Caspofungin Uptake Is Mediated by a High-Affinity Transporter in Candida albicans

The uptake of the echinocandin drug caspofungin acetate in Candidaalbicans was evaluated at drug levels at or near the MIC forthe organism. Maximal uptake was achieved in 10 min and wasenergy independent. A saturable transport system, consistentwith a facilitated-diffusion carrier, was observed with theunlabeled drug competing with the labeled drug for uptake andefflux. More than 90% of the transported drug was observed ina single kinetic compartment that was available for efflux,indicating that the drug was free in the cytoplasm followinguptake. Efflux was also energy independent but was sensitiveto the presence of a fully loaded carrier on both faces of thebilayer. Overall, the data presented are consistent with thepresence of a high-affinity facilitated-diffusion transporterthat mediates caspofungin uptake and could be a potential sourceof transport-related reduced susceptibility.

INTRODUCTION

Top

Introduction

The echinocandin caspofungin acetate (Cancidas) is the firstrepresentative of a new class of ß-(1,3)-D-glucansynthesis inhibitors approved for the treatment of invasiveaspergillosis and esophageal candidiasis. Caspofungin is a water-soluble,semisynthetic polypeptide amine derivative of the natural productpneumocandin B0 with potent activity against Candida spp., Aspergillusspp., and Cryptococcus spp. It targets the 1,3-ß-D-glucansynthase, which is a GTP-dependent enzyme composed of solubleGTP-binding and plasma membrane-bound components (10). The genesencoding the major subunits have been identified from severaldiverse fungi, including Saccharomyces cerevisiae (GSC1 [FKS1]and GSC2 [FKS2]) (5, 11), Candida albicans (GSC1, GSL1, andGSL2) (12), Aspergillus nidulans (FKSA) (7), Paracoccidioidesbrasiliensis (FKS1) (13), and Cryptococcus neoformans (FKS1)(17). In vitro drug resistance has been linked to mutationsin FKS1 (6, 9), supporting it as a potential site of interaction,although little is known about the molecular nature of drug-targetinteractions that lead to inhibition and resistance.

Clinical exposure to caspofungin is growing, and developmentof full resistance or reduced susceptibility to caspofunginappears to be a rare event (3). As a new drug class, cross-resistancewith existing polyene and azole drugs is not expected and hasnot been observed. Echinocandins appear to be poor substratesfor multidrug efflux pumps (15), and other non-FKS1-mediatedresistance mechanisms have not been described. Uptake of caspofungininto the cell is required for inhibition of glucan synthaseactivity, although little or nothing is known about drug uptakein yeasts. In many microorganisms, changes in net uptake eitherby alterations in an uptake transporter or by increasing effluxcan induce drug resistance phenotypes. The presence of a high-affinitytransporter active at or below the MIC of the drug could havesignificance for emergence of potential resistance.

Currently, there is a fundamental gap in our knowledge of echinocandininteractions with the fungal cell. A basic description of cellulartransport of caspofungin would provide a better understandingof drug action. Equally important, an analysis of transportkinetics could help explain differential effects of echinocandinson diverse yeasts and molds. In this report, we present thebasic characteristics of caspofungin transport into cells ofC. albicans and provide evidence for a saturable facilitated-diffusioncarrier mechanism to account for high-affinity transport.

MATERIALS AND METHODS

Top

Materials and Methods

Strains and growth conditions. Wild-type C. albicans strain ATCC 90028 (MIC, 0.25 µg/ml)was used in this study and was grown to mid-log phase in YPDmedium (1% yeast extract, 2% peptone, 2% dextrose, pH 5.7) at37°C for 3 to 4 h following transfer from a confluent overnightculture in the same medium. Cells were harvested by centrifugationat 3,000 x g for 10 min at 4°C and washed twice by resuspensionin ice-cold MES buffer (5 mM morpholineethanesulfonic acid,150 mM NaCl, pH 7.0, sterilely filtered) and centrifugationas described above. The final cell pellet was resuspended in5 ml of MES buffer. The cells were depleted of energy by incubationat 30°C in MES buffer without a carbon source for 1 h withconstant agitation or overnight (14 h) at 4°C and assayedfor medium acidification as previously described (16). The carbon-starvedcells were then diluted to a final concentration of 1.2 x 10⁸/ml.

Transport kinetics. A 5-ml basic reaction medium consisted of MES buffer and Candidacells at 1.2 x 10⁸/ml at 22°C. In some experiments, a portionof the cells was re-energized following a 20-min preincubationwith 2% (wt/vol) glucose. The transport (uptake) reaction wasinitiated by the addition of [³H]caspofungin (1 to 11 µg/ml;kindly provided by Merck Research Labs). Aliquots (250 µl)were removed in triplicate and rapidly centrifuged at top speed(12,000 x g) in a Microfuge for 20 s to pellet the cells. Thesupernatant was rapidly removed, and the cell pellet was washedtwo times by resuspension with an excess (1 ml) of ice-coldMES buffer with or without glucose, depending on the treatmentof the cells, followed by centrifugation as described above.The washed cell pellet was resuspended in 180 µl of MESbuffer and divided into 60-µl aliquots (triplicate) thatwere added to scintillation vials containing 2 ml of scintillationfluid (Ready-Safe liquid scintillation cocktail) and countedin a Beckman LS6500 scintillation counter. Efflux experimentswere performed by first preloading cells with [³H]caspofunginfollowing a 20-min preincubation at 22°C. The cells wereharvested by centrifugation and diluted into a large excess(50- to 100-fold) of MES buffer. Aliquots were removed for centrifugationin the Microfuge and evaluated for residual drug in the pellet.Some transport assays were also conducted by using a filtration-basedprocedure to separate cells from the transport medium in theplace of centrifugation. Aliquots (50 µl) from the transportreaction mixture were added in quadruplicate with a multichannelpipette to a 96-well filtration plate containing Durapore hydrophiliclow-protein-binding membranes with a pore size of 0.65 or 1.2µm in a Millipore Multiscreen filtration apparatus. Thetrapped cells were washed free of excess drug by thrice passingice-cold MES buffer (0.5 ml) through the filter. The maximumconcentration of drug in the cell at the steady state was calculatedby assuming an average cell volume of 76 µm³ (2).

Reagent. The reagent used was caspofungin acetate (MK-0991, L-743,872;Merck Pharmaceuticals). It has a molecular weight of 1,213.42.

RESULTS

Top

Results

Drug aggregation. The uptake of [³H]caspofungin was initially evaluated in a rapidfiltration assay. Unfortunately, this assay could not adequatelydistinguish cell-mediated uptake of the labeled drug from thatof the drug remaining in the reaction medium because of thepresence of a high background level. The principal cause ofthe high background level was a significant population of largedrug complexes formed in free solution that were trapped on1.2-µm-pore-size filters during filtration. These largeaggregates were readily observed following filtration of thetransport medium in the absence of cells. The aggregates appearedto be in equilibrium with a smaller monomer population, sincea portion (5 to 10%) of the labeled drug passed through thefilters. As would be expected, the extent of the aggregateddrug pool was concentration dependent. The aggregation phenomenoncould be minimized below 10 µg/ml, but it was still significant,which prevented a filtration assay from being used. It was foundthat, to separate cells from the labeled drug in solution, rapidcentrifugation at 12,000 x g enabled cells to be effectivelyseparated from all forms (monomeric and multimeric) of the drugin free solution.

Active versus passive drug uptake. The drug uptake was assessed for cells under energized and de-energizedcellular conditions to determine if the transport process wasenergy dependent. Addition of [³H]caspofungin to cells preincubatedwith glucose resulted in rapid uptake of the drug, which reachedsaturation within about 10 min (Fig. 1). To determine whetherthe uptake kinetics were energy dependent, cells were firstde-energized by incubation of cells without a carbon sourceat 30°C for 1 h and then preincubated for 20 min with themetabolic inhibitor sodium azide (1 mM). The energy status ofthe cells was assessed by measuring H⁺-ATPase-mediated protonefflux (medium acidification) from cells in response to theaddition of glucose (14). Addition of glucose to cells resultedin strong medium acidification over 30 min, while in the absenceof an added carbon source and in the presence of the metabolicpoison azide, cells failed to show acid efflux, confirming theirenergy-replete status (data not shown). When energy-depletedcells were challenged with the drug, the initial rate and saturationkinetics for uptake were nearly identical to that of glucose-metabolizingcells, although the final uptake level in energized cells wasconsistently lower (Fig. 1). There was no impact of the drugon cell viability in the time frame examined, which could haveinfluenced uptake. Overall, the nearly identical nature of thenet uptake kinetics for energized and de-energized cells indicatesthat transport is independent of the energy state of the celland is not mediated via active transport. Drug uptake appearedto be diffusion limited, since the calculated concentrationof labeled drug in the cell (1.4 ± 0.21 µM) wasnot statistically significantly different from the startingdrug concentration outside the cell (0.96 µM).

View larger version (9K):
[in this window]
[in a new window]
FIG. 1. Effect of cellular energy status on caspofungin uptake. The uptake of [³H]caspofungin was monitored in 3 ml of reaction medium containing 1.2 x 10⁸ cells/ml. The transport reaction was initiated by the addition of caspofungin at a final concentration of 1 µg/ml to either energized cells maintained in the presence of glucose ( ${diamondsuit}$ ) or de-energized cells starved of a carbon source and pretreated with 1 mM NaN₃ ( ${square}$ ). The assay was performed in triplicate as shown.

Evidence for carrier-mediated transport. A series of experiments were initiated to examine saturationkinetics for uptake as a function of concentration to determinewhether drug uptake was mediated via a facilitated-diffusioncarrier. Because of limited amounts of labeled drug at relativelylow specific activity, it was not possible to adopt a classicalapproach to assess uptake with increasing amounts of labeleddrug over a wide range of concentrations. Two alternative approacheswere used to provide evidence for the presence of a saturablecarrier. First, cells were preloaded with increasing amountsof unlabeled drug (0, 1, 6, and 11 µg/ml) for 30 min andthen rapidly diluted into an excess volume containing labeleddrug at 1.2 µg/ml; uptake was determined as a functionof time. Figure 2 shows that the level of uptake was largelyindependent of the amount of unlabeled drug preloaded in thecells and is consistent with an exchange carrier moving unlabeleddrug out of the cell and labeled drug into the cell. If thesystem were purely a dissipative channel or diffusion throughthe lipid phase, then the high concentration of unlabeled drugwithin the cell would prevent uptake of labeled drug. The reducednet uptake at 30 min with the highest level of unlabeled preloadeddrug can be explained by proposing that as unlabeled drug leakedfrom the cells, it competed with labeled drug at the uptakeface of the carrier.

View larger version (10K):
[in this window]
[in a new window]
FIG. 2. Net uptake of caspofungin into cells preloaded with unlabeled drug. Glucose-energized cells were preloaded with unlabeled drug at 1 ( ${diamondsuit}$ ), 6 ( ${square}$ ), or 11 ( ${blacktriangleup}$ ) µg/ml for 30 min and then diluted into an excess volume containing 1.2 µg of [³H]caspofungin per ml. Uptake of label was monitored in triplicate over 30 min.

To further assess the role of a facilitated-diffusion carrier,uptake of labeled drug (1.2 µg/ml) was monitored as afunction of time in the presence of increasing amounts of externalunlabeled drug (0, 1, 5, 10 µg/ml). Figure 3 shows thatthe presence of unlabeled drug competed with labeled drug, effectivelyreducing the apparent net drug uptake. Both internal loadingand external competition data are consistent with the presenceof a saturable facilitated-diffusion carrier that is operationalat low drug levels. Equilibrium exchange was used to show thatthe putative transporter had a K_m of $≤$ 0.96 µM (data notshown).

View larger version (12K):
[in this window]
[in a new window]
FIG. 3. Effect of competition by external drug on net uptake. Cells were added to a medium containing [³H]caspofungin at 1.2 µg/ml/and unlabeled drug at 0 ( ${diamond}$ ), 1 ( ${square}$ ), 5 (x), and 10 ( ${triangleup}$ ) µg/ml. Uptake was measured in triplicate over 30 min.

Efflux kinetics. Net drug uptake is the sum of unidirectional influx and effluxas a function of time. The efflux component for caspofunginwas determined by preloading cells with saturating amounts ofdrug at 11.2 µg/ml for 10 min and then diluting the cellsinto medium in the absence of the drug to determine the amountof drug left in the cells as a function of time. Efflux wasextremely rapid with a simple declining exponential decay, suggestingthe emptying of a single transport compartment (Fig. 4). Therate of efflux was independent of energy status, as glucose-energizedand carbon-starved and azole-treated cells showed the same apparentrate of efflux (Fig. 4). Importantly, more than 90% of the drugwas removed from the cells, indicating that most of the transportabledrug was in free solution and was not bound significantly tocellular components.

View larger version (12K):
[in this window]
[in a new window]
FIG. 4. Efflux of caspofungin from preloaded cells. Cells were preloaded with [³H]caspofungin at 11.2 µg/ml for 30 min. The cells were diluted into a 50-fold excess volume of buffer containing 5 µg of unlabeled caspofungin per ml, and the amount of labeled drug associated with the cells was determined as a function of time. Efflux was evaluated in both glucose-energized ( ${diamondsuit}$ ) and de-energized (carbon starved and treated with 1 mM NaN₃) ( ${square}$ ) cells.

Effect of external drug on efflux kinetics. To determine whether drug efflux was occurring via reversalof an uptake carrier, energy-replete cells preloaded with labeleddrug were diluted into medium containing increasing amountsof unlabeled drug ranging from 0 to 10 µg/ml. Figure 5shows the exponential efflux of drug plotted as a log functionof time. In the absence of drug or at low levels outside, theinitial rate of efflux was extremely fast, exceeding the zero-timepoint, which was actually between 10 and 15 s. As the levelof drug was increased on the outside, the apparent efflux wasslowed up to a saturation of 5 µg/ml. The second, slowerphase of efflux, especially in the absence of external unlabeleddrug, reflects a basic property of most carriers in that theconformational changes of the loaded carrier are faster thanthose of the unloaded carrier. If the unloaded carrier returnsslowly, this will be the rate-limiting step of the overall transportscheme and entry of labeled drug will be limited by the rateof return of the free carrier.

View larger version (11K):
[in this window]
[in a new window]
FIG. 5. Effect of external drug on efflux kinetics. Cells were preloaded with [³H]caspofungin at 11.2 µg/ml for 30 min and then diluted into a 50-fold excess volume containing unlabeled drug at 0 ( ${diamond}$ ), 1 ( ${square}$ ), 5 ( ${triangleup}$ ), and 10 (x) µg/ml. Labeled drug remaining in the cells was measured in triplicate over a 45-min period.

DISCUSSION

Top

Discussion

The transport of most drugs across the cell membrane representsa critical step in the interaction of a drug with its cellulartarget and hence can influence its overall efficacy. For manydrugs, aqueous diffusion is an important part of the overallmechanism of drug transport, since it is this process that deliversdrug molecules to and from the nonaqueous barrier. In this investigation,we have performed a preliminary characterization of the transportproperties of the new echinocandin drug caspofungin across thecell membrane of C. albicans. Evidence was provided for a saturable,facilitated-diffusion carrier (Fig. 1, 2, and 5). Drug uptakeby this system is energy independent (Fig. 1), as is measuredefflux from preloaded cells (Fig. 4). In each case, drug transportshowed concentration-dependent saturation and was diffusionlimited since the calculated level of the drug approximatedthat in the external medium. Drug entering the cell was notbound and appeared in a single kinetic compartment presumedto be the cytoplasm, since more than 90% of the drug in preloadedcells was recovered as a single declining exponential decay(Fig. 4). The data support the presence of a facilitated-diffusioncarrier that transports the drug at low levels (K_m, <1.2µg/ml) and operates freely in both directions. It cannotbe ruled out that non-carrier-mediated diffusion occurs at higherlevels or that separate transporters exist for influx and efflux.

The presence of a defined facilitated-diffusion-type carriersystem for caspofungin uptake at fungicidal levels is significantbecause it suggests an alternative mechanism that could promotereduced susceptibility. Provided that drug uptake is requiredfor inhibition of glucan synthase, a decrease in the affinityof the drug carrier or a change in the drug translocation ratecould potentially result in reduced susceptibility. The developmentof secondary resistance by Candida spp. following clinical drugexposure is a rare event (3). Yet, limited resistance has beenobserved in in vitro studies and has been linked to mutationsin the FKS1 subunit of the glucan synthase enzyme complex (4-6,9). Non-FKS1-mediated resistance mechanisms have been proposed,including low-level resistance due to overexpression of Cdr2p(15) or Golgi protein Sbe2p (8). Furthermore, microarray-basedscanning of the Saccharomyces cerevisiae genome has revealeda cascade of cell wall biosynthetic genes being overexpressedin response to a caspofungin challenge, as well as many otheressential genes, including putative transporters, suggestingthat numerous genes could influence the development of resistance(1). The presence of a defined high-affinity uptake transporterfor caspofungin may provide another potential mechanism to developresistance if cellular drug uptake is essential for its mechanismof action. Identifying the transporter will be an importantfirst step toward exploring this possibility.

Finally, a dual-uptake model is proposed to account for caspofungintransport. At low drug levels, at or below $~$ 1 µg/ml, ahigh-affinity facilitated-diffusion carrier is suggested tomediate drug uptake into the cell. At higher drug levels, nonspecificdrug uptake could occur through normal diffusion pathways acrossthe bilayer of the plasma membrane. A diffusion coefficientfor caspofungin has not been determined for the low-affinityuptake system, although such measurements will be complicatedby the physical aggregation properties of the drug observedin high-affinity transport experiments.

ACKNOWLEDGMENTS

This work was supported by a Merck Medical School grant to D.S.P.

FOOTNOTES

* Corresponding author. Mailing address: Public Health Research Institute at the International Center for Public Health, 225 Warren St., Newark, NJ 07103. Phone: (973) 854-3200. Fax: (973) 854-3101. E-mail: perlin{at}phri.org.

REFERENCES

Top