The transcription factor CgRPN4 is a determinant of fluconazole resistance in Candida glabrata.
|Drug||MIC (mg/liter) for strain||Category (WT/mutant)|
Transcriptome-wide changes in response to fluconazole stress in C. glabrata.
|Functional group||C. glabrata gene||Description||Log2-fold change|
|WT FLC/WT control||Δcgrpn4 FLC/WT FLC|
|Drug resistance||CDR1||Multidrug transporter of the ABC superfamily, involved in resistance to azoles; expression regulated by Pdr1p; increased abundance in azole-resistant strains; expression increased by loss of the mitochondrial genome||0.81||0.42|
|Sterol metabolism||ERG1b||Squalene epoxidase with role in ergosterol synthesis; involved in growth under conditions of low oxygen tension||0.83||−0.61|
|ERG2b||C-8 sterol isomerase||1.10||−0.71|
|ERG3b||Delta-5,6-sterol desaturase; C-5 sterol desaturase; predicted transmembrane domain and endoplasmic reticulum binding motif; gene used for molecular typing of C. glabrata strain isolates||1.07||−0.63|
|ERG4||Putative C-24 sterol reductase||0.83||−0.46|
|ERG5||Putative C-22 sterol desaturase||0.58||−0.24|
|ERG6||C-24 sterol methyltransferase; mutation confers resistance to amphotericin B and nystatin and increased sensitivity to azoles||0.52||−0.38|
|ERG11b||Putative cytochrome P-450 lanosterol 14-alpha-demethylase; target enzyme of azole antifungal drugs; increased protein abundance in azole-resistant strains||0.96||−0.65|
|ERG24||Ortholog(s) has delta-14-sterol reductase activity and roles in cellular response to drugs, ergosterol biosynthetic processes, filamentous growth of a population of unicellular organisms in response to a biotic stimulus, and pathogenesis||0.53||−0.32|
|ERG25||Ortholog(s) has C-4 methylsterol oxidase activity, role in ergosterol biosynthetic process, and endoplasmic reticulum membrane and plasma membrane localizations||1.02||−0.41|
|ERG29||Ortholog(s) has roles in cellular iron ion homeostasis, ergosterol biosynthetic process, and mitochondrion organization and has endoplasmic reticulum and nuclear envelope localizations||0.84||−0.43|
|HES1b||Ortholog(s) has oxysterol binding, sterol transporter activity, and roles in endocytosis, exocytosis, maintenance of cell polarity, piecemeal microautophagy of the nucleus, and sterol transport||1.48||−0.96|
|CYB5||Ortholog(s) has electron carrier activity, role in ergosterol biosynthetic process, and endoplasmic reticulum membrane localization||0.73||−0.43|
|Lipid and fatty acid metabolism||CSR1b||Ortholog(s) has phosphatidylinositol transporter activity||0.59||−0.90|
|HBN1||Ortholog(s) has oxidoreductase activity acting on NAD(P)H, nitrogenous group as acceptor activity, and roles in cellular response to oxidative stress and negative regulation of fatty acid metabolic process||−0.51||−0.10|
|CAGL0A03740gb||Ortholog(s) has roles in fatty acid beta-oxidation and long-chain fatty acid catabolic processes and has peroxisome localization||−0.53||1.13|
|Stress response||RTA1b||Putative protein involved in 7-aminocholesterol resistance; gene is upregulated in azole-resistant strains||0.80||0.51|
|RAD14b||Ortholog(s) has damaged DNA binding, zinc ion binding activity, and roles in UV damage excision repair, nucleotide excision repair involved in interstrand cross-link repair, nucleotide excision repair, and DNA damage recognition||0.52||−0.53|
|SSA3||Heat shock protein of the HSP70 family||−0.55||0.06|
|Nitrogen metabolism||PUT1b||Ortholog(s) has proline dehydrogenase activity, role in the proline-catabolic process to glutamate, and mitochondrion localization||0.57||0.72|
|MEP2||Ortholog(s) has high-affinity secondary active ammonium transmembrane transporter activity and methylammonium transmembrane transporter activity||−0.50||−0.04|
|Carbon metabolism||PBI1b||Has domain(s) with predicted alcohol O-acetyltransferase activity and role in alcohol metabolic process||2.11||−1.93|
|ATF2||Putative alcohol acetyltransferase involved in steroid detoxification; gene is upregulated in azole-resistant strains||0.70||−0.09|
|MLS1b||Ortholog(s) has malate synthase activity; roles in acetate catabolic process, carbon utilization, fatty acid catabolic process, and glyoxylate cycle; and cytosol, glyoxysome, and peroxisomal matrix localizations||−0.53||0.74|
|Heme biosynthesis||HEM13b||Putative coproporphyrinogen III oxidase; protein differentially expressed in azole-resistant strains||0.81||−0.67|
|HEM14b||Ortholog(s) has oxygen-dependent protoporphyrinogen oxidase activity, role in heme biosynthetic process; and cytosol, mitochondrial inner membrane, and nucleus localizations||0.51||−0.70|
|HMX1||Ortholog(s) has heme oxygenase (decyclizing) activity and roles in cellular iron ion homeostasis, heme catabolic process, response to carbon monoxide, and response to oxidative stress||−0.62||0.40|
|Cytoskeleton/cell cycle||MSC7b||Ortholog(s) has role in reciprocal meiotic recombination and cytosol, endoplasmic reticulum, and nucleus localizations||0.88||−0.56|
|NCE102||Ortholog(s) has roles in actin cytoskeleton organization, eisosome assembly, establishment of protein localization to the plasma membrane, negative regulation of protein phosphorylation, and more||−0.55||0.43|
|XBP1||Ortholog(s) has RNA polymerase II transcription factor activity, sequence-specific DNA binding, and sequence-specific DNA binding activity||−0.69||0.47|
|FMP45||Ortholog(s) has roles in ascospore formation and cellular response to drugs and has fungal-type cell wall organization||−0.69||0.47|
|Mitochondrial function||COX26||Ortholog(s) has mitochondrial respiratory chain complex IV and mitochondrial respiratory chain supercomplex localization||−0.64||0.12|
|Intracellular traffic||SRO7||Ortholog(s) has Rab GTPase binding and SNARE binding activities and roles in Golgi-to-plasma membrane transport, establishment of cell polarity, exocytosis, and small GTPase-mediated signal transduction||0.51||−0.09|
|Unknown function||CAGL0M11660gb||Has domain(s) with predicted hydrolase activity||0.67||1.81|
|CAGL0G05632gb||Ortholog(s) has cytoplasm localization||−0.52||1.32|
|CAGL0K07337gb||Has domain(s) with predicted ion channel activity, role in ion transport, and membrane localization||−0.63||0.66|
|CAGL0A02277g||Protein of unknown function||−0.72||−0.11|
|PET10||Ortholog(s) has lipid particle localization||−0.51||0.45|
|MUP1||Protein of unknown function||−0.78||0.47|
|SET4||Ortholog of S. cerevisiae SET4 and S. cerevisiae S288C YJL105W||1.14||−0.30|
|CAGL0L06776g||Has domain(s) with predicted sequence-specific DNA binding and transcription factor activities, zinc ion binding activity, and role in regulation of transcription, DNA templated||0.85||0.33|
|CAGL0L08547g||Protein of unknown function||0.76||−0.05|
|CAGL0J00297g||Ortholog(s) has endoplasmic reticulum localization||0.63||−0.41|
|CAGL0G00594g||Ortholog(s) has Golgi apparatus and endoplasmic reticulum localization||0.61||−0.31|
Role of CgRpn4 in transcriptome-wide changes occurring in response to fluconazole in C. glabrata.
CgRpn4 is activated upon fluconazole stress, leading to its nuclear accumulation.
CgRPN4 plays a role in the maintenance of ergosterol levels, membrane permeability, and fluconazole accumulation.
Determination of promoter recognition motifs by CgRpn4 and direct regulation of CgERG11 expression.
MATERIALS AND METHODS
Strains, plasmids, and growth media.
Disruption of CgRPN4.
Cloning of the C. glabrata CgRPN4 gene (ORF CAGL0K01727g).
Antifungal susceptibility assays.
CgRpn4 subcellular localization assessment.
Total RNA extraction.
Library preparation and gene expression analysis.
Plasma membrane permeability.
[3H]fluconazole accumulation assays.
In silico prediction of overrepresented sequences in CgRpn4-activated promoters.
Cloning of the CgERG11 promoter and site-directed mutagenesis.
RT-PCR gene expression measurement.
Western blot analysis.
CgRpn4 chromatin immunoprecipitation assays followed by RT-PCR.
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