Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 54
Filtrar
1.
Antimicrob Agents Chemother ; 65(12): e0104421, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34516249

RESUMO

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.


Assuntos
Antifúngicos , Farmacorresistência Fúngica , Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/genética , Candida auris , Farmacorresistência Fúngica/genética , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , Oxirredutases , Esterol 14-Desmetilase/genética
2.
Med Mycol ; 59(8): 763-772, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-33550403

RESUMO

Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility.This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. LAY SUMMARY: A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/genética , Azóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aspergillus fumigatus/efeitos dos fármacos , Fator de Ligação a CCAAT/genética , Membrana Celular/química , Membrana Celular/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Cromatografia Gasosa-Espectrometria de Massas , Proteína HMGB1/genética , Autoantígeno Ku/antagonistas & inibidores , Autoantígeno Ku/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteróis/análise , Transcriptoma , Voriconazol/farmacologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-30455247

RESUMO

Candida glabrata is intrinsically less susceptible to azoles, and resistance to echinocandins and reduced susceptibility (RS) to amphotericin B (AMB) have also been detected. The molecular mechanisms of RS to AMB were investigated in C. glabrata strains in Kuwait by sequence analyses of genes involved in ergosterol biosynthesis. A total of 1,646 C. glabrata isolates were tested by Etest, and results for 12 selected isolates were confirmed by reference broth microdilution. PCR sequencing of three genes (ERG2, ERG6, and ERG11) was performed for all isolates with RS to AMB (RS-AMB isolates) and 5 selected wild-type C. glabrata isolates by using gene-specific primers. The total cell sterol content was analyzed by gas chromatography-mass spectrometry. The phylogenetic relationship among the isolates was investigated by multilocus sequence typing. Wild-type isolates contained only synonymous mutations in ERG2, ERG6, or ERG11, and the total sterol content was similar to that of the reference strains. A nonsynonymous ERG6 mutation (AGA48AAA, R48K) was found in both RS-AMB and wild-type isolates. Four RS-AMB isolates contained novel nonsense mutations at Trp286, Tyr192, and Leu341, and 2 isolates contained a nonsynonymous mutation in ERG6 (V126F or C198F); and the sterol content of these isolates was consistent with ERG6 deficiency. Two other RS-AMB isolates contained a novel nonsynonymous ERG2 mutation (G119S or G122S), and their sterol content was consistent with ERG2 deficiency. Of 8 RS-AMB isolates, 1 fluconazole-resistant isolate also contained nonsynonymous Y141H plus L381M mutations, while 7 isolates contained only synonymous mutations in ERG11 All isolates with ERG6, ERG2, and ERG11 mutations were genotypically distinct strains. Our data show that ERG6 and ERG2 are major targets conferring RS-AMB in clinical C. glabrata isolates.


Assuntos
Anfotericina B/farmacologia , Candida glabrata/efeitos dos fármacos , Candida glabrata/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas Fúngicas/metabolismo , Metiltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Humanos , Metiltransferases/genética , Mutação/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-30783005

RESUMO

Recombinant Candida albicans CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC50) using CYP51 reconstitution assays. The greatest increase in the IC50 compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.3-fold), Y132H+K143R (22.1-fold), Y132F+F145L (10.1-fold), G307S+G450E (13-fold), and D278N+G464S (3.3-fold). The single substitutions K143R, D278N, S279F, S405F, G448E, and G450E conferred at least 2-fold increases in the fluconazole IC50, and the Y132F, F145L, Y257H, Y447H, V456I, G464S, R467K, and I471T substitutions conferred increased residual CYP51 activity at high fluconazole concentrations. In vitro testing of select CaCYP51 mutations in C. albicans showed that the Y132F, Y132H, K143R, F145L, S405F, G448E, G450E, G464S, Y132F+K143R, Y132F+F145L, and D278N+G464S substitutions conferred at least a 2-fold increase in the fluconazole MIC. The catalytic tolerance of the purified proteins to voriconazole, itraconazole, and posaconazole was far lower and limited to increased residual activities at high triazole concentrations for certain mutations rather than large increases in IC50 values. Itraconazole was the most effective at inhibiting CaCYP51. However, when tested against CaCYP51 mutant strains, posaconazole seemed to be the most resistant to changes in MIC as a result of CYP51 mutation compared to itraconazole, voriconazole, or fluconazole.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Esterol 14-Desmetilase/metabolismo , Sequência de Aminoácidos , Candida albicans/genética , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Itraconazol/farmacologia , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Esterol 14-Desmetilase/genética , Triazóis/farmacologia , Voriconazol/farmacologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-29439966

RESUMO

The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (n = 96) with a MIC range of 0.016 to 0.25 µg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 µg/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 µg/ml), especially Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 µg/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo, once-daily intranasal administration of PC1244 (3.2 to 80 µg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans.


Assuntos
Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Triazóis/farmacologia , Administração Intranasal , Animais , Aspergillus fumigatus/isolamento & purificação , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Citocinas/sangue , Farmacorresistência Fúngica , Células Epiteliais/metabolismo , Ergosterol/biossíntese , Proteínas Fúngicas/antagonistas & inibidores , Galactose/análogos & derivados , Humanos , Hifas/metabolismo , Mananas/sangue , Camundongos , Testes de Sensibilidade Microbiana , Rhizopus/efeitos dos fármacos , Trichophyton/efeitos dos fármacos , Voriconazol/farmacologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-28483956

RESUMO

Prior to characterization of antifungal inhibitors that target CYP51, Trichophyton rubrum CYP51 was expressed in Escherichia coli, purified, and characterized. T. rubrum CYP51 bound lanosterol, obtusifoliol, and eburicol with similar affinities (dissociation constant [Kd ] values, 22.7, 20.3, and 20.9 µM, respectively) but displayed substrate specificity, insofar as only eburicol was demethylated in CYP51 reconstitution assays (turnover number, 1.55 min-1; Km value, 2 µM). The investigational agent VT-1161 bound tightly to T. rubrum CYP51 (Kd = 242 nM) with an affinity similar to that of clotrimazole, fluconazole, ketoconazole, and voriconazole (Kd values, 179, 173, 312, and 304 nM, respectively) and with an affinity lower than that of itraconazole (Kd = 53 nM). Determinations of 50% inhibitory concentrations (IC50s) using 0.5 µM CYP51 showed that VT-1161 was a tight-binding inhibitor of T. rubrum CYP51 activity, yielding an IC50 of 0.14 µM, whereas itraconazole, fluconazole, and ketoconazole had IC50s of 0.26, 0.4, and 0.6 µM, respectively. When the activity of VT-1161 was tested against 34 clinical isolates, VT-1161 was a potent inhibitor of T. rubrum growth, with MIC50, MIC90, and geometric mean MIC values of ≤0.03, 0.06, and 0.033 µg ml-1, respectively. With its selectivity versus human CYP51 and drug-metabolizing cytochrome P450s having already been established, VT-1161 should prove to be safe and effective in combating T. rubrum infections in patients.


Assuntos
Antifúngicos/farmacologia , Piridinas/farmacologia , Tetrazóis/farmacologia , Trichophyton/efeitos dos fármacos , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Clotrimazol/farmacologia , Farmacorresistência Fúngica , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Itraconazol/farmacologia , Cetoconazol/farmacologia , Testes de Sensibilidade Microbiana , Esterol 14-Desmetilase/metabolismo , Especificidade por Substrato , Voriconazol/farmacologia
7.
Artigo em Inglês | MEDLINE | ID: mdl-28630186

RESUMO

Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2 Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida parapsilosis/efeitos dos fármacos , Candida parapsilosis/genética , Equinocandinas/farmacologia , Oxirredutases/genética , Azóis/metabolismo , Candida parapsilosis/isolamento & purificação , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Farmacorresistência Fúngica Múltipla/genética , Equinocandinas/metabolismo , Ergosterol/biossíntese , Ergosterol/genética , Fungemia/tratamento farmacológico , Fungemia/microbiologia , Fungemia/prevenção & controle , Dosagem de Genes/genética , Genoma Fúngico/genética , Humanos , Testes de Sensibilidade Microbiana , Polimorfismo de Nucleotídeo Único/genética
8.
Artigo em Inglês | MEDLINE | ID: mdl-28223388

RESUMO

The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 µM and 0.22 µM, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to >8 µg/ml, while those of voriconazole ranged from 0.064 to 4 µg/ml. Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 µg/ml, whereas voriconazole (0.019 to >1 µg/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 µg/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 µg/mouse, while posaconazole showed similar effects (44%) at 14 µg/mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung.


Assuntos
Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Benzamidas/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Triazóis/farmacologia , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/isolamento & purificação , Células Cultivadas , Sistema Enzimático do Citocromo P-450 , Humanos , Itraconazol/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Voriconazol/farmacologia
9.
Antimicrob Agents Chemother ; 60(8): 4530-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27161631

RESUMO

Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [Kd] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (Kd range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (Kd, 4.53 µM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC50] range, 0.14 to 0.20 µM), while it only weakly inhibited human CYP51 activity (IC50, ∼600 µM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes.


Assuntos
Antifúngicos/farmacologia , Cryptococcus/efeitos dos fármacos , Piridinas/efeitos adversos , Piridinas/farmacologia , Esterol 14-Desmetilase/metabolismo , Tetrazóis/efeitos adversos , Tetrazóis/farmacologia , Antifúngicos/efeitos adversos , Clotrimazol/efeitos adversos , Clotrimazol/farmacologia , Cryptococcus/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ergosterol/metabolismo , Fluconazol/efeitos adversos , Fluconazol/farmacologia , Humanos , Itraconazol/efeitos adversos , Itraconazol/farmacologia , Cetoconazol/efeitos adversos , Cetoconazol/farmacologia , Lanosterol/análogos & derivados , Lanosterol/metabolismo , Voriconazol/efeitos adversos , Voriconazol/farmacologia
10.
Antimicrob Agents Chemother ; 59(10): 5942-50, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26169412

RESUMO

While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Candida/genética , Farmacorresistência Fúngica/genética , Regulação Fúngica da Expressão Gênica , Esteróis/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/isolamento & purificação , Candida/metabolismo , Candidíase/microbiologia , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , Análise de Sequência de DNA , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Esteróis/agonistas , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica
11.
Antimicrob Agents Chemother ; 59(12): 7771-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26459890

RESUMO

The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol, purified recombinant Aspergillus fumigatus CPR1 (AfCPR1), and Escherichia coli membrane suspensions containing recombinant A. fumigatus CYP51 proteins, allowing in vitro screening of azole antifungals. Azole-CYP51 studies determining the 50% inhibitory concentration (IC50) showed that A. fumigatus CYP51B (Af51B IC50, 0.50 µM) was 34-fold more susceptible to inhibition by fluconazole than A. fumigatus CYP51A (Af51A IC50, 17 µM) and that Af51A and Af51B were equally susceptible to inhibition by voriconazole, itraconazole, and posaconazole (IC50s of 0.16 to 0.38 µM). Af51A-G54W and Af51A-M220K enzymes were 11- and 15-fold less susceptible to inhibition by itraconazole and 30- and 8-fold less susceptible to inhibition by posaconazole than wild-type Af51A, confirming the azole-resistant phenotype of these two Af51A mutations. Susceptibility to voriconazole of Af51A-G54W and Af51A-M220K was only marginally lower than that of wild-type Af51A. Susceptibility of Af51A-L98H to inhibition by voriconazole, itraconazole, and posaconazole was only marginally lower (less than 2-fold) than that of wild-type Af51A. However, Af51A-L98H retained 5 to 8% residual activity in the presence of 32 µM triazole, which could confer azole resistance in A. fumigatus strains that harbor the Af51A-L98H mutation. The AfCPR1/Af51 assay system demonstrated the biochemical basis for the increased azole resistance of A. fumigatus strains harboring G54W, L98H, and M220K Af51A point mutations.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Aspergillus fumigatus/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Testes de Sensibilidade Microbiana , Mutação Puntual , Proteínas Recombinantes/química
12.
Antimicrob Agents Chemother ; 59(8): 4707-13, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26014948

RESUMO

In this study, we investigate the amebicidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole, and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amebicidal activities of the triazoles were assessed by in vitro minimum inhibition concentration (MIC) determinations using trophozoites of A. castellanii and A. polyphaga. In addition, triazole effectiveness was assessed by ligand binding studies and inhibition of CYP51 activity of purified A. castellanii CYP51 (AcCYP51) that was heterologously expressed in Escherichia coli. Itraconazole and voriconazole bound tightly to AcCYP51 (dissociation constant [Kd] of 10 and 13 nM), whereas fluconazole bound weakly (Kd of 2,137 nM). Both itraconazole and voriconazole were confirmed to be strong inhibitors of AcCYP51 activity (50% inhibitory concentrations [IC50] of 0.23 and 0.39 µM), whereas inhibition by fluconazole was weak (IC50, 30 µM). However, itraconazole was 8- to 16-fold less effective (MIC, 16 mg/liter) at inhibiting A. polyphaga and A. castellanii cell proliferation than voriconazole (MIC, 1 to 2 mg/liter), while fluconazole did not inhibit Acanthamoeba cell division (MIC, >64 mg/liter) in vitro. Voriconazole was an effective inhibitor of trophozoite proliferation for A. castellanii and A. polyphaga; therefore, it should be evaluated in trials versus itraconazole for controlling Acanthamoeba infections.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Amebíase/tratamento farmacológico , Amebicidas/farmacologia , Antifúngicos/farmacologia , Azóis/farmacologia , Esterol 14-Desmetilase/metabolismo , Acanthamoeba castellanii/metabolismo , Amebíase/microbiologia , Proliferação de Células/efeitos dos fármacos , Fluconazol/farmacologia , Humanos , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana/métodos , Triazóis/farmacologia , Voriconazol/metabolismo
13.
Appl Environ Microbiol ; 81(10): 3379-86, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25746994

RESUMO

Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires cytochrome P450 reductase (CPR) as its redox partner for functional activity. The M. graminicola CPR (MgCPR) is able to catalyze the sterol 14α-demethylation of eburicol and lanosterol when partnered with Candida albicans CYP51 (CaCYP51) and that of eburicol only with M. graminicola CYP51 (MgCYP51). The availability of the functional in vivo redox partner enabled the in vitro catalytic activity of MgCYP51 to be demonstrated for the first time. MgCYP51 50% inhibitory concentration (IC50) studies with epoxiconazole, tebuconazole, triadimenol, and prothioconazole-desthio confirmed that MgCYP51 bound these azole inhibitors tightly. The characterization of the MgCPR/MgCYP51 redox pairing has produced a functional method to evaluate the effects of agricultural azole fungicides, has demonstrated eburicol specificity in the activity observed, and supports the conclusion that prothioconazole is a profungicide.


Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Esterol 14-Desmetilase/química , Sequência de Aminoácidos , Ascomicetos/química , Ascomicetos/genética , Candida albicans/enzimologia , Candida albicans/genética , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriais/química , Fungicidas Industriais/metabolismo , Lanosterol/análogos & derivados , Lanosterol/química , Lanosterol/metabolismo , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Oxirredução , Alinhamento de Sequência , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Especificidade por Substrato , Temperatura
14.
Appl Environ Microbiol ; 80(19): 6154-66, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25085484

RESUMO

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 µM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 µM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 µg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 µg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 µg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 µM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 µg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Antifúngicos/farmacologia , Clotrimazol/farmacologia , Doenças dos Peixes/tratamento farmacológico , Saprolegnia/efeitos dos fármacos , Animais , Antifúngicos/química , Azóis/química , Azóis/farmacologia , Vias Biossintéticas , Clotrimazol/química , Doenças dos Peixes/microbiologia , Peixes , Testes de Sensibilidade Microbiana/veterinária , Filogenia , Saprolegnia/enzimologia , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/genética , Esterol 14-Desmetilase/metabolismo , Esteróis/análise
15.
Nat Commun ; 15(1): 4261, 2024 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769341

RESUMO

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.


Assuntos
Antifúngicos , Aspergilose , Aspergillus , Ergosterol , Proteínas Fúngicas , Metiltransferases , Triazóis , Animais , Metiltransferases/metabolismo , Metiltransferases/genética , Antifúngicos/farmacologia , Aspergillus/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Camundongos , Aspergilose/microbiologia , Aspergilose/tratamento farmacológico , Ergosterol/metabolismo , Ergosterol/biossíntese , Triazóis/farmacologia , Regulação Fúngica da Expressão Gênica , Aspergillus fumigatus/genética , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/metabolismo , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Hifas/genética , Hifas/metabolismo , Feminino , Testes de Sensibilidade Microbiana , Virulência/genética
16.
Nat Commun ; 15(1): 3642, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38684680

RESUMO

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.


Assuntos
Antifúngicos , Aspergillus fumigatus , Ergosterol , Proteínas Fúngicas , Hidroximetilglutaril-CoA Redutases , Triazóis , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Antifúngicos/farmacologia , Triazóis/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ergosterol/metabolismo , Ergosterol/biossíntese , Hidroximetilglutaril-CoA Redutases/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Testes de Sensibilidade Microbiana , Esterol 14-Desmetilase/metabolismo , Esterol 14-Desmetilase/genética , Humanos , Mutação
17.
Antimicrob Agents Chemother ; 57(3): 1352-60, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23274672

RESUMO

Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K(s), 14 to 18 µM) and catalyzed the 14α-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K(d)s], 42 to 131 nM) but bound fluconazole (K(d), ~30,500 nM) and voriconazole (K(d), ~2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K(d)s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (K(d)s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (K(d), ~40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC(50)) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC(50), ~150 µM) and did not significantly inhibit Δ60HsCYP51.


Assuntos
Antifúngicos/química , Candida albicans/enzimologia , Proteínas Fúngicas/química , Esterol 14-Desmetilase/química , Candida albicans/química , Clotrimazol/química , Escherichia coli/genética , Fluconazol/química , Proteínas Fúngicas/genética , Humanos , Itraconazol/química , Cetoconazol/química , Cinética , Lanosterol/química , NADP/química , Ligação Proteica , Pirimidinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade da Espécie , Esterol 14-Desmetilase/genética , Triazóis/química , Voriconazol
18.
Antimicrob Agents Chemother ; 57(7): 3182-93, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23629718

RESUMO

Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 µg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 µg/ml), voriconazole (MIC > 16 µg/ml), and amphotericin B (MIC > 32 µg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6ß-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14α-demethylase and sterol Δ(5,6)-desaturase leading to azole-polyene cross-resistance.


Assuntos
Candida albicans/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Candida tropicalis/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Azóis/farmacologia , Sequência de Bases , Candida albicans/genética , Candida albicans/isolamento & purificação , Candida glabrata/genética , Candida glabrata/isolamento & purificação , Candida tropicalis/isolamento & purificação , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/metabolismo , Ergosterol/biossíntese , Ergosterol/genética , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Polienos/farmacologia , Pirimidinas/farmacologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Triazóis/farmacologia , Tunísia , Voriconazol
19.
Appl Environ Microbiol ; 79(5): 1639-45, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23275516

RESUMO

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Inibidores Enzimáticos/farmacologia , Esterol 14-Desmetilase/metabolismo , Triazóis/farmacologia , Antifúngicos/metabolismo , Inibidores Enzimáticos/metabolismo , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Triazóis/metabolismo , Voriconazol
20.
New Phytol ; 198(3): 821-835, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23442154

RESUMO

CYP51 encodes the cytochrome P450 sterol 14α-demethylase, an enzyme essential for sterol biosynthesis and the target of azole fungicides. In Fusarium species, including pathogens of humans and plants, three CYP51 paralogues have been identified with one unique to the genus. Currently, the functions of these three genes and the rationale for their conservation within the genus Fusarium are unknown. Three Fusarium graminearum CYP51s (FgCYP51s) were heterologously expressed in Saccharomyces cerevisiae. Single and double FgCYP51 deletion mutants were generated and the functions of the FgCYP51s were characterized in vitro and in planta. FgCYP51A and FgCYP51B can complement yeast CYP51 function, whereas FgCYP51C cannot. FgCYP51A deletion increases the sensitivity of F. graminearum to the tested azoles. In ΔFgCYP51B and ΔFgCYP51BC mutants, ascospore formation is blocked, and eburicol and two additional 14-methylated sterols accumulate. FgCYP51C deletion reduces virulence on host wheat ears. FgCYP51B encodes the enzyme primarily responsible for sterol 14α-demethylation, and plays an essential role in ascospore formation. FgCYP51A encodes an additional sterol 14α-demethylase, induced on ergosterol depletion and responsible for the intrinsic variation in azole sensitivity. FgCYP51C does not encode a sterol 14α-demethylase, but is required for full virulence on host wheat ears. This is the first example of the functional diversification of a fungal CYP51.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/efeitos dos fármacos , Fusarium/enzimologia , Fusarium/patogenicidade , Esterol 14-Desmetilase/metabolismo , Amidas/farmacologia , Arabidopsis/microbiologia , Azóis/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Ergosterol/genética , Ergosterol/metabolismo , Evolução Molecular , Proteínas Fúngicas/genética , Fusarium/fisiologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Lanosterol/análogos & derivados , Lanosterol/metabolismo , Malus/microbiologia , Mutação , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Esterol 14-Desmetilase/genética , Tricotecenos/metabolismo , Triticum/microbiologia , Virulência/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA