A homologous overexpression system to study roles of drug transporters in Candida glabrata

Abstract

Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters.

Introduction

Candida glabrata, present as a commensal in the human microbiota, is the only second most common infectious agent to cause superficial skin infections to be severe and life-threatening bloodstream infections among genus Candida (Kasper, Seider and Hube 2015). The continuous use of antimycotic drugs, especially azoles, has led to an emergence of drug-resistant clinical strains (Ksiezopolska and Gabaldón 2018). C. glabrata exhibits intrinsically low susceptibility to azoles (Whaley et al. 2016). This clinically rising drug resistance in C. glabrata is a culmination of several mechanisms which also includes overexpression of drug efflux pumps belonging to ATP Binding Cassette (ABC) superfamily and Major Facilitator Superfamily (MFS) of transporters which help cells to rapidly extrude antimycotic drugs to circumvent their lethality (Holmes et al. 2016). The existence of a large number of ABC transporters in C. glabrata genome makes it difficult to dissect the functional relevance of individual transporters (Kumari et al. 2018). Presently, a widely used Saccharomyces cerevisiae based heterologous expression system, that lacks several ABC transporters has been extensively used to study membrane proteins from different yeasts (Lamping et al. 2007). These studies helped in unraveling details of drug efflux, protein localization and directed mutagenesis to dissect the molecular basis of the promiscuity towards substrate recognition (Redhu et al. 2018). Notwithstanding, the immense importance of the S. cerevisiae system that has contributed immensely to the study of ABC transporters, questions are always raised concerning the differences among different species and non-pathogenic host strain’s cellular environment, the lipid content of membrane and protein sorting behavior.

To circumvent these issues, we have developed a system in C. glabrata devoid of any masking effects by dominant drug transporters and an integrative plasmid for the expression of the gene of interest (GOI). For this, a strain of C. glabrata MSY8 was constructed by disrupting seven clinically relevant membrane-localized ABC drug transporter genes that included CgSNQ2, CgAUS1, CgCDR1, CgPDH1, Cgycf1, CgYBT1 and CgYOR1 and introduced a gain of function (GOF) mutation in the CgPDR1 transcription factor (TF) resulting in a hyper-activation of CgCDR1 locus. The integration of GOI by employing an engineered expression plasmid allowed overexpression of fully functional ABC and MFS transporters.

Material And Methods

Chemicals and strains

Antifungal drugs were of analytical grade and were purchased from Sigma-Aldrich, Bengaluru, India. Yeast strains were maintained in YPD media. Bacterial cultures were maintained in LB media with a final concentration of ampicillin at 100 |μg/mL.

Deletion construction

A homologous recombination-based strategy was used to make deletion as described previously (Borah, Shivarathri and Kaur 2011). Sequential deletions were made by using a drug-based recyclable (FRT-NAT1-FRT) cassette. Briefly, 5’ and 3’UTR flanking regions (500-700 bp) of the transporter gene were PCR amplified from wild type genomic DNA. The selection marker NAT1 gene with FRT sites was also PCR amplified in two halves from plasmid pRK625. Both the UTRs were fused to one half of the NAT1 gene, and these fused PCR products were co-transformed into the wild type strain. Positive transformants were selected on YPD agar medium containing 200 μg/mL nourseothricin, and deletions were confirmed by genomic DNA PCR. The primers used in ABC transporter gene deletion and confirmation are listed as File S1 (Supporting Information). The selection marker was then recycled by using plasmid pRK70, which contains the Ura3 selection marker and flippase (FLP1) gene under the constitutive EPA1 promoter. Then after rescuing both the selection marker and plasmid, the strain was used for the next round of deletion. The constructed strains are listed in Table 1.

Table 1. List of strains used in the study.

Mutant NameGenotype		Source
WT (BG14)	ura3Δ::Tn903 NeoR	Lab strain
WTGOF	Cgpdr1::CgPDR1G840C FRT	This study
MSY1	ΔCgsnq2::FRT	This study
MSY2	ΔCgsnq2::FRT, ΔCgaus1::FRT	This study
MSY3	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT	This study
MSY4	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT, ΔCgpdh1::FRT	This study
MSY5	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT, ΔCgpdh1::FRT, ΔCgycf1::FRT	This study
MSY6	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1::FRT	This study
MSY7	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1::FRT, ΔCgyor1::FRT	This study
MSY8	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1::FRT, ΔCgyor1::FRT,Cgpdr1::CgPDR1G840C FRT	This study (Strain accession no. MTCC25307)
MSY9	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT::CgCDR1-GFP::NAT1, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1:: FRT, ΔCgyor1::FRT, Cgpdr1::CgPDR1G840C FRT	This study
MSY10	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT::CgPDH1-GFP::NAT1, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1:: FRT, ΔCgyor1::FRT, Cgpdr1::CgPDR1G840C FRT	This study
MSY11	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT::CaCDR1-GFP::NAT1, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1::FRT, ΔCgyor1::FRT, Cgpdr1::CgPDR1G840C FRT	This study
MSY12	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT::ScPDR5-GFP::NAT1, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1:: FRT, ΔCgyor1::FRT, Cgpdr1::CgPDR1G840C FRT	This study
MSY13	ΔCgsnq2::FRT, ΔCgaus1::FRT, ΔCgcdr1::FRT::CgFLR1-GFP::NAT1, ΔCgpdh1::FRT, ΔCgycf1::FRT, ΔCgybt1:: FRT, ΔCgyor1::FRT, Cgpdr1::CgPDR1G840C FRT	This study

Plasmid constructions

The integration-based plasmid was constructed to integrate mutated CgPDR1 ORF in place of native CgPDR1 locus. An integration-based expression plasmid that can integrate GOI at CgCDR1 locus was constructed that contained NAT1 selection marker, CgCDR1 terminator and promoter regions, YeGFP and four unique restriction sites for cloning the GOI. The details of expression plasmid pMS6 construction and the list of the plasmids used are documented in File S2 (Supporting Information).

Growth analysis

Growth curve analysis was performed by a micro-cultivation method in a 96-well plate using the liquid handling system (Tecan, Grödig, Austria) in YPD broth at 30°C. Briefly, overnight grown cells were inoculated at a dilution of 0.1 OD₆₀₀ in a 96-well plate and allowed to grow at 30°C. OD₆₀₀ was measured every 30 min for an interval to up to 24 h. Doubling times of strains were calculated by measuring the time taken in doubling of logarithms values of the OD600 of the exponential phase.

Drug susceptibility and efflux assays

Susceptibility to antifungal drugs was estimated either by broth micro-dilution or serial dilution spot assays, as described earlier (Shah, Shukla and Prasad 2017). The MIC80 was defined at the lowest concentration inhibiting growth by at least 80% relative to the drug-free YPD control after incubation. Both Rhodamine-6-G (R6G) and Nile Red (NR) efflux assays were performed by spectrofluorometer based assays as per protocols used earlier (Keniya et al. 2015; Gbelska et al. 2017). For R6G efflux, 527 nm excitation and 555 nm emission, and for NR, 552 nm excitation and 636 nm emission were used. A straight line for the relation between fluorescence and concentration was plotted for both the substrates, and the concentration in the supernatant was determined by plotting the fluorescence to this graph.

cDNA synthesis and real-time PCR

Total RNA was extracted by using the RNeasy Mini Kit by following manufacturer instructions (QIAGEN, Hilden, Germany). 5 μg of total RNA was used to synthesize cDNA by using Rever-tAid H minus first-strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania) as per the protocol. The quantitative gene expression profile was evaluated by using iTaq universal SYBR green supermix (BioRad, Gurugram, India) and genespecific primers, including CgPGK1 as an internal control. Comparative expression profiles were analyzed by 2^-ΔΔCt method. qRT-PCR experiments were performed in triplicates. P-values were determined by students t-test with ≥ 0.05 were considered as significant.

Results

Construction of multiple ABC transporters deleted strain of C. glabrata

ABC transporters extrude a wide variety of physiological compounds. Among ABC subfamilies, there is a greater similarity in the sequence and structure, which could make them share partial overlapping substrate profiles (Kolaczkowski et al. 1998). Due to the similarity in the substrate profile, some transporters may function as dominant transporters by masking the function of other transporters. The dominance of ABC transporters such as CaCdr1p and CgCdr1p and CgPdh1p in C. albicans and C. glabrata, respectively, or ScPdr5p in S. cerevisiae, are few examples which are well reported. Therefore, functional analysis of a transporter in the absence of dominant transporters is very important to evaluate its true potential for drug efflux and the development of multiple drug resistance. To generate such a host strain, we selected seven transporters genes (CgSNQ2, CgAUSl, CgCDR1, CgPDH1, Cgycf1, CgYBT1 and Cgyor1) which were earlier shown to be upregulated in azole-resistant clinical isolates of C. glabrata or involved in azole drug resistance (Sanguinetti et al. 2005; Vermitsky et al. 2006; Torelli et al. 2008; Ferrari et al. 2011). For these deletions, a recyclable FRT based dominant selection marker NAT1 was used by employing fusion PCR based homologous recombination (Borah, Shivarathri and Kaur 2011; Srivastava, Suneetha and Kaur 2015). Thus, we sequentially deleted these transporters’ coding sequences, and resulting strains were designated as MSY1-MSY7. These deletions were not in a particular order. Each deletant was designated as MSY1 (Cgsnq2Δ), MSY2 (MSY1 + CgauslΔ), MSY3 (MSY2 + CgCDR1Δ), MSY4 (MSY3 + CgPDH1Δ), MSY5 (MSY4 + Cgycf1Δ), MSY6 (MSY5 + Cgybt1Δ), MSY7 (MSY6 + Cgyor1Δ).

Growth kinetics of MSY1–MSY7 strains

To determine the effect of each transporter deletion on growth, we performed growth kinetics by micro-cultivation method. The cells were grown in YPD broth at 30°C, and OD₆₀₀ was recorded for 24 h. The growth curves of WT and deletion mutants MSY1–MSY7 are depicted in Fig. 1A. Notably, the doubling times of MSY1 to MSY6 were similar to WT strain (60 min); however, the deletion of Cgyor1Δ (MSY7) resulted in a slightly slower growth rate with a doubling time of 68.04 min (Fig. 1A). To further confirm if the slow growth phenotype of the MSY7 is due to Cgyor1 deletion and not due to any other changes, we complemented the CgYOR1 gene by episomal expression in plasmid pGRB2.2. While the episomal complementation reversed the drug sensitivity phenotype in MSY7 to a level up to MSY6 (data not shown), it could not restore the growth defect of MSY7. This growth defect is probably due to the cumulative effect of deletions.

Figure 1. Phenotypic characterization of strains MSY1-MSY7.

(A) Growth kinetics study of MSY1-MSY7 strains was performed by a micro-cultivation method in a 96-well plate using Liquid Handling System (Tecan, Austria) in YPD broth at 30°C. Doubling time for the strains was calculated by quantifying the time taken in doubling of logarithms values of the log phase. Experiments were conducted in triplicates (n = 3), and values are expressed in mean ± standard deviation. (B) Spot assays were performed by serial dilution spot assay to check the effect of deletion on the antifungals tested. (C) R6G and NR efflux in MSY1-MSY7 cells. The experiment was performed in biological triplicates with three technical replicates. Significance was determined by calculating the P-value for all the strains with respect to WT by employing the student’s t-test. P-values < 0.05 = *, <0.0005 = *** and ns = not significant.

Drug susceptibility profiling of MSY1–MSY7 mutants

The drug susceptibility profile of each deletion mutant was determined by spot assays, MIC80 determination by microdilution method and growth kinetics using fluconazole (FLC), ketoconazole (KTC), voriconazole (VRC), miconazole (MCZ) and itraconazole (ITZ), caspofungin (CSF), amphotericin B (AmB) and nystatin (NYS), allylamine terbinafine (TER), protein synthesis inhibitors cycloheximide (CHX) and anisomycin (ANY), DNA damaging agents 4-nitroquinoline (4-NQO) and metal chelator 1,10-phenanthroline (OP) at indicated concentrations (Fig. 1B and File S3, Supporting Information). The initial two deletions MSY1 (Cgsnq2Δ) and MSY2 (Cgsnq2Δ, Cgausl Δ) did not show any alteration in MIC80 with respect to WT on all the tested drugs except for 4-NQO and OP which was supported by spot assays as well (Fig. 1B). However, when CgCDR1A was deleted, in MSY3 strain, as expected, the mutant MIC₈₀ were dropped dramatically for tested azoles, including FLC MIC₈₀ drop from 64 pg/mL to 4 pg/mL. The MIC₈₀ was two doubling dilution lower (1 pg/mL) for FLC when CgPDH1Δ was deleted to make MSY4 strain. The subsequent deletion of Cgycf1Δ, in MSY5, and Cgybt1Δ in MSY6, did not yield any further change in MIC80 of FLC as well as of other tested drugs (except for KTC). The subsequent last deletion of Cgyor1Δ, MSY7, led to another 4-fold drop in MIC of FLC (0.25 μg/mL) in comparison to the MSY6 strain. A similar reduction of MIC80 of MSY7 was also observed with other azoles, protein synthesis inhibitors and allylamine. As expected, the deletion of Cgsnq2Δ, MSY1 displayed dramatic susceptibility towards 4-NQO and OP, which are the known substrates of ScSnq2p; however, no further change in susceptibility towards 4-NQO and OP was noticed in subsequent deletions MSY2-MSY7 (Fig. 1B, File S3, Supporting Information). This indicated that changes in drug susceptibility are transporter dependent. We further confirmed the drug susceptibility profile of each deletion mutant by growth curve analysis in the presence of tested drugs (File S4, Supporting Information). The MIC₈₀ to tested echinocandin and polyenes remained unchanged in all the deletion strains even after the deletion of all the seven transporters (File S3, Supporting Information). Together, these results suggested that the deletion of seven ABC transporters, which generated the MSY7 strain, displayed supersensitive phenotype to a wide range of tested drugs.

Assessment of efflux capacity of MSY1–MSY7 mutants

Fluorescent compounds R6G and NR are the known substrates of some of the ABC transporters and have been commonly used to evaluate the efflux activity. Therefore, we tested the R6G and NR efflux by MSY1-MSY7 deletion mutants to confirm the transport defect in these mutants. Indicated cells were pre-incubated with R6G or NR for 4 h in the absence of energy source to accumulate the dyes inside the cells. The energy-starved cells were then supplemented with 2% glucose to initiate the efflux of fluorescent substrates. After 45 min, the fluorescence was recorded in the supernatant and quantified. As expected, there was no reduction in R6G efflux in MSY1-MSY2 mutant strains. Surprisingly, MSY3 strain (Cgcdr1 Δ) showed only ~10% reduction in R6G efflux and an almost 48% reduction in NR efflux as compared to WT cells. The subsequent Cgpdh1Δ in MSY4 resulted in 33 and 44% reduction of R6G and NR efflux, respectively, which coincided well with a recorded drop in MIC₈₀ of FLC. Subsequent deletions (MSY5-MSY6) did not show any significant changes in R6G and NR efflux. However, the deletion of Cgyor1Δ (MSY7 strain) further decreased R6G efflux by ~93% and NR efflux by ~23% as compared to MSY4 (Fig. 1C).

GOF mutation in WT enhances azole resistance by < 100 fold

In C. glabrata, the upregulation of ABC transporters is linked to alterations in zinc-clustered TF CgPDR1. This TF recognizes PDRE present at promoters of several ABC transporters genes, including CgCDR1, CgPDH1, CgSNQ2, CgYOR1, CgYCF1 and CgYBT1. Several GOF mutations in the CgPDR1 coding region have been linked to an increase in the expression of transporters and resistance towards azoles (Ferrari et al. 2009). A CgPDR1 GOF mutation (CgPDR1 ^G840C) in C. glabrata DSY565 strain showed MIC₈₀ of FLC 128 μg/mL along with approximately 64-fold upregulation of CgCDR1 transcript (Ferrari et al. 2009). We introduced this mutation (CgPDR1^G840C) in WT, and the resulting mutant was designated as WT^GOF (Cgpdr1::CgPDR1^G840C). For this, pMS3 plasmid was linearized with SacI/KpnI and transformed by electroporation, which replaced the native CgPDR1 coding sequence (Fig. 2A). The drug susceptibility of WT^GOF was tested for azoles, and expectedly we observed increased resistance to FLC along with ~114-fold upregulated transcript of the CgCDR1 gene (Fig. 2B and C).

Figure 2. Introduction of GOF mutation (CgPDR1^G840C) in MSY7 and phenotypes tested with of MSY8 strain.

(A) Integration plasmid for GOF TF CgPDR1 was constructed by cloning mutated CgPDR1 ORF (CgPDR1^G840C) from C. glabrata strain DSY565 (Ferrari et al. 2009). The resulting plasmid pMS3 was then digested with KpnI/SacI and transformed in WT and MSY7 strain to introduce the GOF in the genome to construct WT^GOF and MSY8, respectively. (B) Serial dilution spot assay to depicts drug susceptibility of WT^GOF cells. (C) Expression analysis of CgCDR1 in WT^GOF cells as compared with WT cells. (D) Substrate transport assay (R6G and NR) with MSY7 and MSY8 strains.

GOF mutation in MSY8 does not impact growth, susceptibility to antifungals and substrate transport

To construct a supersensitive C. glabrata strain where GOI could be overexpressed without the interference of dominant transporters for functional analysis, we constructed MSY8 strain, with seven transporter deletions and hyperactive CgCDR1 locus in its genome. For this, the above-constructed cassette from digested pMS3 plasmid was transformed in the aboveconstructed MSY7 strain to generate the MSY8 strain. The constructed MSY8 strain has a GOF CgPDR1 (CgPDR1^G840C) and a hyperactive CgCDR1 promoter. Since TF CgPDR1 regulon includes several other genes apart from ABC transporters, we checked the possibility of altered growth and azole susceptibility in MSY8 strain. Interestingly MSY8 did not show a further change in susceptibility towards antifungals and efflux of substrates to MSY7 strain (Fig. 2C and D; File S3, Supporting Information).

Overexpression of ABC transporters validated the appropriateness of MSY8 as an expression system

To validate the expression system, we expressed well known ABC transporters of C. glabrata (CgCDR1 and CgPDH1), C. albicans (CaCDR1) and S. cerevisiae (ScPDR5) in MSY8 strain. These transporters were cloned in pMS6 plasmid upstream of the YeGFP at either SacI/SacII or NotI/PacI sites, as described in File S2 (Supporting Information). The whole cassette containing GOI-GFP chimeric construct was then digested with SacI/KpnI and integrated into the MSY8 strain at CgCDR1 locus. The constructed strains MSY9 (MSY8/CgCDR1), MSY10 (MSY8/CgPDH1), MSY11 (MSY8/CaCDR1) and MSY12 (MSY8/ScPDR5) were tested for the expression and localization. As evident from confocal images, all the GFP-chimeric transporters CgCdr1-GFP, CgPdh1-GFP, CaCdr1-GFP and ScPdr5-GFP showed precise plasma membrane localization (Fig. 3A). The transcripts levels of each transporter CgCDR1, CgPDH1, CaCDR1 and ScPDR5 in MSY9, MSY10, MSY11 and MSY12 cells were confirmed by RT-PCR, which also displayed the overexpression of these transporters at the locus (Fig. 3C).

Figure 3. Overexpression of ABC transporters and their characterization.

(A) Integration of ABC transporters CgCDR1, CgPDH1, CaCDR1 and ScPDR5 was performed as described in Methods and resulting strains designated as MSY9-MSY12. Confocal images of MSY9-MSY12 show the localization at the plasma membrane. Confocal images were taken by using the FITC filter. (B) Drug susceptibility tests were performed by spot assays at indicated concentrations. (C) Transcript levels of integrated transporters into MSY8 was determined by RT-PCR. (D) R6G substrate transport assay with the overexpressed transporters. P-value was determined by the student’s t-test and P-value < 0.0005 represented as (***).

Functional analysis of CgCdr1-GFP, CgPdh1-GFP, CaCdr1-GFP and ScPdr5-GFP

The functionality of the recombinantly expressed ABC transporters in MSY9-MSY12 strains was analyzed by testing their susceptibility towards azoles, allylamines and protein synthesis inhibitors in comparison to parental strain MSY8. We rationalized that the overexpressing transporter proteins should be able to reverse the higher susceptibility of MSY8 cells. Expectedly, the expression of all four transporters drastically decreased the susceptibility of MSY8 cells, albeit to variable levels (Fig. 3B). R6G is a known substrate of ABC transporters, and these four transporters are known to efflux R6G (Lamping et al. 2007). Spectroflu-orometric based efflux assay showed a 25 to 32-fold increased efflux of R6G as compared to MSY8 cells (Fig. 3D). Results indicated that ABC transporters from different fungal species could be expressed and functional in the MSY8 strain.

MSY8 expression system is suitable to study non-ABC transporters

We explored the suitability of our expression system for functional analysis of non-ABC membrane transporters. For this, we transformed and integrated a GFP-chimeric-CgFLR1 transporter belonging to MFS superfamily in MSY8 strain to generate the MSY13 strain. Drug: H⁺ antiporter CgFlr1p is a well-known drug transporter that confers 5-flucytosine (5-FC) antifungal resistance in C. glabrata (Pais et al. 2016). While the homolog CgFlr2p confers resistance to both azoles and 5-FC, the CgflrlΔ deletant imparts susceptibility to 5-FC, but not to azoles (Chen et al. 2007). As expected, MSY13 strain became resistant to 5-FC and interestingly, also demonstrated increased resistance to azoles (Fig. 4A). The increased azole resistance could be attributed to the masking effect of CgFLR2 on azole resistance phenotype of CgFLRl. MSY13 overexpressing CgFlr1-GFP showed proper localization at the plasma membrane, as was evident from confocal images (Fig. 4B), and 2-fold increased efflux of NR, a well-known MFS transporters substrate (Fig. 4C).

Figure 4. Expression of MFS transporter and its phenotypic characterization.

(A) Drug spot assays of MSY13 by serial dilution at indicated concentrations. (B) Confocal images of localization of CgFlr-GFP taken from the FITC filter of the confocal microscope. (C) NR efflux in MSY13 cells. Each experiment was performed in biological triplicates with three technical replicates. P-value was determined by paired student t-test, and P-value < 0.0005 was represented as (***).

Discussion

Several independent studies have called attention to the relevance of drug transporters belonging to ABC and MFS superfamilies in drug resistance and their posing of a serious hindrance to successful antifungal therapy. Such a situation demands an in-depth structure and function analysis of clinically relevant drug transporters. For this, there is always an unmet need for an overexpression system that can act as a platform to facilitate the functional characterization of these transporters to analyze the basis of their substrate poly-specificity, structural analysis and to develop inhibitors/modulators. Over the years, this requirement has been successfully met by the S. cerevisiae overexpression system established by Goffeau’s and Cannon’s groups (Decottignies et al. 1998; Lamping et al. 2007). Using this approach, efflux pumps not only from S. cerevisiae but from different fungal pathogens as well could be structurally and functionally analyzed (Shukla et al. 2003; Lamping et al. 2007; Panapruksachat et al. 2016). Notwithstanding, the extensive use of the S. cerevisiae system by several laboratories for nearly two decades, concerns have been raised pertaining to its artefactual environment posed by heterologous system, particularly applicable to the functionality of drug transporters of pathogenic Candida species. The differences in regulatory circuitry governing drug resistance and subtle differences in membrane lipid environment influencing the functionality of drug transporter proteins are some of the concerns faced by such studies. In this context, it is worth mentioning that membrane lipids do impact drug transporter protein insertion, integrity and overall functioning (Opekarová and Tanner 2003; Pasrija, Panwar and Prasad 2008).

Our present study is focused to resolve challenges imposed by the heterologous system for the overexpression of drug transporter proteins of C. glabrata. We took advantage of a GOF mutation of TF CgPDR1 azole-resistant clinical isolate of C. glabrata and introduced it into an engineered hyper-susceptible MSY7 mutant strain, which was deleted for seven dominant ABC transporters. Thus the resulting strain MSY8 apart from major transporters deleted background also possessed hyperactivated CgCDR1 locus. This followed by the construction of an integration-based plasmid for cloning GOI at the activated CgCDR1 locus. The suitability of the expression system was well demonstrated by overexpressing fully functional clinically relevant ABC drug transporters of C. glabrata (CgCDR1 and CgPDH1), C. albicans (CaCDR1), S. cerevisiae (ScPDR5) and C. glabrata MFS transporter CgFLR1. This MSY8 system thus presents a good platform for detailed substrate profiling and functional analysis of transporter proteins with minimum interference by major drug transporters of C. glabrata.