Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.
We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
Nidhi
N
Puri
Raman
R
Manoharlal
Monika
M
Sharma
Dominique
D
Sanglard
Rajendra
R
Prasad
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D001665:Binding Sites; D041221:Candida glabrata / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism; D025141:Drug Resistance, Fungal; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010078:Oxazines / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012235:Rhodamines / Q000378:metabolism
D000935:Antifungal Agents; D001393:Azoles; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D010078:Oxazines; D012235:Rhodamines; C026188:rhodamine 6G; D049452:Green Fluorescent Proteins; D015725:Fluconazole; C044808:nile red