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Crystal Structures of Full-Length Lanosterol 14?-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provide Tools for Antifungal Discovery.

ABSTRACT: Targeting lanosterol 14?-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

SUBMITTER: Keniya MV

PROVIDER: S-EPMC6201081 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Heterologous Expression of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provides Tools for Antifungal Discovery.

Project description:Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are modest for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed these problems by expressing in Saccharomyces cerevisiae functional, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) for drug discovery purposes and determining their X-ray crystal structures. Compared with S. cerevisiae overexpressing LDM6×His (ScLDM6×His), the reduced susceptibility of CgLDM6×His to all azole drugs tested correlated with its level of overexpression. In contrast, the reduced susceptibility to short-tailed (fluconazole and voriconazole) but not medium-tailed (VT-1161) or long-tailed azoles (itraconazole and posaconazole) indicates CaLDM6×His works best when coexpressed with its cognate NADPH-cytochrome P450 reductase (CaNcp1A) rather than the host reductase (ScNcp1). Overexpression of LDM or Ncp1 modified the ergosterol content of yeast and affected growth inhibition by the polyene antibiotic amphotericin B. Affinity-purified recombinant Candida LDMs bind carbon monoxide and show tight type II binding of a range of azole drugs, including itraconazole, posaconazole, fluconazole, and voriconazole. This study provides a practical basis for the phenotype-, biochemistry-, and structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

| S-EPMC6201088 | biostudies-literature

Computational insights into fluconazole resistance by the suspected mutations in lanosterol 14α-demethylase (Erg11p) of Candida albicans.

Project description:Mutations in the ergosterol biosynthesis gene 11 (ERG11) of Candida albicans have been frequently reported in fluconazole-resistant clinical isolates. Exploring the mutations and their effect could provide new insights into the underlying mechanism of fluconazole resistance. Erg11p_Threonine285Alanine (Erg11p_THR285ALA), Erg11p_Leucine321Phenylalanine (Erg11p_LEU321PHE) and Erg11p_Serine457Proline (Erg11p_SER457PRO) are three fluconazole-resistant suspected mutations reported in clinical isolates of C. albicans. Therefore, our study aims to investigate the role of these suspected mutations in fluconazole resistance using in-silico methods. Molecular dynamics simulation (MDS) analysis of apo-protein for 25ns (nanosecond) showed that suspected mutant proteins underwent slight conformational changes in the tertiary structure. Molecular docking with fluconazole followed by binding free energy analysis showed reduced non-bonded interactions with loss of heme interaction and the least binding affinity for Erg11p_SER457PRO mutation. MDS of suspected mutant proteins-fluconazole complexes for 50ns revealed that Erg11p_SER457PRO and Erg11p_LEU321PHE have clear differences in the interaction pattern and loss or reduced heme interaction compared to wild type Erg11p-fluconazole complex. MDS and binding free energy analysis of Erg11p_SER457PRO-fluconazole complex showed the least binding similar to verified mutation Erg11p_TYR447HIS-fluconazole complex. Taken together, our study concludes that suspected mutation Erg11p_THR285ALA may not have any role whereas Erg11p_LEU321PHE could have a moderate role. However, Erg11p_SER457PRO mutation has a strong possibility to play an active role in fluconazole resistance of C. albicans.

| S-EPMC7731972 | biostudies-literature

Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing.

Project description:Introduction/objectivesAn increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting.MethodsForty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced.ResultsFifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2.ConclusionThis study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.

| S-EPMC6328131 | biostudies-literature

Antifungal Activity of a Hydroethanolic Extract From Astronium urundeuva Leaves Against Candida albicans and Candida glabrata.

Project description:We have previously reported on the activity of different extracts from Astronium sp. against Candida albicans, with the hydroethanolic extract prepared from leaves of A. urundeuva, an arboreal species widely distributed in arid environments of South America and often used in folk medicine, displaying the highest in vitro activity. Here we have further evaluated the antifungal activity of this extract against strains of C. albicans and C. glabrata, the two most common etiological agents of candidiasis. The extract was tested alone and loaded into a nanostructured lipid system (10% oil phase, 10% surfactant and 80% aqueous phase, 0.5% Poloxamer 407®). In vitro susceptibility assays demonstrated the antifungal activity of the free extract and the microemulsion against both Candida species, with increased activity against C. glabrata, including collection strains and clinical isolates displaying different levels of resistance against the most common clinically used antifungal drugs. Checkerboard results showed synergism when the free extract was combined with amphotericin B against C. albicans. Serial passage experiments confirmed development of resistance to fluconazole but not to the free extract upon prolonged exposure. Although preformed biofilms were intrinsically resistant to treatment with the extract, it was able to inhibit biofilm formation by C. albicans at concentrations comparable to those inhibiting planktonic growth. Cytotoxicity assays in different cell lines as well as an alternative model using Artemia salina L. confirmed a good safety profile of the both free and loaded extracts, and an in vivo assay demonstrated the efficacy of the free and loaded extracts when used topically in a rat model of vaginal candidiasis. Overall, these results reveal the promise of the A. urundeuva leaves extract to be further investigated and developed as an antifungal.

| S-EPMC6873212 | biostudies-literature

Biguanides enhance antifungal activity against Candida glabrata.

Project description:Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC50 of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC50 in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections.

| S-EPMC6086317 | biostudies-literature

The Candida albicans lanosterol 14-alpha-demethylase (ERG11) gene promoter is maximally induced after prolonged growth with antifungal drugs.

Project description:The azole antifungal drugs that target lanosterol 14-alpha-demethylase, encoded by the ERG11 gene, are used to treat a variety of infections caused by Candida albicans. Azoles are known to induce expression of ERG11 mRNA. The ERG11 promoter was cloned 5' of the luciferase-coding region, and the induction of ERG11 expression by azoles was monitored by luciferase assays. Maximal induction of the ERG11 promoter by azoles occurs not during logarithmic growth but after the diauxic shift and requires azoles to be present throughout logarithmic growth. The effects of pH, carbon source, and aerobic or anaerobic growth on induction of the ERG11 promoter by azoles were analyzed. Treatment with terbinafine and fenpropimorph, which target other enzymes in the ergosterol biosynthetic pathway, also resulted in a delayed induction of ERG11 promoter activity. Nascent sterol synthesis was shown to parallel ERG11 promoter activity, and total sterols were reduced coincident with the timing of ERG11 promoter activation. These results as a whole suggest that expression of the ERG11 promoter is regulated in response to sterol depletion.

| S-EPMC375326 | biostudies-literature

Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate.

Project description:Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.

| S-EPMC8621857 | biostudies-literature

Candida albicans and Candida glabrata clinical isolates exhibiting reduced echinocandin susceptibility.

Project description:A recognized hotspot for mutations conferring reduced echinocandin susceptibility (RES) is residue S645 of Candida albicans Gsc1(Fks1). We report that the mutation F641Y is associated with RES in a C. albicans isolate. The analogous Fks2 residue is mutated F to V in a Candida glabrata RES isolate; the introduction of this mutation into susceptible C. glabrata confirmed its role in RES. Y641-equivalent Fks residues were identified in intrinsically RES Fusarium species and Candida guilliermondii.

| S-EPMC1538661 | biostudies-literature

Propargyl-linked antifolates are dual inhibitors of Candida albicans and Candida glabrata.

Project description:Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized. Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values less than 1 μg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in achieving dual antifungal activity.

| S-EPMC3983340 | biostudies-literature

Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis.

Project description:Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

| S-EPMC4814137 | biostudies-literature

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