Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

Rapid Evolution of Multidrug Resistance in a Candida lusitaniae Infection during Micafungin Monotherapy.

Candida (Clavispora) lusitaniae is a rare, emerging non-albicans Candida species that can cause life-threatening invasive infections, spread within hospital settings, and rapidly acquire antifungal drug resistance, including multidrug resistance. The frequency and spectrum of mutations causing antifungal drug resistance in C. lusitaniae are poorly understood. Analyses of serial clinical isolates of any Candida species are uncommon and often analyze a limited number of samples collected over months of antifungal therapy with multiple drug classes, limiting the ability to understand relationships between drug classes and specific mutations. Here, we performed comparative genomic and phenotypic analysis of 20 serial C. lusitaniae bloodstream isolates collected daily from an individual patient treated with micafungin monotherapy during a single 11-day hospital admission. We identified isolates with decreased micafungin susceptibility 4 days after initiation of antifungal therapy and a single isolate with increased cross-resistance to micafungin and fluconazole, despite no history of azole therapy in this patient. Only 14 unique single nucleotide polymorphisms (SNPs) were identified between all 20 samples, including three different FKS1 alleles among isolates with decreased micafungin susceptibility and an ERG3 missense mutation found only in the isolate with increased cross-resistance to both micafungin and fluconazole. This is the first clinical evidence of an ERG3 mutation in C. lusitaniae that occurred during echinocandin monotherapy and is associated with cross-resistance to multiple drug classes. Overall, the evolution of multidrug resistance in C. lusitaniae is rapid and can emerge during treatment with only first-line antifungal therapy.

Species-Specific Differences in C-5 Sterol Desaturase Function Influence the Outcome of Azole Antifungal Exposure.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol diol that disrupts membrane function. In Candida albicans, sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3. Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensities of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans (CaERG3), Candida glabrata (CgERG3), Candida auris (CaurERG3), Cryptococcus neoformans (CnERG3), Aspergillus fumigatus (AfERG3A-C) and Rhizopus delemar (RdERG3A/B) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored C-5 sterol desaturase activity and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant, confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the MIC and residual growth observed at supra-MICs. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced various levels of 14α-methylergosta-8,24(28)-dien-3ß,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C-5 sterol desaturase may be an important determinant of intrinsic azole sensitivity.

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles.

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.

Loss of C-5 Sterol Desaturase Activity in Candida albicans: Azole Resistance or Merely Trailing Growth?

Increased expression of drug efflux pumps and changes in the target enzyme Erg11p are known to contribute to azole resistance in Candida albicans, one of the most prevalent fungal pathogens. Mutations that inactivate ERG3, which encodes sterol Δ5,6-desaturase, also confer in vitro azole resistance. However, it is unclear whether the loss of Erg3p activity is sufficient to confer resistance within the mammalian host, and relatively few erg3 mutants have been reported among azole-resistant clinical isolates. Trailing growth (residual growth in the presence of the azoles) is a phenotype observed with many C. albicans isolates and, in its extreme form, can be mistaken for resistance. The purpose of this study was to determine whether the growth of Erg3p-deficient C. albicans mutants in the presence of the azoles possesses the characteristics of azole resistance or of an exaggerated form of trailing growth. Our results demonstrate that, similar to trailing isolates, the capacity of an erg3Δ/Δ mutant to endure the consequences of azole exposure is at least partly dependent on both temperature and pH. This contrasts with true azole resistance that results from enhanced drug efflux and/or changes in the target enzyme. The erg3Δ/Δ mutant and trailing isolates also appear to sustain significant membrane damage upon azole treatment, further distinguishing them from resistant isolates. However, the insensitivity of the erg3Δ/Δ mutant to azoles is unaffected by the calcineurin inhibitor cyclosporin A, distinguishing it from trailing isolates. In conclusion, the erg3 mutant phenotype is qualitatively and quantitatively distinct from both azole resistance and trailing growth.

Regulatory Role of ERG3 and Efg1 in Azoles-Resistant Strains of Candida albicans Isolated from Patients Diagnosed with Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, affects women's health and life. We aimed to explore the correlation between ERG3 as well as Efg1 mutation/overexpression and azoles-resistance, and the correlation between ERG3 and Efg1 mRNA expression in C. albicans. First, C. albicans was isolated from clinical VVC patients. ERG3 and Efg1 mutations were detected by polymerase chain reaction (PCR) and sequencing, and the expression levels of these two genes were also identified by qRT-PCR. Correlations between mutation/overexpression of ERG3/Efg1 and azoles-resistance as well as ERG3 and Efg1 mRNA expression were analyzed. Based on the ERG3 sequencing, the results showed that there were 2 missense mutation sites, 1 nonsense mutation site, and 4 silent mutation sites, while 1 missense mutation sites, 1 nonsense mutation site, and 12 silent mutation sites were found in Efg1. Furthermore, the mRNA levels of ERG3 gene in the strains sensitive to FCA, ITR or VRC were higher than those in the strains resistant to FCA, ITR, VRC (P < 0.05). While for the mRNA levels of Efg1, susceptible strains were lower than resistant strains. Besides, there was a significant linear negative correlation between ERG3 and Efg1 mRNA expression (r = - 0.614, P < 0.001).

Alternative ergosterol biosynthetic pathways confer antifungal drug resistance in the human pathogens within the Mucor species complex.

Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance. With this new model, phenotypic analyses of gene deletion mutants were conducted to define Erg3 and Erg6a as key biosynthetic enzymes in the M. circinelloides ergosterol pathway. Erg3 is a C-5 sterol desaturase involved in growth, sporulation, virulence, and azole susceptibility. In other fungal pathogens, erg3 mutations confer azole resistance because Erg3 catalyzes the production of a toxic diol upon azole exposure. Surprisingly, M. circinelloides produces only trace amounts of this toxic diol and yet, it is still susceptible to posaconazole and isavuconazole due to alterations in membrane sterol composition. These alterations are severely aggravated by erg3Δ mutations, resulting in ergosterol depletion and, consequently, hypersusceptibility to azoles. We also identified Erg6a as the main C-24 sterol methyltransferase, whose activity may be partially rescued by the paralogs Erg6b and Erg6c. Loss of Erg6a function diverts ergosterol synthesis to the production of cholesta-type sterols, resulting in resistance to amphotericin B. Our findings suggest that mutations or epimutations causing loss of Erg6 function may arise during human infections, resulting in antifungal drug resistance to first-line treatments against mucormycosis. IMPORTANCE: The Mucor species complex comprises a variety of opportunistic pathogens known to cause mucormycosis, a potentially lethal fungal infection with limited therapeutic options. The only effective first-line treatments against mucormycosis consist of liposomal formulations of amphotericin B and the triazoles posaconazole and isavuconazole, all of which target components within the ergosterol biosynthetic pathway. This study uncovered M. circinelloides Erg3 and Erg6a as key enzymes to produce ergosterol, a vital constituent of fungal membranes. Absence of any of those enzymes leads to decreased ergosterol and consequently, resistance to ergosterol-binding polyenes such as amphotericin B. Particularly, losing Erg6a function poses a higher threat as the ergosterol pathway is channeled into alternative sterols similar to cholesterol, which maintain membrane permeability. As a result, erg6a mutants survive within the host and disseminate the infection, indicating that Erg6a deficiency may arise during human infections and confer resistance to the most effective treatment against mucormycoses.

Candida glabrata maintains two Hap1 homologs, Zcf27 and Zcf4, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

Candida glabrata exhibits innate resistance to azole antifungal drugs but also has the propensity to rapidly develop clinical drug resistance. Azole drugs, which target Erg11, is one of the three major classes of antifungals used to treat Candida infections. Despite their widespread use, the mechanism controlling azole-induced ERG gene expression and drug resistance in C. glabrata has primarily revolved around Upc2 and/or Pdr1. In this study, we determined the function of two zinc cluster transcription factors, Zcf27 and Zcf4, as direct but distinct regulators of ERG genes. Our phylogenetic analysis revealed C. glabrata Zcf27 and Zcf4 as the closest homologs to Saccharomyces cerevisiae Hap1. Hap1 is a known zinc cluster transcription factor in S. cerevisiae in controlling ERG gene expression under aerobic and hypoxic conditions. Interestingly, when we deleted HAP1 or ZCF27 in either S. cerevisiae or C. glabrata, respectively, both deletion strains showed altered susceptibility to azole drugs, whereas the strain deleted for ZCF4 did not exhibit azole susceptibility. We also determined that the increased azole susceptibility in a zcf27Δ strain is attributed to decreased azole-induced expression of ERG genes, resulting in decreased levels of total ergosterol. Surprisingly, Zcf4 protein expression is barely detected under aerobic conditions but is specifically induced under hypoxic conditions. However, under hypoxic conditions, Zcf4 but not Zcf27 was directly required for the repression of ERG genes. This study provides the first demonstration that Zcf27 and Zcf4 have evolved to serve distinct roles allowing C. glabrata to adapt to specific host and environmental conditions.

An expanded toolkit of drug resistance cassettes for Candida glabrata, Candida auris, and Candida albicans leads to new insights into the ergosterol pathway.

IMPORTANCE: The increasing problem of drug resistance and emerging pathogens is an urgent global health problem that necessitates the development and expansion of tools for studying fungal drug resistance and pathogenesis. Prior studies in Candida glabrata, Candida auris, and Candida albicans have been mainly limited to the use of NatMX/SAT1 and HphMX/CaHyg for genetic manipulation in prototrophic strains and clinical isolates. In this study, we demonstrated that NatMX/SAT1, HphMX, KanMX, and/or BleMX drug resistance cassettes when coupled with a CRISPR-ribonucleoprotein (RNP)-based system can be efficiently utilized for deleting or modifying genes in the ergosterol pathway of C. glabrata, C. auris, and C. albicans. Moreover, the utility of these tools has provided new insights into ERG genes and their relationship to azole resistance in Candida. Overall, we have expanded the toolkit for Candida pathogens to increase the versatility of genetically modifying complex pathways involved in drug resistance and pathogenesis.

Titration of C-5 Sterol Desaturase Activity Reveals Its Relationship to Candida albicans Virulence and Antifungal Susceptibility Is Dependent upon Host Immune Status.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), which depletes cellular ergosterol and promotes synthesis of the dysfunctional lipid 14α-methylergosta-8,24(28)-dien-3ß,6α-diol, ultimately arresting growth. Mutations that inactivate sterol Δ5,6-desaturase (Erg3p), the enzyme that produces the sterol-diol upon S14DM inhibition, enhances Candida albicans growth in the presence of the azoles. However, erg3 null mutants are sensitive to some physiological stresses and can be less virulent than the wild type. These fitness defects may disfavor the selection of null mutants within patients. The objective of this study was to investigate the relationship between Erg3p activity, C. albicans pathogenicity, and the efficacy of azole therapy. An isogenic panel of strains was constructed that produce various levels of the ERG3 transcript. Analysis of the sterol composition confirmed a correspondingly wide range of Erg3p activity. Phenotypic analysis revealed that even moderate reductions in Erg3p activity are sufficient to greatly enhance C. albicans growth in the presence of fluconazole in vitro without impacting fitness. Moreover, even low levels of Erg3p activity are sufficient to support full virulence of C. albicans in the mouse model of disseminated infection. Finally, while the antifungal efficacy of fluconazole was similar for all strains in immunocompetent mice, there was an inverse correlation between Erg3p activity and the capacity of C. albicans to endure treatment in leukopenic mice. Collectively, these results establish that relative levels of Erg3p activity determine the antifungal efficacy of the azoles upon C. albicans and reveal the critical importance of host immunity in determining the clinical impact of this resistance mechanism. IMPORTANCE Mutations that completely inactivate Erg3p enable the prevalent human pathogen C. albicans to endure the azole antifungals in vitro. However, such null mutants are less frequently identified in azole-resistant clinical isolates than other resistance mechanisms, and previous studies have reported conflicting outcomes regarding antifungal resistance of these mutants in animal models of infection. The results of this study clearly establish a direct correlation between the level of Erg3p activity and the antifungal efficacy of fluconazole within a susceptible mammalian host. In addition, low levels of Erg3p activity are apparently more advantageous for C. albicans survival of azole therapy than complete loss of function. These findings suggest a more nuanced but more important role for Erg3p as a determinant of the clinical efficacy of the azole antifungals than previously appreciated. A revised model of the relationship between Erg3p activity, host immunity, and the antifungal susceptibility of C. albicans is proposed.

Molecular Mechanisms of Azole Resistance in Four Clinical Candida albicans Isolates.

Azole resistance constitutes a serious clinical problem in the management of infections caused by Candida albicans. This study aimed to explore azole-resistant mechanisms in clinical C. albicans isolates collected in Jinan, Shandong, China. In total, 22 samples were collected and analyzed. Among these, four isolates (28A, 28D, 28I, and 28J) exhibited high level of pan-azole-resistance that was Hsp90 dependent. Gene sequencing revealed that the four Hsp90-dependent strains contained different ERG3 mutations that led to four novel amino acid substitutions (S265Y, N322D, N324S, and E355D) in Erg3. The role of these substitutions in azole resistance development was determined by constructing one copy of the mutated ERG3 from the 28A, 28D, and 28I strains into C. albicans CAI4, respectively. The minimum inhibitory concentration value of fluconazole (FLC) against C. albicans CAI4-ERG328I increased fourfold compared with the wild-type C. albicans strain, suggesting that the novel combination of substitutions S265Y, N322D, and N324S played an important role in mediating azole resistance in 28I. Besides, we identified several different mechanisms in other three isolates. Strains 28A and 28D displayed increased efflux ability and overexpression of MDR1. Strain 28J showed high level of ERG11 expression, but no mutation in its regulator Upc2 was observed. Our study revealed that multiple factors confer azole resistance in clinical C. albicans isolates and combination therapy should be conducted clinically.

Role of Calcium/Calcineurin Signalling in Regulating Intracellular Reactive Oxygen Species Homeostasis in Saccharomyces cerevisiae.

The calcium/calcineurin signalling pathway is required for cell survival under various environmental stresses. Using Saccharomyces cerevisiae, we explored the mechanism underlying calcium-regulated homeostasis of intracellular reactive oxygen species (ROS). We found that deletion of acyltransferase Akr1 and C-5 sterol desaturase Erg3 increased the intracellular ROS levels and cell death, and this could be inhibited by the addition of calcium. The hexose transporter Hxt1 and the amino acid permease Agp1 play crucial roles in maintaining intracellular ROS levels, and calcium induced the expression of the HXT1 and AGP1 genes. The cytosolic calcium concentration was decreased in both the akr1Δ and erg3Δ mutants relative to wild-type cells, potentially lowering basal expression of HXT1 and AGP1. Moreover, the calcium/calcineurin signalling pathway also induced the expression of AKR1 and ERG3, indicating that Akr1 and Erg3 might perform functions that help yeast cells to survive under high calcium concentrations. Our results provided mechanistic insight into how calcium regulated intracellular ROS levels in yeast.

ERG3-Encoding Sterol C5,6-DESATURASE in Candida albicans Is Required for Virulence in an Enterically Infected Invasive Candidiasis Mouse Model.

Gastrointestinal colonization by Candida species is considered the main source of candidemia. The ERG3 gene in Candida albicans encodes a sterol C5,6-desaturase, which is essential for ergosterol biosynthesis. Although ERG3 inactivation shows reduced virulence in mouse models of disseminated candidiasis, the role of ERG3 in intestinal infections is unknown. Here, we infected mice with the C. albicans strains CAE3DU3 and CAF2-1, containing mutant and wild-type ERG3, respectively, and studied gut infection and colonization by these strains. We found that the CAE3DU3 strain showed reduced colonization, pathogenesis, damage to gut mucosa, and chemokine production in the mouse model of invasive candidiasis. Additionally, mice inoculated with CAE3DU3 showed lower mortality than mice inoculated with CAF2-1 (p < 0.0001). Chemokines were less induced in the gut inoculated with CAE3DU3 than in the gut inoculated with CAF2-1. Histopathologically, although the wild-type gene was associated with a higher pathogenicity and invasion of the gut mucosa and liver tissues causing remarkable tissue necrosis, the erg3/erg3 mutant was associated with a higher accumulation of cells and lower damage to surrounding tissues than wild-type ERG3. These results establish that the ergosterol biosynthetic pathway may be associated with C. albicans gut colonization and subsequent dissemination.

Phenotypic Analysis of Mutants of Ergosterol Biosynthesis Genes (ERG3 and ERG4) in the Red Yeast Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid used in aquaculture. Astaxanthin is synthesized from metabolites of the mevalonate pathway, which are also precursors for sterols biosynthesis. The interruption of the CYP61 gene, which is involved in the synthesis of ergosterol (mutant CBS.cyp61 -), resulted in a phenotype that overproduces carotenoids due to the activation of the SREBP pathway. In this work, we constructed other mutants of ergosterol biosynthesis in this yeast to evaluate whether they have the same phenotype as mutant CBS.cyp61 -. By bioinformatic analysis, the ERG3 and ERG4 genes of X. dendrorhous were identified, and each gene was deleted in the wild-type strain. Mutants CBS.Δerg3 and CBS.Δerg4 did not produce ergosterol; CBS.Δerg3 primarily accumulated episterol, and CBS.Δerg4 primarily accumulated ergosta-5,7,22,24(28)-tetraenol. The transcription levels of the HMGS gene of the mevalonate pathway were evaluated by RT-qPCR, which showed a slight increase in CBS.Δerg4, but the transcription levels were still 10-fold lower than in strain CBS.cyp61 -. Both CBS.Δerg3 and CBS.Δerg4 did not overproduce carotenoids, even though they do not produce ergosterol. Thus, the results of this study indicate that the absence of ergosterol does not activate the SREBP pathway in X. dendrorhous, but rather it depends on other alterations in sterol composition.

Comparative structure analysis of the ETSi domain of ERG3 and its complex with the E74 promoter DNA sequence. Corrigendum.

The article by Sharma et al. [(2018), Acta Cryst. F74, 656-663] is corrected.

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity.

Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicanserg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance.IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.

Comparative structure analysis of the ETSi domain of ERG3 and its complex with the E74 promoter DNA sequence.

ERG3 (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors, which contain a highly conserved DNA-binding domain. The ETS family of transcription factors differ in their binding to promoter DNA sequences, and the mechanism of their DNA-sequence discrimination is little known. In the current study, crystals of the ETSi domain (the ETS domain of ERG3 containing a CID motif) in space group P41212 and of its complex with the E74 DNA sequence (DNA9) in space group C2221 were obtained and their structures were determined. Comparative structure analysis of the ETSi domain and its complex with DNA9 with previously determined structures of the ERGi domain (the ETS domain of ERG containing inhibitory motifs) in space group P65212 and of the ERGi-DNA12 complex in space group P41212 were performed. The ETSi domain is observed as a homodimer in solution as well as in the crystallographic asymmetric unit. Superposition of the structure of the ETSi domain on that of the ERGi domain showed a major conformational change at the C-terminal DNA-binding autoinhibitory (CID) motif, while minor changes are observed in the loop regions of the ETSi-domain structure. The ETSi-DNA9 complex in space group C2221 forms a structure that is quite similar to that of the ERG-DNA12 complex in space group P41212. Upon superposition of the complexes, major conformational changes are observed at the 5' and 3' ends of DNA9, while the conformation of the core GGA nucleotides was quite conserved. Comparison of the ETSi-DNA9 structure with known structures of ETS class 1 protein-DNA complexes shows the similarities and differences in the promoter DNA binding and specificity of the class 1 ETS proteins.