The recent completion of the sequencing project of the opportunistic human pathogenic yeast, Candida albicans (http://www.ncbi.nlm.nih.gov/), led us to analyze and classify its ATP-binding cassette (ABC) proteins, which constitute one of the largest superfamilies of proteins. Some of its members are multidrug transporters responsible for the commonly encountered problem of antifungal resistance. TBLASTN searches together with domain analysis identified 81 nucleotide-binding domains, which belong to 51 different putative open reading frames. Considering that each allelic pair represents a single ABC protein of the Candida genome, the total number of putative members of this superfamily is 28. Domain organization, sequence-based analysis and self-organizing map-based clustering led to the classification of Candida ABC proteins into 6 distinct subfamilies. Each subfamily from C. albicans has an equivalent in Saccharomyces cerevisiae suggesting a close evolutionary relationship between the two yeasts. Our searches also led to the identification of a new motif to each subfamily in Candida that could be used to identify sequences from the corresponding subfamily in other organisms. It is hoped that the inventory of Candida ABC transporters thus created will provide new insights into the role of ABC proteins in antifungal resistance as well as help in the functional characterization of the superfamily of these proteins.

Background: The major facilitator superfamily (MFS) is one of the two largest superfamilies of membrane transporters present ubiquitously in bacteria, archaea, and eukarya and includes members that function as uniporters, symporters or antiporters. We report here the complete transportome of MFS proteins of a human pathogenic yeast Candida albicans.

The incidences of human pathogenic yeast Candida albicans and its related species acquiring resistance to antifungals have increased considerably, which poses serious problems towards its successful chemotherapy. The resistance of these pathogenic fungi is not restricted to the commonly used triazole compounds but is even encountered, though not often, with polyene derivatives as well. The efflux pump proteins belonging to ABC (ATP Binding Cassette) and MFS (Major Facilitators) super family are the most prominent contributors of multidrug resistance (MDR) in yeasts. The abundance of the drug transporters and their wider specificity suggest that these transporters may not be exclusively drug exporters in yeasts and may have other cellular functions. In this article we focus on some of the recent advances on the structure and function, evolution and transcriptional control of drug efflux proteins of Candida. A short discussion on the physiological relevance of drug transporters is also included.

In view of the importance of Candida Drug Resistance Protein (Cdr1p) of pathogenic Candida albicans in azole resistance, we have characterized its ability to efflux variety of substrates by subjecting its entire transmembrane segment (TMS) 5 to site directed mutagenesis. All the mutant variants of putative 21 amino acids of TMS 5 and native CaCdr1p were over expressed as a GFP-tagged protein in a heterologous host Saccharomyces cerevisiae. Based on the drug susceptibility pattern, the mutant variants could be grouped into two categories. The variants belonging to first category were susceptible to all the tested drugs, as compared to those belonging to second category which exhibited resistance to selective drugs. The mutant variants of both the categories were analyzed for their ATP catalysis and drug efflux properties. Irrespective of the categories, most of the mutant variants of TMS 5 showed an uncoupling between ATP hydrolysis and drug efflux. The mutant variants such as M667A, F673A, I675A and P678A were an exception since they reflected a sharp reduction in both Km and Vmax values of ATPase activity when compared with WT CaCdr1p-GFP. Based on the competition experiments, we could identify TMS 5 residues which are specific to interact with select drugs. TMS 5 residues of CaCdr1p thus not only impart substrate specificity but also selectively act as a communication link between ATP hydrolysis and drug transport.

The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have in the past shown that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all the mutant variants showed characteristics similar to those of wild type with respect to cell surface expression and photoaffinity drug analogue [ 125 I] IAAP and [ 3 H] azidopine labeling. While Cdr1p D327N mutant variant showed comparable binding with [α-32 P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. Cdr1p D327N mutant variant showed ∼40% enhancement of its residual ATPase activity at acidic pH while no such pH effect was seen with Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.One of the most clinically significant mechanisms of azoles resistance in the pathogenic fungi, C. albicans is an over expression of the drug efflux pumps encoding genes CDR1 and CDR2 belonging to the ABC (ATP-Binding Cassette) (1-7) and CaMDR1 belonging to MFS (Major Facilitator Superfamily) transporters (8)(9)(10). Among the ABC transporters, high level of expression of CDR1 invariably contributes to an increased efflux of fluconazole and thus corroborates its direct involvement in drug efflux (6,11,12). Hence, Cdr1p has not only acquired significant clinical importance but is also considered an important target in any design of strategies to combat antifungal resistance (7,13,14). *Corresponding author: E-mail: rp47@hotmail.com; Telephone: 91-11-26704509; Fax: 91-11-26717081. NIH Public Access Media chemical and strainsPlasmids were maintained in Escherichia coli DH5α. E. coli was cultured in Luria-Bertani medium (Difco, BD Biosciences, NJ, USA) to which ampicillin was added (0.1 mg/ml). The yeast strains were cultured in YEPD broth (Bio101, Vista, CA, USA) or SD-ura -(Bio101). Table 2 lists all the strains used in this study. MethodsSite-specific mutagenesis-Site directed mutagenesis was performed using quick-change mutagenesis system as described ...