Do efflux pumps hold the key to better antifungal treatment?

Although resistance to yeast pathogens used to be avery rare event, during the early 1990s, hospitalsbegan to report cases of increased resistance toantifungal drugs, particularly when Candidaalbicans, C. glabrata and C. krusei were involved.Candida infections can give rise to a variety ofconditions including acute pseudomembraneouscandidiasis (oral thrush), chronic atrophiccandidiasis (denture stomatitis), chronichyperplastic candidiasis, angular cheilitis andvaginal candidiasis (vaginal thrush). In addition,systemic disease can be spread via the blood(candidaemia) to infect the liver, kidneys andcentral nervous system. Although all species ofCandida can cause these diseases, C. albicans is themost common, while C. glabrata is often associatedwith vaginitis. An added factor is that the incidenceof opportunistic fungal infections is increasing andthis is particularly evident in immunocompromisedindividuals (for example, individuals with AIDS orthose undergoing bone marrow transplants).

One major worry for clinicians is that cross-resistance is emerging between some previouslyvery effective, established antifungal drugs suchas fluconazole and itraconazole. Dr DominiqueSanglard (Institut de Microbiologie, CentreHospitalier Universitaire Vaudois, Lausanne,Switzerland) and his colleagues have shown thatresistance in clinical isolates of C. albicans candevelop by overexpression of different multidrugefflux transporter genes. These belong to one oftwo classes: the ABC transporters (Cdr1p andCdr2p) or the major facilitators (BENr), whichhave different, but sometimes overlapping,substrate specificity. High levels of resistance areassociated with increased drug efflux from thecell and, put at its simplest, drug moleculesentering the cell from the bloodstream are rapidlypumped out before high enough intracellularlevels can be reached to kill the pathogen. DrSanglard reported his group’s findings at the 4thCongress of the European Confederation of MedicalMycology (Glasgow, UK, 11–13 May 1998).

Fortunately, there is as yet no evidence thatresistance can be transferred from one strain toanother. C. albicans has no sexual cycle andthere are no documented cases of gene transferbetween strains. Therefore, individual strains of C. albicans develop resistance independentlywhen exposed to antifungal drugs (acquiredresistance). This resistance is stable so that,even in the absence of drugs, such strains retaintheir resistance. However, there is nowworrying evidence emerging that these resistantstrains are entering the ‘commensal’ population,

i.e. those strains carried by normal, healthyindividuals.

Hygiene surveys in many hospitals have shownthat resistant strains are passed from patient topatient via the hands of healthcare workers. This issupported by a number of recent studies showingthat, in some hospitals, standards of hygiene havefallen below minimum acceptable levels.

So far, it is not known whether these effluxpumps have a normal role to play in cellular function,although it would seem reasonable that they areinvolved in the transport of other (i.e. non-drug)compounds out of cells. However, no endogenouspump substrates have yet been identified.

At the same meeting, Dr Richard Cannon(Department of Oral Sciences and Orthodontics,University of Otago, New Zealand) described thework his group have been carrying out on Cdr1p.This protein is similar to P-glycoprotein, which isresponsible for the multiple drug resistanceobserved in tumour cells, but it possesses aunique pumping specificity. Previous studies haveestablished a correlation between drug resistancein C. albicans and the expression of plasmamembrane proteins homologous to energy-dependent pumps from the ABC protein family.

Dr Cannon’s group has adapted a secretoryvesicle assay to investigate the specificity of drugtranslocation by the C. albicans protein Cdr1p(Fig. 1). This in vitro system uses a secretoryvesicle preparation of Saccharomyces cerevisiaeSY1, which has been previously used toinvestigate the pumping behaviour by human P-glycoprotein. Using this assay, they have foundthat pumping is not inhibited by verapamil (aknown P-glycoprotein inhibitor) or severalantifungal agents (Fig. 1).

Using this technique, they have been able toshow that Cdr1p specifically transports anumber of azole drugs such as fluconazole,itraconazole and ketoconazole. More detailedinformation about the pumping activity of effluxpumps from C. albicans should allow thedevelopment of pump inhibitors, or at leastpoint the way towards novel antifungal agentsthat are not substrates for these efflux pumps.Dr Cannon has already identified somecompetitive pump inhibitors but is currentlyreluctant to give any more information becausethey are not patented.

David Jack

N e w sMOLECULAR MEDICINE TODAY, JULY 1998

Figure 1. The use of secretory vesicles (sv) to identify drug pump inhibitors. (a) Shows the normal cellularlocation for Cdr1 in the plasma membrane, where it translocates the antifungal agent fluconazole (Fcz) outof drug-resistant cells. (b) Secretory vesicles can be isolated in large numbers from S. cerevisiaesec6-4 cells incubated at the non-permissive temperature of 37°C. (c) Isolated secretory vesicles can beused to identify compounds that inhibit Cdr1 and prevent [3H]-fluconazole accumulation by the vesicles.