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

crossmark Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans Arturo Luna-Tapia, Hélène Tournu, Tracy L. Peters, Glen E. Palmer Department of Clinical Pharmacy, Division of Clinical and Experimental Therapeutics, College of Pharmacy, University of Tennessee Health Sciences Center, Memphis, Tennessee, USA The azole antifungals arrest fungal growth through inhibition of ergosterol biosynthesis. We recently reported that a Candida albicans vps21⌬/⌬ mutant, deficient in membrane trafficking through the late endosome/prevacuolar compartment (PVC), continues to grow in the presence of the azoles despite the depletion of cellular ergosterol. Here, we report that the vps21⌬/⌬ mutant exhibits less plasma membrane damage upon azole treatment than the wild type, as measured by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21⌬/⌬ mutant has abnormal levels of intracellular Ca2ⴙ and, in the presence of fluconazole, enhanced expression of a calcineurin-responsive RTA2-GFP reporter. Furthermore, the azole tolerance phenotype of the vps21⌬/⌬ mutant is dependent upon both extracellular calcium levels and calcineurin activity. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment and indicate that this may occur through modulation of calcium- and calcineurin-dependent responses. S everal important antifungal drugs inhibit the synthesis of ergosterol, a lipid that modulates the thickness, fluidity, and permeability of fungal cell membranes (1). These drugs include the azoles, which inhibit lanosterol demethylase (Erg11p). Both the depletion of cellular ergosterol and the accumulation of intermediate sterol species are thought to cause plasma membrane dysfunction and, ultimately, growth arrest following azole treatment (2). The emergence of azole-resistant fungal isolates is a problem of increasing medical significance (3). Several specific mechanisms that contribute to the development of azole resistance in the prevalent human fungal pathogen Candida albicans have been described. These mechanisms include increased expression of the target enzyme, point mutations that alter the target enzyme’s affinity for the azoles (4, 5), and increased expression of efflux pumps, such as Cdr1p and Mdr1p, that export the azoles out of the fungal cell (6–8). 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. 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). The objective of this study was to define the mechanism(s) underlying the azole tolerance of the C. albicans vps21⌬/⌬ mutant. 7170 aac.asm.org MATERIALS AND METHODS Growth conditions. C. albicans was routinely grown at 30°C in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) supplemented with uridine (50 ␮g/ml) when necessary. Transformant selection was carried out on minimal YNB medium (6.75 g/liter yeast nitrogen base without amino acids, 2% dextrose, 2% Bacto agar) supplemented with the appropriate auxotrophic requirements, as described previously for Saccharomyces cerevisiae (11), or 50 ␮g/ml uridine. Plasmid construction. Plasmid pLUX (12) was kindly provided by William Fonzi (Georgetown University). Plasmids pLUXVPS21 (13), pKE1 (14), and pKE1-NLUC (15) were described previously. All oligonucleotides used in this study are listed in Table S1 in the supplemental material. The VPH1 open reading frame (ORF) with 5=- and 3=-untranslated region (UTR) sequences was amplified from SC5314 genomic DNA (gDNA) with HiFi Platinum Taq (Invitrogen) and primer set VPH1AMPF and VPH1AMPR-KpnI and cloned between the SacI and KpnI restriction sites of pLUX to produce plasmid pLUXVPH1. For the construction of reporter plasmid pRTA2prGFP␥, 1,000 bp of the RTA2 promoter was amplified from SC5314 gDNA with primer pair RTA2prF-KpnI and RTA2prR-SalI and cloned between the KpnI and SalI sites of pKE1 in place of the ACT1 promoter. The GFP␥ (green fluorescent protein) coding sequence was then amplified by using primers GFPAMPF-SalI and GFPAMPR-MluI and cloned downstream of the RTA2 promoter between the SalI and MluI sites. C. albicans strains. All strains used in this study are listed in Table S2 in the supplemental material. The vps21⌬/⌬, aps3⌬/⌬, and vps21⌬/⌬ Received 13 May 2016 Returned for modification 22 June 2016 Accepted 11 September 2016 Accepted manuscript posted online 19 September 2016 Citation Luna-Tapia A, Tournu H, Peters TL, Palmer GE. 2016. Endosomal trafficking defects can induce calcium-dependent azole tolerance in Candida albicans. Antimicrob Agents Chemother 60:7170 –7177. doi:10.1128/AAC.01034-16. Address correspondence to Glen E. Palmer, . Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.01034-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved. Antimicrobial Agents and Chemotherapy December 2016 Volume 60 Number 12 Azole Tolerance of C. albicans vps21Δ/Δ Mutant aps3⌬/⌬ mutants were constructed in previous studies (13, 14, 16, 17). Control strain YJB6284 (18) was kindly provided by Judith Berman (Tel Aviv University). Transformation of C. albicans with DNA constructs was performed by using the lithium acetate method (19). Gene deletion strains were constructed by a PCR-based approach described previously by Wilson et al. (20), using ura3⌬/⌬ his1⌬/⌬ arg4⌬/⌬ strain BWP17 (kindly provided by Aaron Mitchell, Carnegie Mellon University). Strain CAI4 was kindly provided by William Fonzi (Georgetown University). VPH1 deletion cassettes were amplified by PCR with primers VPH1DISF and VPH1DISR, using pRS-ARG4⌬SpeI, pGEM-HIS1, or pDDB57 (containing a recyclable URA3-dpl200 marker) (20, 21) as the template. Each VPH1 allele was sequentially deleted by using HIS1 and ARG4 markers to generate vph1⌬/⌬ ura3⌬/⌬ gene deletion mutants. The correct integration of the deletion cassettes was confirmed at each step by PCR with the following primers sets: AR (...truncated)