Karl W. Henry

Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors.

ABSTRACT Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals. Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations. We hypothesized that upregulation ofERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candidaspecies. RNA analysis revealed that ERG11 expression in C. albicans is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase. Incubation with fluconazole, however, resulted in a two- to fivefold increase in ERG11 RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth.ERG11 upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH. Within 1 h of drug removal ERG11 upregulation was reversed. Azole-dependent upregulation was not limited to ERG11: five of five ERG genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated. Similarly, ERG11upregulation occurred following treatment of C. albicanscultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for globalERG upregulation, e.g., in response to ergosterol depletion. Finally, azole-dependent ERG11 upregulation was demonstrated in three additional Candida species (C. tropicalis, C. glabrata, and C. krusei), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.

Histone H3 phosphorylation can promote TBP recruitment through distinct promoter-specific mechanisms.

Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Previously, we showed that histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex‐mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. Here we examine the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA‐bound activators, and in its role in modifying histones and recruiting TATA‐binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10s role in recruiting SAGA may substitute for recruitment by DNA‐bound activator. Our results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter‐specific requirements.

Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast

PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5′ ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent ≤2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes (SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicansLEM3 and RHO1, specificity was 79–95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity (slt2Δ and RHO1/rho1Δ), cycloheximide hypersensitivity (pdr1Δ), or miltefosine resistance (lem3Δ and lem3Δ/lem3Δ). PRODIGE application to the S. cerevisiaePDR5 gene in strains with and without the Pdr1–Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter-URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond.

ROX1 and ERG Regulation in Saccharomyces cerevisiae: Implications for Antifungal Susceptibility

ABSTRACT Yeasts respond to treatment with azoles and other sterol biosynthesis inhibitors by upregulating the expression of the ERG genes responsible for ergosterol production. Previous studies on Saccharomyces cerevisiae implicated the ROX1 repressor in ERG regulation. We report that ROX1 deletion resulted in 2.5- to 16-fold-lower susceptibilities to azoles and terbinafine. In untreated cultures, ERG11 was maximally expressed in mid-log phase and expression decreased in late log phase, while the inverse was observed for ROX1. In azole-treated cultures, ERG11 upregulation was preceded by a decrease in ROX1 RNA. These inverse correlations suggest that transcriptional regulation of ROX1 is an important determinant of ERG expression and hence of azole and terbinafine susceptibilities.