PDR16-mediated azole resistance in Candida albicans

Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.     

A mutation in intracellular loop 4 affects the drug-efflux activity of the yeast multidrug resistance ABC transporter Pdr5p

Multidrug resistance protein Pdr5p is a yeast ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance by active efflux of intracellular drugs. However, the highly polymorphic Pdr5p from clinical strain YJM789 loses its ability to expel azole and cyclohexmide. To investigate the role of amino acid changes in this functional change, PDR5 chimeras were constructed by segmental replacement of homologous BY4741 PDR5 fragments. Functions of PDR5 chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, ATPase activity, and efflux efficiency for other substrates were also analyzed. Using multiple lines of evidence, we show that an alanine-to-methionine mutation at position 1352 located in the predicted short intracellular loop 4 significantly contributes to the observed transport deficiency. The degree of impairment is likely correlated to the size of the mutant residue.     

The role of PDR13 in tolerance to high copper stress in budding yeast.

PDR13 in Saccharomyces cerevisiae contributes to drug resistance via sequential activation of PDR1 and PDR5. In this study, we found that a PDR13 deletion mutant was hypersensitive to Cu(2+) compared to the wild-type counterpart. The Cu(2+) tolerance mechanism mediated by Pdr13 does not seem to involve Pdr1 or Pdr5, since mutants harboring a deletion of either the PDR1 or PDR5 gene did not show elevated Cu(2+) sensitivity. Instead, we found that the PDR13 null mutant could not express CUP1 or CRS5 metallothionein at wild-type levels when subjected to high Cu(2+) stress. These results suggest that Pdr13 contributes to high Cu(2+) tolerance of S. cerevisiae, at least in part, via a mechanism involving metallothionein expression.     

Yeast Pdr13p and Zuo1p molecular chaperones are new functional Hsp70 and Hsp40 partners

The deletion of the TOM1 gene encoding a putative ubiquitin ligase causes a temperature sensitive cellular growth in Saccharomyces cerevisiae. The arrested cells exhibit pleiotropic defects in nuclear division, maintenance of nuclear structure and heat stress responses. We previously identified a zuo1 mutation as an extragenic suppressor of the tom1 mutant. ZUO1 encodes a DnaJ-related Hsp40. Here we show that a recessive cold sensitive mutation in PDR13 coding for an Hsp70 suppressed the tom1 mutation. The pdr13 deletion mutant was sensitive to high osmolarity, just like the zuo1 deletion mutant. A zuo1 pdr13 double deletion mutant did not show additive phenotypes. Furthermore, a tagged-Zuo1p was co-immunoprecipitated with a tagged-Pdr13p. Taken together, we propose that Pdr13p and Zuo1p are a new pair of Hsp70:Hsp40 molecular chaperones. In addition, Pdr13p co-sedimented with translating ribosomes and this association was independent of the presence of Zuo1p.     

The ABC transporter Pdr5p mediates the efflux of nonsteroidal ecdysone agonists in Saccharomyces cerevisiae.

We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH-5992) is actively excluded by resistant cells of insects. To identify the transporter that could be involved in the efflux of RH-5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter-deletion mutants of yeast Saccharomyces cerevisiae. PDR5 (pleiotropic drug resistance 5) deletion mutants (Deltapdr5 and Deltapdr5Deltasnq2) retained significantly higher levels of 14C-radiolabeled RH-5992 within the cells when compared to wild-type strain or single deletion mutants of SNQ2 (Deltasnq2) and YCF1 (Deltaycf1). Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of [14C]RH-5992 from these cells as efficiently as the wild-type cells. These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of [14C]RH-5992 in yeast. This exclusion was temperature-dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process. The mutants with the PDR5 deletion can also selectively accumulate [14C]RH-0345 and [14C]RH-2485, but not [14C]RH-5849, indicating that these three compounds share the same transporter Pdr5p for efflux.