Azole drugs are imported by facilitated diffusion in Candida albicans and other pathogenic fungi.
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ABSTRACT:
SUBMITTER: Mansfield BE
PROVIDER: S-EPMC2947996 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The tetracycline (TET) promoter has been used in several systems as an inducible regulator of gene expression. In control analyses, the standard Candida albicans laboratory strain SC5314 was found to have altered susceptibility to a variety of antifungal drugs in the presence of relatively high concentrations (50-200 microg ml(-1)) of TET. Altered susceptibility was most notable with exposure to amphotericin B (AMB), with a 32-fold increase in susceptibility, and terbinafine (TRB), with a 32-fold decrease in susceptibility. The TET/AMB synergy was observed in several clinical isolates of C. albicans and in the distantly related species Aspergillus fumigatus and Cryptococcus neoformans. The TET/AMB synergy is not related to efflux pump activity, as determined by FACS analyses and by analysis of a strain containing efflux pump deletions. Gene expression analyses by luciferase and by quantitative real-time reverse transcriptase PCR failed to identify significant alterations in expression of any genes associated with resistance. C. albicans grown with TET for 48 h does show a reduction in total cellular ergosterol. Analysis of growth curves suggests that the TET effect is associated with lack of a diauxic shift, which is related to a loss of mitochondrial function. MitoTracker fluorescent dye was used to demonstrate that TET has a direct effect on mitochondrial function. These results demonstrate the need for careful analysis of TET effects when using a TET-inducible promoter, especially in studies that involve antifungal drugs. This study defines some limits to the use of the TET-inducible promoter, and identifies effects on cells that are the result of TET exposure alone, not the result of expression of a targeted gene.
| S-EPMC2615396 | biostudies-literature
Project description:We have shown earlier that fluconazole (FLC) stress induces global changes in the lipidome of Candida albicans in clinically adapted isolates. However, several laboratories have developed adapted in vitro FLC resistant strains of C. albicans to study azole resistance mechanisms. This study aimed to identify the lipid changes associated with FLC resistance in these in vitro adapted isolates. Using comparative lipidomics and principal component and discriminant analyses, we observed gradual changes in several lipid classes and molecular species upon FLC exposure of in vitro resistant C. albicans strains. Although the lipid imprint of FLC in vitro resistant isolates was very distinct from that of clinical isolates of C. albicans, the overall changes in lipid class compositions were similar in both cases. For example, an increased sterol content and depleted sphingolipid levels were the salient features of FLC resistance in both conditions. Taken together, it appears that the overall cellular lipid homeostasis is a critical factor in the observed FLC resistance and in handling FLC stress in both clinical and laboratory situations. The new observations reported herein have implications for more efficacious antifungal drug development as well as understanding host-infectious agent interactions in postgenomics microbiology practice.
| S-EPMC3567621 | biostudies-literature
Project description:Fungal infections by drug-resistant Candida albicans pose a global public health threat. However, the pathogen's diploid genome greatly hinders genome-wide investigations of resistance mechanisms. Here, we develop an efficient piggyBac transposon-mediated mutagenesis system using stable haploid C. albicans to conduct genome-wide genetic screens. We find that null mutants in either gene FEN1 or FEN12 (encoding enzymes for the synthesis of very-long-chain fatty acids as precursors of sphingolipids) exhibit resistance to fluconazole, a first-line antifungal drug. Mass-spectrometry analyses demonstrate changes in cellular sphingolipid composition in both mutants, including substantially increased levels of several mannosylinositolphosphoceramides with shorter fatty-acid chains. Treatment with fluconazole induces similar changes in wild-type cells, suggesting a natural response mechanism. Furthermore, the resistance relies on a robust upregulation of sphingolipid biosynthesis genes. Our results shed light into the mechanisms underlying azole resistance, and the new transposon-mediated mutagenesis system should facilitate future genome-wide studies of C. albicans.
| S-EPMC6206040 | biostudies-literature
Project description:With some advances in modern medicine (such as cancer chemotherapy, broad exposure to antibiotics, and immunosuppression), the incidence of opportunistic fungal pathogens such as Candida albicans has increased. Cases of drug resistance among these pathogens have become more frequent, requiring the development of new drugs and a better understanding of the targeted enzymes. Sterol 14?-demethylase (CYP51) is a cytochrome P450 enzyme required for biosynthesis of sterols in eukaryotic cells and is the major target of clinical drugs for managing fungal pathogens, but some of the CYP51 key features important for rational drug design have remained obscure. We report the catalytic properties, ligand-binding profiles, and inhibition of enzymatic activity of C. albicans CYP51 by clinical antifungal drugs that are used systemically (fluconazole, voriconazole, ketoconazole, itraconazole, and posaconazole) and topically (miconazole and clotrimazole) and by a tetrazole-based drug candidate, VT-1161 (oteseconazole: (R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol). Among the compounds tested, the first-line drug fluconazole was the weakest inhibitor, whereas posaconazole and VT-1161 were the strongest CYP51 inhibitors. We determined the X-ray structures of C. albicans CYP51 complexes with posaconazole and VT-1161, providing a molecular mechanism for the potencies of these drugs, including the activity of VT-1161 against Candida krusei and Candida glabrata, pathogens that are intrinsically resistant to fluconazole. Our comparative structural analysis outlines phylum-specific CYP51 features that could direct future rational development of more efficient broad-spectrum antifungals.
| S-EPMC5399120 | biostudies-literature
Project description:The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why "delocalized" Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.
| S-EPMC1790725 | biostudies-literature
Project description:In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2?/? mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2?/? mutant was unable to grow on a solid medium containing 10 ?g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2?/? mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 ?g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2?/? mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity.
| S-EPMC4135735 | biostudies-literature
Project description:Purified Candida albicans sterol 14-? demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K(s) values of 11 and 25 ?M, respectively, and a K(m) value of 6 ?M for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (?A(max)) and the azole concentration required to obtain a half-?A(max) being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K(d) values of 10 to 26 ?M under oxidative conditions, compared with 47 ?M for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min(-1)), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.
| S-EPMC2944560 | biostudies-literature
Project description:TAC1 (for transcriptional activator of CDR genes) is critical for the upregulation of the ABC transporters CDR1 and CDR2, which mediate azole resistance in Candida albicans. While a wild-type TAC1 allele drives high expression of CDR1/2 in response to inducers, we showed previously that TAC1 can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of CDR1/2. High azole resistance levels are achieved when C. albicans carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the TAC1 locus on chromosome 5 (Chr 5), which is linked to the mating-type-like (MTL) locus. Both are located on the Chr 5 left arm along with ERG11 (target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analyzed for the TAC1 and ERG11 alleles and for Chr 5 alterations. While recovered ERG11 alleles contained known mutations, 17 new TAC1 alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism analysis of Chr 5 revealed that azole-resistant strains acquired TAC1 hyperactive alleles and, in most cases, ERG11 mutant alleles by LOH events not systematically including the MTL locus. TAC1 LOH resulted from mitotic recombination of the left arm of Chr 5, gene conversion within the TAC1 locus, or the loss and reduplication of the entire Chr 5. In one case, two independent TAC1 hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them TAC1 and ERG11. Our work shows that azole resistance was due not only to the presence of specific mutations in azole resistance genes (at least ERG11 and TAC1) but also to their increase in copy number by LOH and to the addition of extra Chr 5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in C. albicans is therefore a powerful instrument for generating genetic diversity.
| S-EPMC2043391 | biostudies-literature
Project description:Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol ?(5,6)-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ?256, 16, 16, 8, and 1 ?g ml?¹, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 ?g ml?¹); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 ?g ml?¹). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14?-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.
| S-EPMC2976150 | biostudies-literature
Project description:The azole antifungals arrest fungal growth through inhibition of ergosterol biosynthesis. We recently reported that a Candida albicans vps21Δ/Δ mutant, deficient in membrane trafficking through the late endosome/prevacuolar compartment (PVC), continues to grow in the presence of the azoles despite the depletion of cellular ergosterol. Here, we report that the vps21Δ/Δ mutant exhibits less plasma membrane damage upon azole treatment than the wild type, as measured by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21Δ/Δ mutant has abnormal levels of intracellular Ca2+ and, in the presence of fluconazole, enhanced expression of a calcineurin-responsive RTA2-GFP reporter. Furthermore, the azole tolerance phenotype of the vps21Δ/Δ mutant is dependent upon both extracellular calcium levels and calcineurin activity. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment and indicate that this may occur through modulation of calcium- and calcineurin-dependent responses.
| S-EPMC5118996 | biostudies-literature