Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

Details

Serval ID
serval:BIB_52CB446D0F70
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.
Journal
Biochemical and Biophysical Research Communications
Author(s)
Puri N., Manoharlal R., Sharma M., Sanglard D., Prasad R.
ISSN
1090-2104[electronic], 0006-291X[linking]
Publication state
Published
Issued date
2011
Volume
404
Number
1
Pages
357-363
Language
english
Abstract
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.
Keywords
Amino Acid Sequence, Antifungal Agents/metabolism, Antifungal Agents/pharmacology, Azoles/pharmacology, Binding Sites, Candida glabrata/drug effects, Candida glabrata/isolation & purification, Drug Resistance, Fungal, Fluconazole/metabolism, Fluconazole/pharmacology, Fluorescent Dyes/metabolism, Fungal Proteins/chemistry, Fungal Proteins/genetics, Green Fluorescent Proteins/genetics, Green Fluorescent Proteins/metabolism, Humans, Membrane Transport Proteins/chemistry, Membrane Transport Proteins/genetics, Molecular Sequence Data, Oxazines/metabolism, Promoter Regions, Genetic, Rhodamines/metabolism
Pubmed
Web of science
Create date
08/03/2011 8:46
Last modification date
20/08/2019 14:08
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