Trafficking through the Late Endosome Significantly Impacts Candida albicans Tolerance of the Azole Antifungals

Abstract

The azole antifungals block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of "toxic" sterol intermediates are both thought to compromise plasma membrane function. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole and to determine whether, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole can be observed as an early consequence of azole treatment, and it precedes significant growth inhibition. In addition, a C. albicans vps21⌬/⌬ mutant, blocked in membrane trafficking through the late endosomal prevacuolar compartment (PVC), is able to grow significantly more than the wild type in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21⌬/⌬ mutant is able to grow despite the depletion of cellular ergosterol. This phenotype resembles an exaggerated form of "trailing growth" that has been described for some clinical isolates. In contrast, the vps21⌬/⌬ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. On the basis of these results, we propose that endosomal trafficking defects may lead to the cellular "redistribution" of the sterol intermediates that accumulate following inhibition of ergosterol biosynthesis. Furthermore, the destination of these intermediates, or the precise cellular compartments in which they accumulate, may be an important determinant of their toxicity and thus ultimately antifungal efficacy.

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

“…1B), indicating that fluconazole induces similar levels of membrane permeabilization in either strain at the lower temperature. This is consistent with our previously reported observation that the continued growth of the vps21⌬/⌬ mutant in the presence of the azoles (as measured by the OD 600 ) occurs at 35°C but not at 30°C (9). To further confirm the temperature dependence of the azole tolerance of the vps21⌬/⌬ mutant, we compared its growth with and without fluconazole using two additional measures, dry weight and CFU (Fig.…”

“…We previously reported that the vps21⌬/⌬ mutant exhibits enhanced growth in the presence of the azole antifungals versus wild-type (WT) controls, despite ergosterol depletion (9). We therefore examined plasma membrane integrity following azole treatment using a luciferasebased assay (15) to determine if the vps21⌬/⌬ mutant exhibited signs of membrane damage.…”

“…In a previous study, we established that the C. albicans vps21⌬/⌬ vacuolar trafficking mutant, deficient in transport through the PVC, is able to grow in the presence of fluconazole at 35°C despite ergosterol depletion (9). In contrast, a C. albicans vps15⌬/⌬ mutant, deficient in retrograde trafficking from the PVC back to the Golgi apparatus (37), as well as two mutants unable to form multivesicular bodies within the PVC (38) are sensitive to fluconazole.…”

“…However, the azole resistance of many fungal isolates is not fully accounted for by these well-characterized mechanisms. We recently reported that membrane trafficking through the late endosome/prevacuolar compartment (PVC) has a significant impact upon C. albicans azole tolerance (9). Specifically, we found that a deletion of VPS21, which encodes a Rab GTPase required for PVC trafficking, substantially enhanced C. albicans growth in the presence of the azoles.…”

“…Specifically, we found that a deletion of VPS21, which encodes a Rab GTPase required for PVC trafficking, substantially enhanced C. albicans growth in the presence of the azoles. The azole tolerance of the vps21⌬/⌬ mutant does not depend upon established mechanisms of azole resistance such as the activity of the wellcharacterized drug efflux pumps Mdr1p and Cdr1p, or increased target protein expression, but occurs despite the depletion of cellular ergosterol (9). This phenotype resembles an exaggerated form of "trailing growth," a phenomenon that is observed using standard CLSI antifungal susceptibility testing protocols (10), in which a subset of C. albicans isolates appear to be azole susceptible at early time points but display significant growth at later time points (10).…”