Targeting the Oxidative Stress Response System of Fungi with Redox-Potent Chemosensitizing Agents

Msn2p and the partially redundant factor Msn4p are key regulators of stress-responsive gene expression in Saccharomyces cerevisiae. They are required for the transcription of a number of genes coding for proteins with stress-protective functions. Both Msn2p and Msn4p are Cys2His2 zinc finger proteins and bind to the stress response element (STRE). In vivo footprinting studies show that the occupation of STREs is enhanced in stressed cells and dependent on the presence of Msn2p and Msn4p. Both factors accumulate in the nucleus under stress conditions, such as heat shock, osmotic stress, carbon-source starvation, and in the presence of ethanol or sorbate. Stress-induced nuclear localization was found to be rapid, reversible, and independent of protein synthesis. Nuclear localization of Msn2p and Msn4p was shown to be correlated inversely to cAMP levels and protein kinase A (PKA) activity. A region with significant homologies shared between Msn2p and Msn4p is sufficient to confer stress-regulated localization to a SV40-NLS-GFP fusion protein. Serine to alanine or aspartate substitutions in a conserved PKA consensus site abolished cAMP-driven nuclear export and cytoplasmic localization in unstressed cells. We propose stress and cAMP-regulated intracellular localization of Msn2p to be a key step in STRE-dependent transcription and in the general stress response.

Introduction Microbial survival depends critically upon coordination of sensing environmental stimuli with control of the appropriate cellular responses. As a consequence, microbes have evolved elaborate mechanisms to sense and respond to diverse environmental stresses, including oxidative stress, osmotic stress, thermal stress, changes in pH, and nutrient limitation [1], [2]. Signal transduction cascades integrate recognition and response to these stresses as well as to challenges imposed by exposure to various small molecules that are a ubiquitous presence in the environment. Small molecules can have a dramatic effect on cellular signaling, mediate communication between microbes, or exert potentially lethal toxicity [3], [4], [5], [6], [7]. Many natural products are produced by microbes in competitive communities and can lead to selection for enhanced capacity to tolerate these agents. Since natural products and their derivatives are extensively used in medicine and agriculture [8], [9], the evolution of resistance to these agents can have profound consequences for human health. The evolution of drug resistance in fungal pathogens poses considerable concern given that invasive fungal infections are a leading cause of human mortality worldwide, especially among immunocompromised individuals. The frequency of such infections is on the rise in concert with the growing population of patients with compromised immune systems due to chemotherapy, transplantation of organs or hematopoietic stem cells, or infection with HIV [10], [11]. The leading fungal pathogen of humans is Candida albicans, which ranks as the fourth most common cause of hospital acquired infectious disease and is associated with mortality rates approaching 50% [12], [13], [14]. There is a very limited repertoire of antifungal drugs with distinct targets for the treatment of fungal infections, in part due to the close evolutionary relationships between these eukaryotic pathogens and their hosts [15], [16]. Most of the antifungal drugs in clinical use target the biosynthesis or function of ergosterol, the main sterol of fungal membranes [2], [17], [18]. The therapeutic efficacy of most antifungal drugs is compromised by the emergence of drug resistant strains, superinfection with resistant strains, and by static rather than cidal activities that block fungal growth but do not eradicate the pathogen population. To improve clinical outcome it will be necessary to develop new antifungal drugs with different mechanisms of action and to discover drugs that improve the fungicidal activity of current antifungals. The molecular basis of antifungal drug resistance is best characterized in the context of the azoles through studies with C. albicans and the model yeast Saccharomyces cerevisiae. The azoles have been the most widely deployed class of antifungal drugs for decades and inhibit lanosterol 14α-demethylase, encoded by ERG11, resulting in a block in ergosterol biosynthesis, the accumulation of a toxic sterol intermediate, and cell membrane stress [2], [17], [18]. The azoles are generally fungistatic against Candida species and many patients are on long-term therapy, creating favorable conditions for the emergence of resistance. Despite the evolutionary distance between C. albicans and S. cerevisiae, mechanisms of azole resistance are largely conserved [19]. Resistance can arise by mechanisms that minimize the impact of the drug on the fungus, such as the overexpression of multidrug transporters or alterations of the drug target that prevent the drug from inhibiting its target. Alternatively, resistance can arise by mechanisms that minimize drug toxicity, such as loss of function of the ergosterol biosynthetic enzyme Erg3, which blocks the production of the toxic sterol that would otherwise accumulate when the azoles inhibit Erg11. Recent studies have established that basal tolerance of wild-type strains and resistance due to mechanisms that mitigate drug toxicities without blocking the effect of the drug on the cell are often dependent upon stress responses that are critical for survival of azole-induced cell membrane stress [2], [18]. The key regulator of cellular stress responses implicated in both basal tolerance and resistance to azoles is Hsp90 [2], [18], [20]. Hsp90 is an essential molecular chaperone that regulates the stability and function of a diverse set of client proteins, many of which are regulators of cellular signaling [21], [22], [23]. In S. cerevisiae and C. albicans, inhibition of Hsp90 function blocks the rapid evolution of azole resistance and abrogates resistance that was acquired by diverse mutations [24], [25]. A central aspect of Hsp90's role in the emergence and maintenance of azole resistance is that it enables calcineurin-dependent stress responses that are required to survive the membrane stress exerted by azoles. In both yeast species, Hsp90 physically interacts with calcineurin keeping it in a stable conformation that is poised for activation [26], [27]. Inhibition of calcineurin function phenocopies inhibition of Hsp90 function, abrogating azole resistance of diverse mutants [24], [25]. This has led to the model that calcineurin is the key mediator of Hsp90-dependent azole resistance. Notably, in C. albicans both Hsp90 and calcineurin have recently been demonstrated to regulate resistance to the echinocandins, the only new class of antifungals to reach the clinic in decades; they inhibit the synthesis of (1,3)-β-D-glucan, a key component of the fungal cell wall [20], [27]. Another key cellular stress response pathway implicated in basal tolerance to antifungal drugs is the protein kinase C (PKC) cell wall integrity pathway, though it has only been implicated in tolerance to drugs targeting the cell wall. Central to the core of this signaling cascade is Pkc1, the sole PKC isoenzyme in S. cerevisiae that is essential under standard growth conditions and regulates maintenance of cell wall integrity during growth, morphogenesis, and response to cell wall stress [28], [29], [30], [31]. Signals are initiated by a family of cell surface sensors that are coupled to the small G-protein Rho1, which activates a set of effectors including Pkc1. Pkc1 signaling has been the focus of extensive study in S. cerevisiae where it is known to regulate multiple targets, most notably the mitogen-activated protein kinase (MAPK) cascade comprised of a linear series of protein kinases including the MAPKKK Bck1, the MAPKKs Mkk1/2, and the MAPK Slt2 that relays signals to the terminal transcription factors Rlm1 and Swi4/Swi6. While Pkc1 is not essential in C. albicans [32], the Pkc1-activated MAPK cascade is conserved in C. albicans with Bck1, Mkk2, and the Slt2 homolog Mkc1 [33]. In both species, components of the Pkc1 signaling cascade have been implicated in mediating tolerance to the stress exerted by the echinocandins that target the fungal cell wall [34], [35], [36], [37]. Here, we embarked on a drug screen of 1,280 pharmacologically active compounds to identify molecules that abrogate azole resistance of both an S. cerevisiae resistant mutant and a C. albicans clinical isolate. We identified a key role for PKC signaling in mediating crucial responses to azoles as well as to other drugs targeting the ergosterol biosynthesis pathway, including allylamines and morpholines. Pkc1 regulated responses to azoles at least in part via the MAPK cascade in both species via multiple downstream effectors. Strikingly, inhibition of Pkc1 function phenocopied inhibition of Hsp90 or calcineurin. In S. cerevisiae, compromise of PKC signaling blocked calcineurin activation in response to ergosterol biosynthesis inhibitors, providing a compelling mechanism for the impact on drug resistance. In C. albicans, we found that Pkc1 and calcineurin independently regulate resistance via a common target. The complexity of interactions linking PKC signaling, Hsp90, and calcineurin was further illuminated as genetic reduction of C. albicans Hsp90 resulted in destabilization of Mkc1 thereby blocking its activation. Deletion of C. albicans PKC1 rendered the fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic disease. Our findings establish an entirely new role for PKC signaling in basal tolerance and resistance to ergosterol biosynthesis inhibitors, a novel mechanism through which Hsp90 regulates drug resistance, and that targeting Pkc1 provides a promising therapeutic strategy for life-threatening fungal infections. Results A screen of 1,280 pharmacologically active compounds identifies hits that abrogate azole resistance To identify compounds that enhance the efficacy of the azole fluconazole we screened the LOPAC1280 Navigator library. Our initial screen used an S. cerevisiae strain with azole resistance due to deletion of ERG3. This resistance phenotype is exquisitely sensitive to perturbation of stress response pathways [24], [25]. To enhance the activity of library compounds, this azole-resistant mutant also harbored deletion of PDR1 and PDR3, transcription factors that regulate the expression of numerous multidrug transporters which efflux structurally diverse compounds from the cell [38]. The library was initially screened at 25 µM in defined RPMI medium at 30°C in the presence of 8 µg/ml fluconazole, which reduces growth of this strain by less than 50% . The compounds that reduced growth by greater than or equal to 50% relative to the fluconazole-only controls were re-screened at 12.5 µM in the presence and absence of fluconazole to distinguish those that enhance the activity of fluconazole from those that are simply toxic on their own. This screen identified 185 compounds that enhanced the efficacy of fluconazole (data not shown). To prioritize compounds with synergistic activity with fluconazole against a clinical isolate of C. albicans, we then screened the 185 compounds at 12.5 µM for activity against an isolate from an HIV-infected patient undergoing fluconazole treatment, both in the presence and absence of fluconazole at 8 µg/ml. The capacity of this clinical isolate to grow in the presence of high concentrations of azole is critically dependent upon cellular stress responses [25], despite the fact that it has increased expression of the multidrug transporter Mdr1 relative to a drug-sensitive isolate recovered from the same patient at an earlier time point [39], [40], [41]. This secondary screen identified seven compounds that had little toxicity on their own but which enhanced the efficacy of fluconazole (Figure 1A). One hit from our screen, brefeldin A, was recently confirmed to exhibit potent synergy with antifungals against Candida and Aspergillus [42]. Strikingly, three of the seven hits were characterized as inhibitors of protein kinase C (PKC). 10.1371/journal.ppat.1001069.g001 Figure 1 Pharmacological inhibition of PKC signaling enhances the efficacy of antifungal drugs targeting the cell membrane. (A) A drug screen identifies compounds that abrogate fluconazole (FL) resistance of a Candida albicans clinical isolate (CaCi-2). Seven compounds from the LOPAC1280 Navigator library had little toxicity on their own but enhanced the efficacy of FL against CaCi-2 when tested at 12.5 µM in RPMI medium with 2% glucose in the presence or absence of 8 µg/ml FL. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no compound control (-) or FL-only control. Data was quantitatively displayed with colour using Treeview (see colour bar). The target or mode of action of each compound is indicated in blue. 1CAT = choline acetyltransferase; 2JAK3 = Janus kinase family protein; and 3KDO-8-P = 3-deoxy-D-manno-2-octulosonate-8-phosphate. (B) Pharmacological inhibition of Pkc1 with cercosporamide abrogates azole resistance and reduces echinocandin tolerance of CaCi-2 in an MIC assay. Assays were done in yeast peptone dextrose (YPD) with a fixed concentration of 2 µg/ml micafungin (MF) or 8 µg/ml FL, as indicated. Data was analyzed after 48 hours at 30°C as in part A. (C) Pkc1 inhibitors confer increased sensitivity to other ergosterol biosynthesis inhibitors. A fixed concentration of CaCi-2 cells was incubated in YPD with no antifungal (-), 2 µg/ml MF, 8 µg/ml FL, 2.5 µg/ml fenpropimorph (FN), or 2 µg/ml terbinafine (TB) and with the PKC inhibitors cercosporamide (100 µg/ml) or staurosporine (37.5 ng/ml), as indicated. Data was analyzed after 48 hours at 30°C as in part A. Pharmacological inhibition of PKC enhances the efficacy of antifungal drugs targeting the cell membrane PKC governs the cell wall integrity signaling pathway so named for its role in regulating cell wall integrity during growth, morphogenesis, and exposure to stress in fungi [29], [30], [31]. In both S. cerevisiae and C. albicans, the PKC signaling cascade is known to regulate cellular responses crucial for survival of exposure to antifungal drugs targeting the cell wall, such as the echinocandins [34], [35], [36], [37]. Since the PKC inhibitors identified in our screen were characterized in mammalian cells [43], [44], we next turned to other pharmacological inhibitors of PKC whose mode of action had been validated in fungi. Cercosporamide was identified as a selective Pkc1 inhibitor through C. albicans Pkc1-based high-throughput screening and was shown to exhibit potent synergy with echinocandins [45]. We purified cercosporamide from the fungus Cercosporidium henningsii following standard protocols [46]. As a positive control, we tested the impact of a concentration gradient of cercosporamide on growth in the presence of a fixed concentration of the echinocandin micafungin that causes less than 50% inhibition of growth on its own and confirmed that cercosporamide had the expected synergistic activity with micafungin against the clinical C. albicans isolate (Figure 1B). Using a comparable assay, we determined that cercosporamide also enhanced the activity of fluconazole (Figure 1B), validating the results from our screen. We further confirmed our pharmacological findings with another PKC inhibitor characterized in fungi, staurosporine [47], [48]. Both cercosporamide and staurosporine enhanced the efficacy of antifungals targeting the cell wall, micafungin, and those targeting the cell membrane (Figure 1C), including fluconazole and the morpholine fenpropimorph, which inhibits Erg2 and Erg24 [49]. While staurosporine enhanced the efficacy of another ergosterol biosynthesis inhibitor that inhibits Erg1 [49], the allylamine terbinafine, cercosporamide did not (Figure 1C). The lack of effect of cercosporamide on terbinafine tolerance is likely an artifact of an inactivating drug-drug interaction given that mutants that are hypersensitive to terbinafine are rendered resistant by cercosporamide (data not shown). Genetic validation that Pkc1 enables tolerance to drugs that affect the cell membrane via the MAPK cascade in S. cerevisiae In S. cerevisiae, PKC1 is essential [50], thus we used a strain harboring only a temperature-sensitive (ts) pkc1-3 allele [51] and assayed tolerance to three ergosterol biosynthesis inhibitors fluconazole, fenpropimorph, and terbinafine. Growth of the wild-type strain and the pkc1-3 ts mutant was assayed over a gradient of drug concentrations relative to a drug-free control at either the permissive temperature (30°C) or at a more restrictive temperature, but where the pkc1-3 ts mutant was still able to thrive in the absence of antifungals (35°C). At the permissive temperature, the wild type and the pkc1-3 ts mutant had comparable tolerance to all three drugs tested (Figure S1A). At the restrictive temperature, the pkc1-3 ts mutant was hypersensitive to all three drugs (Figure 2A). The same trend was observed when a dilution series of cells was spotted on solid medium with a fixed concentration of drug (Figure S1B). To determine if reduction of Pkc1 function rendered the fungistatic ergosterol biosynthesis inhibitors fungicidal we used tandem assays with an antifungal susceptibility test performed at the restrictive temperature followed by spotting onto rich medium without any inhibitors. The wild-type strain was able to grow on rich medium following exposure to all concentrations of drug tested (Figure 2B); compromise of Pkc1 function in the pkc1-3 ts mutant enhanced cidality of all three drugs with the most severe effect for fluconazole and fenpropimorph. Thus, reduction of Pkc1 activity increases sensitivity to drugs targeting the cell membrane and enhances cidality of these otherwise fungistatic agents. 10.1371/journal.ppat.1001069.g002 Figure 2 Pkc1 enables basal tolerance to ergosterol biosynthesis inhibitors via the MAPK cascade in Saccharomyces cerevisiae. (A) Drug tolerance of a wild-type (WT) strain (BY4741), a derivative (pkc1-ts) with a temperature sensitive PKC1 allele, and derivatives with deletions of BCK1 and SLT2 are compared in MIC assays. Assays were performed in synthetic defined (SD) medium at 35°C. Data was analyzed after 48 hours as in Figure 1A. The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC80) is indicated for each strain. (B) Genetic compromise of Pkc1 creates a fungicidal combination with ergosterol biosynthesis inhibitors. MIC assays with two-fold dilutions of fluconazole (FL), fenpropimorph (FN), and terbinafine (TB) were performed in SD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed. (C) Schematic of the S. cerevisiae Pkc1 cell wall integrity pathway. Despite the simple linear schematic commonly used to illustrate the architecture of the Pkc1 cell wall integrity pathway (Figure 2C), there is evidence for additional Pkc1 targets [30] and multiple cases of cross talk with other stress response pathways [28]. We next sought to determine if the effects of Pkc1 on tolerance to ergosterol biosynthesis inhibitors are due to signaling via the downstream MAPK cascade. S. cerevisiae mutants lacking the MAPKKK Bck1 or the terminal MAPK Slt2 were hypersensitive to all three ergosterol biosynthesis inhibitors tested in both a liquid antifungal susceptibility assay measuring growth of a fixed concentration of cells across a gradient of drug concentrations (Figure 2A) and a spotting assay of a dilution of cells on solid medium with a fixed concentration of drug (Figure S1B). Deletion of the MAPK components also rendered these fungistatic drugs fungicidal. Thus, Pkc1 enables tolerance to ergosterol biosynthesis inhibitors via the MAPK cascade in S. cerevisiae. Pkc1 enables tolerance to drugs that affect the cell membrane in part via the MAPK cascade in C. albicans In C. albicans, PKC1 is not essential though it does share a high degree of sequence conservation with S. cerevisiae PKC1 and has a conserved role in regulating cell wall integrity through a conserved MAPK cascade [32], [33]. To genetically validate the role of C. albicans PKC1 in tolerance to drugs affecting the cell membrane, we constructed a pkc1Δ/pkc1Δ mutant. Homozygous deletion of PKC1 rendered the strain hypersensitive to all three ergosterol biosynthesis inhibitors tested in liquid static susceptibility assays (Figure 3A) as well as on solid medium (Figure S2A). Comparable results were obtained in well-aerated shaking liquid cultures (data not shown). Restoring a wild-type PKC1 allele under the control of the native promoter to the native locus restored drug tolerance (Figure S2). To determine if deletion of C. albicans PKC1 renders the ergosterol biosynthesis inhibitors fungicidal, we used tandem assays with an antifungal susceptibility test followed by spotting onto rich medium without inhibitor. A strain with wild-type PKC1 levels was able to grow on rich medium following exposure to all drug concentrations tested (Figure 3B). Homozygous deletion of C. albicans PKC1 was cidal in combination with any dose of ergosterol biosynthesis inhibitor tested; no cells were able to grow on rich medium following exposure to the treatments. Thus, Pkc1 regulates crucial cellular responses for surviving the cell membrane stress exerted by antifungal drugs. 10.1371/journal.ppat.1001069.g003 Figure 3 Pkc1 enables basal tolerance to ergosterol biosynthesis inhibitors in part via the MAPK cascade in Candida albicans. (A) Deletion of PKC1, BCK1 or MKC1 reduces tolerance to fluconazole (FL), fenpropimorph (FN), and terbinafine (TB) in MIC assays. Assays were performed in YPD medium at 35°C with strains derived from the WT SN95. Data was analyzed after 72 hours growth as in Figure 1A. The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC80) is indicated for each strain. (B) Deletion of PKC1, but not MAPK components, creates a fungicidal combination with the ergosterol biosynthesis inhibitors in C. albicans. MIC assays with four-fold dilutions of FL, FN, and TB were performed in YPD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed. As an initial approach to assess whether the MAPK cascade was implicated in responses to drugs targeting the cell membrane, we monitored activation of the terminal MAPK in C. albicans. Mkc1 is known to be activated in response to distinct stress conditions including oxidative stress, changes in osmotic pressure, cell wall damage, and cell membrane perturbation [52]. To determine if Mkc1 is activated in response to ergosterol biosynthesis inhibitors we monitored Mkc1 phosphorylation using an antibody that detects dual phosphorylation on conserved threonine and tyrosine residues. Exposure to fluconazole, fenpropimorph, and terbinafine led to Mkc1 activation comparable to exposure to the cell wall damaging antifungal micafungin (Figure S3A). However, activation of signal transducers is not always coupled with functional consequences of their deletion. For example, Mkc1 is activated by exposure to hydrogen peroxide but is not required for survival of this stress [52]. To determine if the role of the MAPK cascade was conserved in C. albicans, we constructed homozygous deletion mutants lacking either the MAPKKK Bck1 or the terminal MAPK Mkc1 (homolog of S. cerevisiae Slt2). Homozygous deletion of either BCK1 or MKC1 rendered strains hypersensitive to fluconazole, fenpropimorph, and terbinafine (Figure 3A) but had negligible effect at elevated temperatures (Figure S3B). This stands in contrast to our results with S. cerevisiae that demonstrated an equivalent role of the MAPK cascade at all temperatures tested (Figure 2, Figure S1 and S4). While deletion of C. albicans PKC1 rendered the ergosterol biosynthesis inhibitors fungicidal, deletion of BCK1 or MKC1 did not (Figure 3B). These results not only implicate the MAPK cascade in C. albicans but also suggest that alternate effectors downstream of Pkc1 are more important at elevated temperature and enable survival in the presence of ergosterol biosynthesis inhibitors. The role of targets downstream of the terminal MAPK in tolerance to ergosterol biosynthesis inhibitors Effectors downstream of the terminal MAPK of the PKC signaling cascade have been well studied in S. cerevisiae and include both nuclear and cytoplasmic proteins. Slt2 is known to regulate activation of two transcription factors Rlm1 and SBF, which is comprised of Swi4 and Swi6 [30]. Rlm1 mediates the majority of the transcriptional output of cell wall integrity signaling, largely genes involved in cell wall biogenesis [53]. SBF drives cell cycle-specific transcription and is also regulated by Slt2 in response to cell wall stress (reviewed in [30]). Swi4 interacts directly with Slt2 and has additional roles in transcriptional regulation independent of the regulatory subunit Swi6 [54]. Slt2 translocates from the nucleus to the cytoplasm in response to cell wall stress [55]. Cytoplasmic Slt2 is required for activation of a high-affinity Ca2+ influx system in the plasma membrane that is comprised of two subunits, Cch1 and Mid1, in response to endoplasmic reticulum stress [56]. Activation of the Cch1-Mid1 channel leads to the accumulation of intracellular Ca2+ and activation of the protein phosphatase calcineurin [57]. To dissect the role of downstream effectors of Slt2 in ergosterol biosynthesis inhibitor tolerance, we tested the impact of their deletion individually and in combination on antifungal susceptibility. For reference, we included a strain lacking the regulatory subunit of calcineurin, CNB1, which is hypersensitive to ergosterol biosynthesis inhibitors [24]. For fluconazole, deletion of RLM1, CCH1, or MID1 had negligible impact on tolerance while deletion of SWI4 or SWI6 rendered strains almost as sensitive as the slt2Δ mutant (Figure 4). To determine if there was redundancy among the downstream effectors, we constructed strains harboring deletion of multiple effectors. Deletion of CCH1 phenocopies deletion of the entire channel and deletion of SWI4 abolishes SBF function as well as Swi4-dependent transcription independent of SBF. Thus, combined deletion of CCH1, SWI4, and RLM1 should eliminate the four known targets of Slt2 phosphorylation. No additional increase in sensitivity was observed in double or triple mutants. This suggests that the SBF transcription factor is of central importance for enabling responses to fluconazole. For fenpropimorph, deletion of RLM1, CCH1, or MID1 had no impact on tolerance individually while deletion of SWI4 or SWI6 caused a partial increase in sensitivity (Figure 4). Deletion of RLM1 in the context of the swi4Δ or swi6Δ mutants further increased fenpropimorph sensitivity. Deletion of CCH1 in the mutant backgrounds had little additional impact. This suggests that SBF is the major determinant of fenpropimorph tolerance with RLM1 enabling additional responses important in the absence of SBF. For tolerance to terbinafine, deletion of RLM1 had no impact while deletion of SWI4 caused a partial increase in sensitivity (Figure 4). Unlike tolerance to fluconazole and fenpropimorph, deletion of SWI6 had negligible impact on terbinafine tolerance while deletion of CCH1 or MID1 caused a partial increase in sensitivity. Deletion of both RLM1 and CCH1 in the swi4Δ mutant caused an incremental increase in sensitivity (Figure 4). These results suggest that Swi4 enables terbinafine tolerance independent of the SBF complex and that Rlm1 and Cch1 mediate responses that are important in the absence of Swi4. Thus, distinct downstream effectors are important for tolerance of S. cerevisiae to different ergosterol biosynthesis inhibitors. 10.1371/journal.ppat.1001069.g004 Figure 4 Distinct downstream effectors are important for tolerance of S. cerevisiae to different ergosterol biosynthesis inhibitors. To dissect the role of downstream effectors of Slt2 in tolerance to ergosterol biosynthesis inhibitors, we tested the impact of their deletion individually and in combination on drug susceptibility in an MIC assay. Data was analyzed after 72 hours at 35°C in SD medium as in Figure 1A. The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC80) is indicated for each strain. Next, we tested a set of C. albicans mutants to determine if the role of the effectors downstream of the terminal MAPK of the PKC signaling cascade was conserved. As was the case with S. cerevisiae, deletion of RLM1 on its own had no impact on tolerance to the ergosterol biosynthesis inhibitors (Figure 5), consistent with recent findings [58]. Deletion of SWI4 rendered strains hypersensitive to all three ergosterol biosynthesis inhibitors tested (Figure 5). Deletion of SWI6 or combined deletion of both SWI4 and SWI6 conferred a comparable increase in sensitivity (data not shown; unpublished strains generously provided by Catherine Bachewich), implicating the SBF complex in responses to drug-induced membrane stress. Deletion of CCH1 or MID1 individually or in combination had a comparable effect to deletion of SWI4 rendering the strain hypersensitive to all three ergosterol biosynthesis inhibitors tested (Figure 5). Notably, C. albicans cch1Δ/cch1Δ and mid1Δ/mid1Δ mutants share some but not all phenotypes of a calcineurin mutant [59]. In terms of ergosterol biosynthesis inhibitor sensitivity, deletion of the gene encoding the catalytic subunit of calcineurin, CNA1, caused hypersensitivity akin to that of the cch1Δ/cch1Δ and mid1Δ/mid1Δ mutants for fluconazole and fenpropimorph but caused slightly greater sensitivity to terbinafine (Figure 5). Thus, in C. albicans both the SBF complex and the Cch1-Mid1 channel play critical roles in tolerance to drugs that target the cell membrane. 10.1371/journal.ppat.1001069.g005 Figure 5 Swi4 and Cch1-Mid1 play critical roles in ergosterol biosynthesis inhibitor tolerance of C. albicans. Deletion of SWI4 or components of the Cch1-Mid1 channel confer increased sensitivity to the ergosterol biosynthesis inhibitors in a MIC assay. Deletion of Rlm1 had no impact on drug sensitivity. A strain lacking the catalytic subunit of calcineurin (Cna1) is included for reference. Data was analyzed after 48 hours in YPD at 35°C as in Figure 1A. The minimum drug concentration that inhibits growth by 80% relative to the drug-free growth control (MIC80) is indicated for each strain. PKC signaling enables calcineurin activation in response to ergosterol biosynthesis inhibitors via a mechanism distinct from Cch1-Mid1 in S. cerevisiae Given calcineurin's established role in mediating drug-induced membrane stress responses [24], [25], [57] and that Slt2 has been shown to enable calcineurin activation by phosphorylating Cch1 [56], we tested whether calcineurin was activated in response to ergosterol biosynthesis inhibitors and whether deletion of Slt2 blocked this activation. To monitor calcineurin activation, we used a well-established reporter system that exploits the downstream effector Crz1, a transcription factor that is dephosphorylated upon calcineurin activation [60], [61]. Dephosphorylated Crz1 translocates to the nucleus, driving expression of genes with calcineurin-dependent response elements (CDREs) in their promoters. We used a reporter containing four tandem copies of CDRE and a CYC1 minimal promoter driving lacZ [61]. We confirmed previous findings that fluconazole activates calcineurin ([27], [62] and Figure 6A). We also found that the other ergosterol biosynthesis inhibitors terbinafine and fenpropimorph activate calcineurin (P 0.05, ANOVA, Bonferroni's Multiple Comparison Test, Figure S5). Thus, PKC signaling enables calcineurin activation in response to ergosterol biosynthesis inhibitors by a mechanism that is largely distinct from the Cch1-Mid1 channel or transcriptional control of calcineurin. PKC signaling and calcineurin independently regulate tolerance to ergosterol biosynthesis inhibitors via a common target in C. albicans In contrast to the minor impact of deletion of the S. cerevisiae Cch1-Mid1 channel, deletion of the C. albicans Cch1-Mid1 channel had nearly as great an effect as deletion of the catalytic subunit of calcineurin in response to fluconazole and fenpropimorph; for terbinafine the effect was partial (Figure 5). To test if the ergosterol biosynthesis inhibitors activate calcineurin and if inhibition of PKC signaling blocks this activation, we monitored transcript levels of two calcineurin-dependent genes, PLC3 and UTR2 [63]. In a wild-type strain, fluconazole activated calcineurin as measured by an increase in PLC3 and UTR2 transcript levels (P 0.05, ANOVA, Bonferroni's Multiple Comparison Test, Figure S8), confirming that the chaperone influences Mkc1 stability at the protein level. Thus, Hsp90 stabilizes Mkc1 independent of its activation status and thereby regulates PKC signaling, providing a new mechanism through which Hsp90 regulates drug-induced membrane stress responses (Figure 9B). 10.1371/journal.ppat.1001069.g009 Figure 9 Hsp90 stabilizes the terminal MAPK Mkc1 in C. albicans. (A) Genetic reduction of Hsp90 levels results in depletion of Mkc1. In strains where the sole allele of HSP90 is under the control of a tetracycline repressible promoter (tetO), transcription of HSP90 can be repressed by tetracycline or the analog doxycycline (DOX). One allele of MKC1 was C-terminally 6xHis-FLAG tagged for monitoring total levels of Mkc1. The MAPKKK Bck1 was deleted to block phosphorylation of Mkc1. Cells were grown with or without DOX (20 µg/ml) before being treated for 3 hours with 50 µg/ml terbinafine (TB) to elicit phosphorylation of Mkc1. Total protein was resolved by SDS-PAGE and blots were hybridized with α-Hsp90, α-His6 to monitor total Mkc1 levels, α-phospho p44/42 MAPK to monitor dually phosphorylated Mkc1 levels, and α-H3 as a loading control. (B) Simplified schematic of how C. albicans Hsp90 governs responses to ergosterol biosynthesis inhibitors (EBIs) important for basal tolerance and resistance by regulating both Pkc1-MAPK signaling and calcineurin signaling. Deletion of C. albicans PKC1 attenuates virulence in a murine model of systemic disease Given that deletion of PKC1 enhances the efficacy of antifungal drugs, we next explored the therapeutic efficacy in a well-established murine model in which fungal inoculum is delivered by tail vein injection and progresses from the bloodstream to deep-seated infection of major organs, most notably the kidney [27], [65], [66]. We compared kidney fungal burden of mice infected with either a wild-type strain or a pkc1Δ/pkc1Δ mutant. The average kidney fungal burden in mice infected with 1×105 CFUs of the wild-type parental strain was 4.34+/−0.54 log CFU per gram of kidney (Figure 10A). In stark contrast, the kidneys of mice infected with 1×105 CFUs of the pkc1Δ/pkc1Δ mutant were sterile (Figure 10A). To determine if infection with higher inocula of the pkc1Δ/pkc1Δ mutant would lead to sufficient kidney fungal burden to enable assessment of antifungal efficacy in vivo, we tested the impact of infection with 10-fold and 100-fold higher inocula. Mice infected with 1×106 or 1×107 CFUs of the pkc1Δ/pkc1Δ mutant demonstrated significantly reduced fungal burden relative to those infected with only 1×105 CFUs of the wild-type strain (P 0.5 but <2 indicate no interaction and those ≥2 indicate antagonism. Spotting assays Strains were grown overnight to saturation in indicated media and cell concentrations were determined based on cell counts using a hemacytometer (Hausser Scientific). Five-fold serial dilutions of cell suspensions starting at indicated concentrations (105 or 107cells/ml) were performed in sterile ddH2O or sterile phosphate buffered saline. Cell suspensions were spotted onto indicated media using a spotter (Frogger, V&P Scientific, Inc). Plates were photographed after 3 days in the dark at indicated temperature. Cidality assay For S. cerevisiae, MIC assays with two-fold dilutions of FL, FN, or TB were performed in SD as described above. For FL the gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 128, 64, 32, 16. FN gradients were from 100 µg/ml down to 0 with the following concentration steps in µg/ml: 100, 50, 25, 12.5, 6.25. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 125, 62.5, 31.25, 15.62. Plates were incubated for two days at 35°C. Cells from the MIC assay were spotted onto solid YPD medium and incubated at 30°C for two days before they were photographed. For C. albicans, MIC assays with FL, FN, or TB were performed in YPD as described above with the following modification; four-fold dilutions of each drug were tested. For FL the gradients were from 256 µg/ml down to 0 with the following concentration steps in µg/ml: 256, 64, 16, 4, 1. FN gradients were from 25 µg/ml down to 0 with the following concentration steps in µg/ml: 25, 6.25, 1.5625, 0.390625, 0.09765625. TB gradients were from 250 µg/ml with the following concentration steps in µg/ml: 250, 62.5, 15.625, 3.90625, 0.9765625. Plates were incubated for two days at 35°C. Cells from the MIC assay were spotted onto solid YPD medium and incubated at 30°C for two days before they were photographed. β-galactosidase assays S. cerevisiae cultures were grown overnight at 25°C in SD medium supplemented for auxotrophies. Cells were diluted to OD600 of 0.05 and were either left untreated or were treated with FL (16 µg/ml), FN (1 µg/ml), or TB (25 µg/ml) for 24 hours at 25°C. When STS was used as an inhibitor in the assay, cultures were grown overnight in SD at 25°C and diluted to OD600 of 0.05 in SD with or without STS (2.5 µg/ml) for 24 hours at 25°C. Cells were then diluted to OD600 of 0.05 in SD with or without STS and with or without FL (32 µg/ml) for an additional 24 hours at 25°C. Cells were harvested, washed, protein was extracted, and protein concentrations were determined by Bradford analysis as described [27]. Protein samples were diluted to the same concentration and β-galactosidase activity was measured using the substrate ONPG (O-nitrophenyl-β-D-galactopyranosidase, Sigma Aldrich Co.) as described [27]. β-galactosidase activity is given in units of nanomoles ONPG converted per minute per milligram of protein. Statistical significance was evaluated using GraphPad Prism 4.0. Immune blot analysis For the Mkc1 activation assay, yeast cultures were grown overnight in YPD at 30°C. In the morning, cells were diluted to OD600 of 0.2 in 50 mL YPD and were grown to mid-log (∼3 hours) at 30°C and then cultures were split into 5×10 mL cultures and were either left untreated or were treated with FL (8 µg/mL), FN (1 µg/mL), MF (30 ng/mL), or TB (25 µg/ml) for 2 hours at 30°C. Cells were harvested by centrifugation at 1308×g for 10 minutes at 4°C and were washed with sterile cold phosphate buffered saline (PBS). Cell pellets were resuspended in lysis buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM EDTA, 1%Triton ×100, 50 mM NaF, 10 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktail (complete, EDTA-free tablet, Roche Diagnostics). For the Mkc1 destabilization assay, cultures were grown overnight in YPD at 30°C. In the morning, cells were diluted to OD600 of 0.2 in 10 mL YPD with or without doxycycline (20 µg/mL; BD Biosciences) and left at 30°C for 24 hours. Cells were diluted once again to OD600 of 0.2 in the same treatment conditions as overnight and were grown at 30°C until mid-log phase (∼4 hours). Doxycycline reduces the growth rate of strains with the repressible promoter driving expression of the only HSP90 allele but does not affect stationary phase cell density [65]. Cells were then treated with 50 µg/mL TB for 3 hours at 30°C to elicit phosphorylation of Mkc1. Cells were harvested after TB treatment at 1308×g at 4°C and washed with ice-cold ddH2O. Cell pellets were flash frozen in liquid N2, resuspended in lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton ×100, 100 mM NaF, 20 mM Na3VO4, 1 mM PMSF and protease inhibitor cocktail complete, EDTA-free tablet, Roche Diagnostics). Cells suspended in lysis buffer were mechanically disrupted by adding acid-washed glass beads and bead beating for 1 minute for six cycles with 1 minute on ice between each cycle. Protein concentrations were determined by Bradford analysis. Protein samples were mixed with one-sixth volume of 6× sample buffer containing 0.35 M Tris-HCl, 10% (w/w) SDS, 36% glycerol, 5% β-mercaptoethanol, and 0.012% bromophenol blue for SDS-PAGE. Samples were boiled for 5 minutes and then separated by 10% SDS-PAGE. Protein was electrotransferred to PVDF membrane (Bio-Rad Laboratories, Inc.) and blocked with 5% skimmed milk in PBS with 0.1% tween or 5% bovine serum albumin in phosphate buffered saline with 0.1% tween. Blots were hybridized with antibodies against CaHsp90 (1∶10000 dilution, generously provided by Brian Larsen, [84]), histone H3 (1∶3000 dilution; Abcam ab1791), His6 (1∶10, P5A11, generously provided by Elizabeth Wayner) and phospho-p44/42 MAPK (Thr202/Tyr204) (1∶2000, Cell Signaling). Quantitative reverse transcription-PCR (qRT-PCR) To monitor gene expression changes in response to FL treatment in S. cerevisiae, cells were grown overnight in SD supplemented for auxotrophies at 30°C. Cells were diluted to OD600 of 0.1 in SD and grown for 2 hours in duplicate at 25°C. After 2 hours of growth 16 µg/mL FL was added to one of the two duplicate cultures and left to grow for an additional 4 hours at 25°C. Cell pellets were frozen at −80°C immediately. To monitor gene expression changes in response to FL treatment in C. albicans, cells were grown overnight in YPD at 30°C. Cells were diluted to OD600 of 0.1 in YPD and grown for 2 hours in duplicate at 35°C. After 2 hours of growth 16 µg/mL FL was added to one of the two duplicate cultures and left to grow for an additional 4 hours at 35°C. Cell pellets were frozen at −80°C immediately. To monitor MKC1 transcript levels in response to decreased levels of Hsp90, cultures were grown overnight in YPD at 30°C. In the morning, cells were diluted to OD600 of 0.2 in 10 mL YPD with or without 20 µg/mL doxycycline (BD Biosciences) and left at 30°C for 24 hours. The next morning, cells were diluted once again to OD600 of 0.2 in the same treatment conditions and were grown at 30°C until mid-log phase (∼4 hours). Cell pellets were collected and immediately frozen at −80°C. RNA was isolated using the QIAGEN RNeasy kit and RNAase-free DNase (QIAGEN), and cDNA synthesis was performed using the AffinityScript cDNA synthesis kit (Stratagene). PCR was performed using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich Co.) with the following cycling conditions: 94°C for 2 minutes, 94°C for 15 seconds, 60°C for 1 minute, 72°C for 1 minute, for 30 or 40 cycles. All reactions were performed in triplicate, using primers for the following genes: CaGPD1 (oLC752/753), CaHSP90 (oLC754/755), ScACT1 (oLC1015/1016), ScCNA1 (oLC1286/1287), ScCNA2 (oLC1288/1289), ScCNB1(oLC1290/1291), CaCNB1 (oLC1292/1293), CaCNA1 (oLC1294/1295), ScCRZ1 (oLC1328/1329), CaCRZ1(oLC1330/1331), CaMKC1(oLC1332/1333), CaPLC3(oLC1432/1433), and CaUTR2(oLC1434/1435). Data were analyzed using iQ5 Optical System Software Version 2.0 (Bio-Rad Laboratories, Inc.). Statistical significance was evaluated using GraphPad Prism 4.0. Murine model of C. albicans infection Inoculum was prepared as described [20], [27], [65]. Cultures were started from frozen stocks onto Sabouraud dextrose agar plates and incubated at 35°C for 48 hours. Colonies were suspended in sterile pH 7.4 PBS, centrifuged at 324×g for 5 minutes, washed with sterile PBS one time and diluted to the desired concentration as verified by counting on a Neubauer hematocytometer as well as by serial dilution and culture. Male CD1 mice (Charles River Laboratories, Wilmington, MA) age 8 weeks (weight 30–34 g) were infected via the tail vein with 100 µL of a 1×106 CFU/mL suspension of the wild type strain (CaLC239, 1×105 CFU per mouse, n = 9 mice), an inoculum previously determined to produce morbidity but not mortality when using C. albicans strain SC5314 at 4 days following tail vein injection (Zaas et al. unpublished data). We observed discordance between cell counts and CFU measurements for the pk1cΔ/pkc1Δ mutant, such that CFU values were ∼50% lower than expected; thus, inocula for the pk1cΔ/pkc1Δ mutant were prepared at higher concentrations based on cell counts and the effective concentrations in CFUs were confirmed by dilution plating. For infection with the pk1cΔ/pkc1Δ mutant, we used an inoculum equivalent to that for the wild type (1×105 CFU, n = 8 mice) as well a 10-fold and 100-fold increase in inoculum (1×106, n = 11 mice and 1×107 CFU, n = 8 mice). Mice were observed three times daily for signs of illness and weighed daily. At day 4 following injection, mice were sacrificed using CO2 asphyxiation and the left kidney was removed aseptically, placed in sterile PBS, homogenized using a FastPrep 120 (QBiogene) using 0.5 mm zirconium beads (Biospec, Inc.) for 1 minute and serial dilutions plated for determination of kidney fungal burden. The CFU values in kidneys were expressed as CFU/g of tissue and log-transformed. Statistical significance was evaluated using GraphPad Prism 4.0. Accession numbers for genes and proteins mentioned in text (NCBI Entrez gene ID number) S. cerevisiae: PKC1 (852169); HSC82 (855224); HSP82 (855836); CNA1 (851153); CNA2 (854946); CNB1 (853644); ERG11 (856398); ERG3 (850745); RHO1 (856294); BCK1 (853350); MKK1 (854406); MKK2 (855963); SLT2 (856425); SWI4 (856847); SWI6 (850879); ERG2 (855242); ERG24 (855441); ERG1 (853086); RLM1 (856016); CCH1 (853131); MID1 (855425); CRZ1 (855704); PDR5 (854324); PDR1 (852871); PDR3 (852278); ACT1 (850504). C. albicans PKC1 (3635298); HSP90 (3637507); CNA1 (3639406); CNB1 (3636463); MKC1 (3639710); ERG11 (3641571); ERG3 (3644776); RHO1 (3642564); BCK1 (3641434); MKK2 (3645580); MDR1 (3639260); ERG2 (3639416); ERG24 (3648198); ERG1 (3646509); CEK1 (3642789); CEK2 (3642459); RLM1 (3635703); SWI4 (3645507); SWI6 (3634957); CCH1 (3639950); MID1 (3647441); CRZ1 (3641722); PLC3 (3635941); UTR2 (3636747); CDR1 (3635385); GPD1 (3643986). Supporting Information Figure S1 Pkc1-MAPK signaling enables tolerance to ergosterol biosynthesis inhibitors in S. cerevisiae. (A) At the permissive temperature, the pkc1-ts mutant and the wild-type (WT) strain (BY4741) have comparable tolerance to all three ergosterol biosynthesis inhibitors tested. Assays were performed in synthetic defined (SD) medium at 35°C. Data was analyzed after 48 hours as in Figure 1A. (B) Genetic compromise of Pkc1 function reduces ergosterol biosynthesis inhibitor tolerance on solid SD medium. Drug tolerance of a WT strain (BY4741) and a derivative (pkc1-ts) with a temperature sensitive PKC1 allele spotted in fivefold dilutions (from 1×107 cells/ml) onto plates with no antifungal (-) or with a fixed concentration of fluconazole (FL, 16 µg/mL), terbinafine (TB, 30 µg/mL), or fenpropimorph (FN, 0.5 µg/mL), as indicated. Plates were incubated at 30°C or 35°C and were photographed after 72 hours at the indicated temperatures. (C) Deletion of components of the MAPK cascade, BCK1 and SLT2, reduces ergosterol biosynthesis inhibitor tolerance on solid SD medium. Assays were performed as indicated in (A) and plates were incubated 35°C and photographed after 72 hours. (2.41 MB TIF) Click here for additional data file. Figure S2 Restoring a wild-type PKC1 allele restores basal tolerance to ergosterol biosynthesis inhibitors in C. albicans. (A) Homozygous deletion of PKC1 reduces ergosterol biosynthesis inhibitor tolerance on solid YPD medium and restoring a wild-type PKC1 allele restores tolerance. Cells were spotted in fivefold dilutions (from 1×107 cells/ml for pkc1Δ/pkc1Δ and from 1×105 cells/ml for other strains) onto plates with no antifungal (-) or with a fixed concentration of fluconazole (FL, 4µg/mL), terbinafine (TB, 2.5µg/mL), or fenpropimorph (FN, 0.25µg/mL). Plates were photographed after 72 hours growth at 35°C. (B) Homozygous deletion of PKC1 confers hypersensitivity to all three ergosterol biosynthesis inhibitors tested in MIC assays; restoring a wild-type PKC1 allele under the control of the native promoter to the native locus restores basal tolerance. Assays were performed in YPD medium at 30°C with strains derived from the WT SN95. Data was analyzed after 72 hours growth as in Figure 1A. (C) Homozygous deletion of PKC1 renders the ergosterol biosynthesis inhibitors fungicidal against C. albicans and restoring a wild-type PKC1 allele restores the fungistatic activity. MIC assays with four-fold dilutions of FL, FN, and TB were performed in YPD and incubated for 48 hours at 35°C. Cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed. (1.85 MB TIF) Click here for additional data file. Figure S3 Involvement of the C. albicans MAPK cascade in responses to ergosterol biosynthesis inhibitors at 30°C but not 35°C. (A) Exposure of C. albicans with ergosterol biosynthesis inhibitors activates the MAPK cascade leading to the accumulation of phosphorylated Mkc1. One allele of MKC1 was C-terminally 6xHis–FLAG tagged in this strain. Cells were grown to mid-log before being treated for 2 hours at 30°C as follows: untreated (-); FN, 1 µg/mL; FL, 8 µg/mL; TB, 25 µg/mL; or MF, 30 ng/mL. Total protein resolved by SDS-PAGE was blotted with α-His6 to monitor total Mkc1 levels and α-phospho p44/42 MAPK to monitor dually phosphorylated Mkc1 levels. (B) Deletion of BCK1 and MKC1 does not increase sensitivity to fluconazole (FL), fenpropimorph (FN), or terbinafine (TB) in MIC assays performed in YPD medium at 35°C. Data was analyzed after 72 hours as in Figure 1A. (0.31 MB TIF) Click here for additional data file. Figure S4 Pkc1 enables tolerance to ergosterol biosynthesis inhibitors in S. cerevisiae via the MAPK cascade at 30°C. Deletion of components of the MAPK cascade in the BY4741 background confers hypersensitivity to ergosterol biosynthesis inhibitors in MIC assays conducted in SD at 30°C. Data was analyzed after 48 hours as in Figure 1A. (0.18 MB TIF) Click here for additional data file. Figure S5 Genetic perturbation of PKC signaling in S. cerevisiae does not compromise expression of calcineurin subunits or CRZ1. Deletion of SLT2 does not reduce expression of calcineurin subunits or CRZ1 in the presence or absence of ergosterol biosynthesis inhibitors. Transcript levels of the genes encoding the catalytic subunit (CNA1 and CNA2) and the regulatory subunit (CNB1) of calcineurin and CRZ1 were measured by quantitative RT-PCR after growth in SD at 25°C for 6 hours without any antifungal (U) or for two hours untreated followed by 4 hours with 16 µg/mL fluconazole (FL), as indicated. Transcripts were normalized to ACT1. Levels are expressed relative to the untreated wild-type samples, which were set to 1. Data are means ± SD for triplicate samples. (0.46 MB TIF) Click here for additional data file. Figure S6 Inhibition of Pkc1 signaling phenocopies inhibition of Hsp90 reducing azole resistance of specific mutants. Azole resistance of matched sets of C. albicans clinical isolates (CaCi) taken from HIV-infected patients early (E) and late (L) during the course of fluconazole (FL) treatment is tested in MIC assays. Assays were conducted in YPD medium with no inhibitor (-), with the Hsp90 inhibitor geldanamycin (5 µM), or with the Pkc1 inhibitor staurosporine (0.5 µg/ml). Mutations implicated in azole resistance for each isolate are indicated. Data was analyzed after growth for 48 hours at 30°C as in Figure 1A. (0.26 MB TIF) Click here for additional data file. Figure S7 The C-terminal 6xHis-FLAG epitope tag does not disrupt functionality of C. albicans Mkc1. When MKC1-6xHis-FLAG is expressed as the sole copy MKC1, it is sufficient to confer WT tolerance to the ergosterol biosynthesis inhibitors in MIC assays. Assays were performed in YPD and growth was measured after 48 hours at 30°C. Data was analyzed and plotted as in Figure 1A. (0.26 MB TIF) Click here for additional data file. Figure S8 Genetic depletion of Hsp90 does not affect MKC1 transcript levels. In strains where the sole allele of HSP90 is under the control of a tetracycline repressible promoter (tetO), transcription of HSP90 can be repressed by tetracycline or the analog doxycycline. Cells were grown in YPD with or without doxycycline (20 µg/ml) and MKC1 transcript levels were measured by quantitative RT-PCR. Transcripts were normalized to GPD1. Levels are expressed relative to the untreated wild-type samples, which were set to 1. Data are means ± SD for triplicate samples. (0.40 MB TIF) Click here for additional data file. Figure S9 Structure of cercosporamide. The structure of cercoposamide was made using ChemDraw Pro (CyberChem, Inc.). (0.05 MB TIF) Click here for additional data file. Table S1 Strains used in this study. (0.17 MB DOC) Click here for additional data file. Table S2 Plasmids used in this study (0.03 MB DOC) Click here for additional data file. Table S3 Primers used in this study. (0.04 MB DOC) Click here for additional data file. Text S1 Supporting Materials and Methods. (0.06 MB DOC) Click here for additional data file.