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

Abstract

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 Ca 2؉ 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.

Gene Expression Analysis of Key Players Associated with Fluconazole Resistance in Candida albicans

“…Several lines of evidence suggest that fluconazole resistance may involve many unknown mechanisms that have yet to be elucidated. Recently, studies found that calcium signaling plays an important role in the development of drug resistance and it may be a target for overcoming drug resistance (41,42). A novel gene, RTA2, which mediates calcineurin-dependent resistance to azoles, was found to contribute to the development of fluconazole resistance (23,43).…”

Endosomal Trafficking as a Determinant of Antifungal Tolerance in the Pathogenic Fungus Candida albicans

“…For construction of the reporter plasmid pRTA2prGFPγ (228), 1000 bp of the RTA2 promoter were amplified from SC5314 gDNA with HiFi Platinum Taq (Invitrogen) and primer pairs RTA2prF-KpnI + RTA2prR-SalI, and cloned between the KpnI and SalI sites of pKE1 in place of the ACT1 promoter. The GFPγ coding sequence was then amplified using GFPAMPF-SalI + GFPAMPR-MluI, and cloned downstream of the RTA2 promoter between SalI and MluI sites.…”

“…VPH1 deletion cassettes were amplified by PCR with primers VPH1DISF + VPH1DISR, using pRS-ARG4∆SpeI, pGEM-HIS1, or pDDB57 (containing a recyclable URA3-dpl200 marker) (190,230) as templates. Each VPH1 allele was sequentially deleted using HIS1 and ARG4 markers to generate vph1Δ/Δura3Δ/Δ gene deletion mutants (228). Correct integration of the deletion cassettes was confirmed at each step by PCR with the following primers sets: ARG4INTF2 + VPH1DETR2 and ARG4INTR2 + VPH1DETF2 for ARG4 integration or HIS1INTR2 + VPH1DETF2 for HIS1 integration.…”

“…Construction of a vps21Δ/Δvph1Δ/Δ double mutant from a vps21Δ/Δura3Δ/Δarg4Δ/Δ strain previously described (51) was carried out by deleting the first allele of VPH1 with the URA3-dpl200 selection marker and the second allele with the ARG4 marker (228). Replacement of the first VPH1 allele with the URA3-dpl200 selection marker was confirmed using primer pairs URA3INTF2 + VPH1DETR2 and URA3INTR2 + VPH1DETF2.…”

“…Correct excision of the URA3 marker from the vph1∆:URA3-dpl200 loci was confirmed using the flanking primer pair VPH1AMPF + VPH1AMPR-KpnI. Prototrophic and isogenic mutant and reconstituted control strains were then made by transforming the vps21Δ/Δvph1Δ/Δ double mutant with pLUX, pLUXVPS21, or pLUXVPH1 digested with NheI (228). Correct plasmid integration and restoration of URA3 and IRO1 loci was determined as previously described.…”