirkpatrickW. R.,
WhiteT. C.,
SanglardD.,
PattersonT. F.1998; Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother 42:2932–2937[PubMed]
LuoG.,
SamaranayakeL. P.,
CheungB. P. K.,
TangG.2004; Reverse transcriptase polymerase chain reaction (RT-PCR) detection of HLP gene expression in Candida glabrata and its possible role in in vitro haemolysin production. APMIS 112:283–290 [View Article][PubMed]
MarrK. A.,
CarterR. A.,
CrippaF.,
WaldA.,
CoreyL.2002; Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis 34:909–917 [View Article][PubMed]
MehraR. K.,
GareyJ. R.,
ButtT. R.,
GrayW. R.,
WingeD. R.1989; Candida glabrata metallothioneins. Cloning and sequence of the genes and characterization of proteins. J Biol Chem 264:19747–19753[PubMed]
MiyazakiH.,
MiyazakiY.,
GeberA.,
ParkinsonT.,
HitchcockC.,
FalconerD. J.,
WardD. J.,
MarsdenK.,
BennettJ. E.1998; Fluconazole resistance associated with drug efflux and increased transcription of a drug transporter gene, PDH1, in Candida glabrata
. Antimicrob Agents Chemother 42:1695–1701[PubMed]
NiimiM.,
NagaiY.,
NiimiK.,
WadaS.,
CannonR. D.,
UeharaY.,
MonkB. C.2002; Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole. J Chromatogr B Analyt Technol Biomed Life Sci 782:245–252 [View Article][PubMed]
OgurM.,
St JohnR.,
NagaiS.1957; Tetrazolium overlay technique for population studies of respiration deficiency in yeast. Science 125:928–929 [View Article][PubMed]
PfallerM. A.,
DiekemaD. J.International Fungal Surveillance Participant Group2004; Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida
. Clin Microbiol Infect 10:Suppl. 111–23 [View Article][PubMed]
PfallerM. A.,
DiekemaD. J.,
RexJ. H.,
Espinel-IngroffA.,
JohnsonE. M.,
AndesD.,
ChaturvediV.,
GhannoumM. A.,
OddsF. C.other authors2006; Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints. J Clin Microbiol 44:819–826 [View Article][PubMed]
PitarchA.,
SánchezM.,
NombelaC.,
GilC.2003; Analysis of the Candida albicans proteome. II. Protein information technology on the Net (update 2002). J Chromatogr B Analyt Technol Biomed Life Sci 787:129–148 [View Article][PubMed]
PosteraroB.,
SanguinettiM.,
SanglardD.,
La SordaM.,
BocciaS.,
RomanoL.,
MoraceG.,
FaddaG.2003; Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole. Mol Microbiol 47:357–371 [View Article][PubMed]
RaadI.,
HannaH.,
BoktourM.,
GirgawyE.,
DanawiH.,
MardaniM.,
KontoyiannisD.,
DarouicheR.,
HachemR.,
BodeyG. P.2004; Management of central venous catheters in patients with cancer and candidemia. Clin Infect Dis 38:1119–1127 [View Article][PubMed]
ReddingS. W.,
KirkpatrickW. R.,
SavilleS.,
CocoB. J.,
WhiteW.,
FothergillA.,
RinaldiM.,
EngT.,
PattersonT. F.,
Lopez-RibotJ.2003; Multiple patterns of resistance to fluconazole in Candida glabrata isolates from a patient with oropharyngeal candidiasis receiving head and neck radiation. J Clin Microbiol 41:619–622 [View Article][PubMed]
RobertsR. L.,
MöschH. U.,
FinkG. R.1997; 14-3-3 proteins are essential for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae
. Cell 89:1055–1065 [View Article][PubMed]
SafdarA.,
ChaturvediV.,
CrossE. W.,
ParkS.,
BernardE. M.,
ArmstrongD.,
PerlinD. S.2001; Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob Agents Chemother 45:2129–2133 [View Article][PubMed]
SafdarA.,
ChaturvediV.,
KollB. S.,
LaroneD. H.,
PerlinD. S.,
ArmstrongD.2002; Prospective, multicenter surveillance study of Candida glabrata: fluconazole and itraconazole susceptibility profiles in bloodstream, invasive, and colonizing strains and differences between isolates from three urban teaching hospitals in New York City (Candida Susceptibility Trends Study, 1998 to 1999). Antimicrob Agents Chemother 46:3268–3272 [View Article][PubMed]
SaimanL.,
LudingtonE.,
PfallerM.,
Rangel-FraustoS.,
WiblinR. T.,
DawsonJ.,
BlumbergH. M.,
PattersonJ. E.,
RinaldiM.other authors2000; Risk factors for candidemia in neonatal intensive care unit patients. The National Epidemiology of Mycosis Survey study group. Pediatr Infect Dis J 19:319–324 [View Article][PubMed]
SamaranayakeY. H.,
DassanayakeR. S.,
JayatilakeJ. A. M. S.,
CheungB. P. K.,
YauJ. Y. Y.,
YeungK. W. S.2005; Phospholipase B enzyme expression is not associated with other virulence attributes in Candida albicans isolates from patients with human immunodeficiency virus infection. J Med Microbiol 54:583–593 [View Article][PubMed]
SamaranayakeY. H.,
DassanayakeR. S.,
CheungB. P. K.,
JayatilakeJ. A. M. S.,
YeungK. W. S.,
YauJ. Y.,
SamaranayakeL. P.2006; Differential phospholipase gene expression by Candida albicans in artificial media and cultured human oral epithelium. APMIS 114:857–866 [View Article][PubMed]
SanglardD.,
IscherF.,
CalabreseD.,
MajcherczykP. A.,
BilleJ.1999; The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob Agents Chemother 43:2753–2765[PubMed]
SanguinettiM.,
PosteraroB.,
FioriB.,
RannoS.,
TorelliR.,
FaddaG.2005; Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance. Antimicrob Agents Chemother 49:668–679 [View Article][PubMed]
TramaJ. P.,
MordechaiE.,
AdelsonM. E.2005; Detection and identification of Candida species associated with Candida vaginitis by real-time PCR and pyrosequencing. Mol Cell Probes 19:145–152 [View Article][PubMed]
VicenteF.,
BasilioA.,
PlatasG.,
ColladoJ.,
BillsG. F.,
González del ValA.,
MartínJ.,
TormoJ. R.,
HarrisG. H.other authors2009; Distribution of the antifungal agents sordarins across filamentous fungi. Mycol Res 113:754–770 [View Article][PubMed]
WhiteT. C.1997; Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob Agents Chemother 41:1482–1487[PubMed]
YanJ. X.,
WaitR.,
BerkelmanT.,
HarryR. A.,
WestbrookJ. A.,
WheelerC. H.,
DunnM. J.2000; A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672 [View Article][PubMed]