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. 2015 Apr;59(4):2410-20.
doi: 10.1128/AAC.04239-14. Epub 2015 Feb 9.

Trafficking through the late endosome significantly impacts Candida albicans tolerance of the azole antifungals

Arturo Luna-Tapia  1 , Morgan E Kerns  1 , Karen E Eberle  1 , Branko S Jursic  2 , Glen E Palmer  3
Affiliations

Trafficking through the late endosome significantly impacts Candida albicans tolerance of the azole antifungals

Arturo Luna-Tapia et al. Antimicrob Agents Chemother. 2015 Apr.

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 vps21Δ/Δ 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 vps21Δ/Δ 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 vps21Δ/Δ 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.

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Figures

FIG 1
FIG 1
Inhibition of Erg11p causes vacuolar fragmentation in C. albicans. (A to C) A C. albicans strain expressing the GFP-Ypt72p fusion protein was subcultured into YNB broth supplemented with either 1 μg/ml fluconazole (FLU) or 0.5% DMSO (no-drug control) at ∼1 × 106 cells/ml and incubated at 30°C with shaking. Samples were taken at 30-min intervals, and growth was measured as OD600 (A) and cell viability was determined by the number of CFU. Vacuolar integrity was also observed by fluorescence microscopy using a FITC filter set (C), and matching phase-contrast (PC) images were also obtained. The values in panels A and B are the means ± standard deviations (error bars) from two independent experiments, and the images in panel C are representative of each time point in the same two experiments. Growth and CFU were compared in the presence and absence of fluconazole for each time point using a two-tailed t test. Values that are significantly different are indicated as follows: *, P < 0.05; §, P < 0.001. (D) The C. albicans ERG11 gene was placed under the transcriptional control of a doxycycline-repressible promoter, and the GFP-Ypt72p expression construct was introduced into the tetO-ERG11 strain. The tetO-ERG11 strain was then subcultured into YNB medium in the presence of 5 μg/ml of doxycycline (+ Dox) or in the absence of doxycycline at 1 × 105 cells/ml and incubated at 30°C with shaking. After 8 h, vacuole morphology was observed as described above.
FIG 2
FIG 2
Endosomal trafficking affects C. albicans growth in the presence of ergosterol biosynthesis inhibitors. (A to D) The susceptibility of several C. albicans vacuolar trafficking mutants to fluconazole was compared to “wild-type” (WT) (YJB6284) and “reconstituted” control strains using the standard CLSI broth microdilution protocol. After 48-h incubation, growth was measured as OD600 and expressed as a percentage of the growth in the no-drug (DMSO alone) control wells. (A) An atg9Δ/Δ mutant blocked in autophagy. (B) An aps3Δ/Δ mutant blocked in a direct Golgi-to-vacuole trafficking pathway. (C) A vps21Δ/Δ mutant blocked in membrane fusion and trafficking through the PVC. (D) An ypt72Δ/Δ mutant blocked in membrane fusion at the vacuolar membrane. (E and F) The susceptibility of the C. albicans vps21Δ/Δ mutant to the allylamine terbinafine (E) and the morpholine amorolfine (F) was also determined as described above, except that growth was measured after 24-h incubation. The means ± standard deviations (error bars) from three independent experiments are presented in each panel. The growth of each mutant was compared to that of the “wild-type” control at each drug concentration using a two-tailed t test. Values that are significantly different (P < 0.05) are indicated by an asterisk.
FIG 3
FIG 3
The Vps21p paralogs Ypt52p and Ypt53p have minimal effect upon C. albicans growth in the presence of fluconazole. (A to D) C. albicans ypt52Δ/Δ (A), ypt53Δ/Δ (B), vps21Δ/Δ ypt52Δ/Δ ypt53Δ/YPT53-YPT53T27N (triple GTPase mutant) (C), and pep12Δ/Δ (D) mutants and their isogenic control strains were tested for their susceptibility to fluconazole as described in the legend to Fig. 2. The values in each panel are the means ± standard deviations from three independent experiments. The growth of each mutant was compared to that of the “wild-type” control at each drug concentration using a two-tailed t test. *, P < 0.05.
FIG 4
FIG 4
Growth of the C. albicans vps21Δ/Δ mutant in the presence of fluconazole does not depend upon the Cdr1p efflux pump. (A) Both alleles of the CDR1 gene were deleted from either “wild-type” (VPS21/VPS21) or the vps21Δ/Δ mutant, and the susceptibility of the resulting strains to fluconazole was tested as described in the legend to Fig. 2. The values are the means ± standard deviations (error bars) from three independent experiments. No statistically significant differences were found between the growth of the vps21Δ/Δ and vps21Δ/Δ cdr1Δ/Δ mutants. (B) C. albicans strains expressing GFP-Ypt72p were subcultured into YNB medium with and without 1 μg/ml fluconazole at ∼1 × 106 cells/ml and incubated at 30°C with shaking for 6 h. Vacuole morphology was then observed by fluorescence microscopy as described in the legend to Fig. 1.
FIG 5
FIG 5
The C. albicans vps21Δ/Δ mutant shows features consistent with an exaggerated trailing growth-like phenotype. (A and B) The susceptibility of the vps21Δ/Δ mutant and control strains to fluconazole was tested using the standard CLSI protocol, and growth was compared at either 24 h (A) or 48 h (B) as described in the legend to Fig. 2. (C to E) The same experiment was also conducted with RPMI 1640 medium that was adjusted to a pH of 3 (C) or pH 7 with incubation at either 25°C (D) or 42°C (E), and growth was compared after 48 h of incubation. Values in panels A to E are the means ± standard deviations from three independent experiments. The growth of the vps21Δ/Δ mutant was compared to the WT control at each concentration of fluconazole using a two-tailed t test; *, P < 0.05. (F) The vps21Δ/Δ mutant and isogenic control strain were cultured in YNB broth with and without 4 μg/ml of fluconazole at 35°C for 24 h, before total cellular sterols were extracted and ergosterol content was quantified by NMR analysis. Ergosterol content is expressed as a percentage of the sample weight (wet weight). A C. albicans erg2Δ/Δ mutant (no fluconazole) was used as a negative control for ergosterol. (G) Both VPS21 alleles were deleted from the tetO-ERG11 strain. The growth of the resulting vps21Δ/Δ, “reconstituted,” and “wild-type” (YJB6284) control strains was then compared using a broth microdilution assay in YNB medium supplemented with a range of doxycycline concentrations. Growth was measured as OD600 and expressed as a percentage of growth occurring in the no-doxycycline control wells. The means ± standard deviations of three independent experiments are indicated, and statistical analysis was performed as described above.
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