Substitution of threonine-1351 in the multidrug transporter Cdr1p of Candida albicans results in hypersusceptibility to antifungal agents and threonine-1351 is essential for synergic effects of calcineurin inhibitor FK520.
OBJECTIVE
Functional characterization of a mutant Candida albicans drug resistance protein (Cdr1p) by overexpression in Saccharomyces cerevisiae.


METHODS
We overexpressed green fluorescent protein-tagged Cdr1p in S. cerevisiae AD1-8u- host and introduced a point mutation to substitute T1351 with F in Cdr1p. The cells expressing T1351F mutant Cdr1p were analysed for their functional activity using minimum inhibitory concentration, spot assay, and fluconazole efflux. The binding activity of photoaffinity analogues 8-azidoATP, iodoarylazidoprazosin and azidopine to the mutant T1351F Cdr1p was also characterized.


RESULTS
The T1351F mutant Cdr1p-expressing cells were susceptible to anisomycin, cycloheximide, fluconazole, miconazole and nystatin. The mutant protein was expressed to the same level as that of native Cdr1p in S. cerevisiae cells and was properly localized to the cell surface. There was also no difference between the mutant variant and the native protein's ability to bind a photoaffinity analogue of ATP, 8-azidoATP, or the radiolabelled photoaffinity agents iodoarylazidoprazosin and azidopine. However, the substitution of T1351 resulted in considerable reduction in its ability to export fluorescent substrate rhodamine 6G. The synergy between calcineurin inhibitors FK520 and azoles was abrogated in cells expressing the T1351F mutant variant of Cdr1p.


CONCLUSIONS
The results from this study suggest that the T1351 in the predicted transmembrane domain (TMD) 11 of Cdr1p is not only important for drug-substrate transport but also has a role in governing synergy of FK520.
Shukla
Suneet
S
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Ambudkar
Suresh V
SV
Prasad
Rajendra
R
D000345:Affinity Labels; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology*; D065095:Calcineurin Inhibitors; D002176:Candida albicans / Q000187:drug effects* / Q000235:genetics*; D002462:Cell Membrane / Q000737:chemistry; D026141:Drug Resistance, Multiple, Fungal / Q000235:genetics; D004357:Drug Synergism; D004926:Escherichia coli / Q000187:drug effects; D005656:Fungal Proteins / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013912:Threonine / Q000235:genetics*; D014169:Transformation, Bacterial
D000345:Affinity Labels; D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D065095:Calcineurin Inhibitors; D005656:Fungal Proteins; D007166:Immunosuppressive Agents; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D049452:Green Fluorescent Proteins; D013912:Threonine; C058028:immunomycin; D016559:Tacrolimus