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1.
Braz J Microbiol ; 53(2): 727-738, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35122657

RESUMO

BACKGROUND: The Candida glabrata does not develop into a pathogenic hiphal form; however, it has become the second most common pathogen of fungal infections in humans, partly because of its adhesion ability and virulence. OBJECTIVES: The present study aimed to determine whether Flo8, a transcription factor that plays an important role in the virulence and drug resistance in Candida albicans, has a similar role in C. glabrata. METHODS: We constructed FLO8 null strains of a C. glabrata standard strain and eight clinical strains from different sources, and a FLO8 complemented strain. Real-time quantitative PCR, biofilm formation assays, hydrophobicity tests, adhesion tests, Caenorhabditis elegans survival assay, and drug-susceptibility were then performed. RESULTS: Compared with the wild-type strains, the biofilm formation, hydrophobicity, adhesion, and virulence of the FLO8-deficient strains decreased, accompanied by decreased expression of EPA1, EPA6, and EPA7. On the other hand, it showed no changes in antifungal drug resistance, although the expression levels of CDR1, CDR2, and SNQ2 increased after FLO8 deletion. CONCLUSIONS: These results indicated that Flo8 is involved in the adhesion and virulence of C. glabrata, with FLO8 deletion leading to decreased expression of EPA1, EPA6, and EPA7 and decreased biofilm formation, hydrophobicity, adhesion, and virulence.


Assuntos
Candida glabrata , Proteínas Fúngicas , Antifúngicos/farmacologia , Biofilmes , Candida albicans/metabolismo , Candida glabrata/genética , Candida glabrata/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Virulência
2.
Theor Appl Genet ; 135(1): 351-365, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34665265

RESUMO

KEY MESSAGE: YrFDC12 and PbcFDC, co-segregated in chromosome 4BL, and significantly interacted with Yr30/Pbc1 to enhance stripe rust resistance and to promote pseudo-black chaff development. Cultivars with durable resistance are the most popular means to control wheat stripe rust. Durable resistance can be achieved by stacking multiple adult plant resistance (APR) genes that individually have relatively small effect. Chinese wheat cultivars Ruihua 520 (RH520) and Fengdecun 12 (FDC12) confer partial APR to stripe rust across environments. One hundred and seventy recombinant inbred lines from the cross RH520 × FDC12 were used to determine the genetic basis of resistance and identify genomic regions associated with stripe rust resistance. Genotyping was carried out using 55 K SNP array, and eight quantitative trait loci (QTL) were detected on chromosome arms 2AL, 2DS, 3BS, 4BL, 5BL (2), and 7BL (2) by inclusive composite interval mapping. Only QYr.nwafu-3BS from RH520 and QYr.nwafu-4BL.2 (named YrFDC12 for convenience) from FDC12 were consistent across the four testing environments. QYr.nwafu-3BS is likely the pleiotropic resistance gene Sr2/Yr30. YrFDC12 was mapped in a 2.1-cM interval corresponding to 12 Mb and flanked by SNP markers AX-111121224 and AX-89518393. Lines harboring both Yr30 and YrFDC12 displayed higher resistance than the parents and expressed pseudo-black chaff (PBC) controlled by loci Pbc1 and PbcFDC12, which co-segregated with Yr30 and YrFDC12, respectively. Both marker-based and pedigree-based kinship analyses revealed that YrFDC12 was inherited from founder parent Zhou 8425B. Fifty-four other wheat cultivars shared the YrFDC12 haplotype. These results suggest an effective pyramiding strategy to acquire highly effective, durable stripe rust resistance in breeding.


Assuntos
Cromossomos de Plantas , Resistência à Doença/genética , Genes de Plantas , Doenças das Plantas/genética , Puccinia/fisiologia , Triticum/genética , Mapeamento Cromossômico , Técnicas de Genotipagem , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Puccinia/imunologia , Locos de Características Quantitativas , Triticum/imunologia , Triticum/microbiologia
3.
Braz J Microbiol ; 51(4): 2183, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32794079

RESUMO

Unfortunately, an error occurred in the author affiliations.

4.
Braz J Microbiol ; 51(4): 1553-1561, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32648240

RESUMO

The wide use of antifungal agents has led to the development of resistance in the pathogenic yeast strain Candida albicans. Gain-of-function mutations in transcription factors such as Tac1p demonstrated their ability to control expression of the ABC transporter genes CDR1 and CDR2, and mediation of azole resistance. Previously, we obtained a series of azole-resistant isolates with high-level expression of CDR1 or/and CDR2, and identified the novel H741D mutation in Tac1p. In the present study, the TAC1 alleles from isolate C13 were introduced into tac1Δ/Δ mutant. The H741D change was seen in TAC1C13 in addition to several other amino acid differences. Hyperactive alleles TAC1C13 exhibited higher minimum inhibitory concentrations (MICs) of fluconazole and itraconazole than that observed in SN152 containing the wild-type TAC1 allele. And alleles TAC1C13 conferred constitutively high levels of Cdr1p and Cdr2p. Moreover, the importance of H741D in conferring hyperactivity to TAC1 was also confirmed by site-directed mutagenesis. Compared with SN152, the presence of H741D resulted in > 2-fold increase in CDR1 and CDR2 gene and protein expression, > 4-fold increase in fluconazole and itraconazole MICs and higher rates of Rhodamine 6G efflux by 43.24%.


Assuntos
Candida albicans/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fatores de Transcrição/genética , Fluconazol/farmacologia , Regulação Fúngica da Expressão Gênica , Itraconazol/farmacologia , Mutação
5.
Braz J Microbiol ; 51(4): 1665-1672, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32557281

RESUMO

With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Candida albicans/enzimologia , Reposicionamento de Medicamentos , Proteínas Fúngicas/antagonistas & inibidores , Testes de Sensibilidade Microbiana
6.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544776

RESUMO

Genetic information of polymerase chain reaction (PCR)-based markers, one of the main tools of genetics and genomics research in wheat, have been well documented in wheat. However, the physical position in relation to these markers has not yet been systematically characterized. Aim of this study was to characterize the physical information of thousands of widely used molecular markers.We first assigned 2705 molecular markers to wheat physical map, of which 86.1% and 84.7% were the best hits to chromosome survey sequencing (CSS) project (CSS-contigs) and International Wheat Genome Sequencing Consortium Reference Sequence v1.0 (IWGSC RefSeq v1.0), respectively. Physical position of 96.2% markers were predicated based on BLAST analysis, were in accordance with that of the previous nullisomic/aneuploidy/linkage analysis. A suggestive high-density physical map with 4643 loci was constructed, spanning 14.01 Gb (82.4%) of the wheat genome, with 3.02 Mb between adjacent markers. Both forward and reverse primer sequences of 1166 markers had consistent best hits to IWGSC RefSeq v1.0 based on BLAST analysis, and the corresponding allele sizes were characterized. A detailed physical map with 1532 loci was released, spanning 13.93 Gb (81.9%) of the wheat genome, with 9.09 Mb between adjacent markers. Characteristic of recombination rates in different chromosomal regions was discussed. In addition, markers with multiple sites were aligned to homoeologous sites with a consistent order, confirming that a collinearity existed among A, B and D subgenomes. This study facilitates the integration of physical and genetical information of molecular markers, which could be of value for use in genetics and genomics research such as gene/QTL map-based cloning and marker-assisted selection.


Assuntos
Mapeamento Físico do Cromossomo , Triticum/genética , Ligação Genética , Marcadores Genéticos , Genoma de Planta , Reação em Cadeia da Polimerase , Recombinação Genética/genética
7.
Res Microbiol ; 170(6-7): 272-279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31449848

RESUMO

Candida albicans has the ability to switch reversibly between budding yeast, filamentous, pseudohypha, and hyphal forms, a process in which the transcription factor Flo8 plays an important role. This ability is important for the virulence and pathogenicity of C. albicans. To determine whether Flo8 plays a role in the regulation of drug sensitivity, we constructed a FLO8 null mutant flo8/flo8 from the parental strain SN152 and a Flo8-overexpressing strain, flo8/flo8::FLO8. The susceptibility of the isolates to antifungal agents was then evaluated using the agar dilution and broth microdilution methods. Expression of drug resistance-related genes by the isolates was investigated by real-time PCR. The flo8/flo8 mutation isolates exhibited increased resistance to fluconazole, voriconazole, and itraconazole compared with the wild-type and drug sensitivity was restored by FLO8 overexpression (flo8/flo8∷FLO8). Of seven drug resistance-related genes, the FLO8 null mutation resulted in increased CDR1 and CDR2 expression (1.60-fold and 5.27-fold, respectively) compared with SN152, while FLO8 overexpression resulted in decreased CDR1 expression (0.63-fold). These results suggest that Flo8 is involved in the susceptibility of C. albicans to antifungal azoles, with FLO8 deletion leading to constitutive overexpression of CDR1 and CDR2 and resistance to antifungal azoles.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/genética , Farmacorresistência Fúngica/genética , Transativadores/genética , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Proteínas Fúngicas/genética , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , /genética
8.
Braz J Microbiol ; 50(1): 157-163, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30617866

RESUMO

This study aimed to establish the influence of biofilm from clinical isolates of Candida albicans on fluconazole resistance, focusing on efflux pumps and azole-targeted enzymes. Twenty-three C. albicans clinical isolates were collected from two hospitals in Shanghai, China. Antifungal susceptibility tests were performed on biofilm and planktonic cells. A crystal violet assay was used to monitor biofilm growth. Real-time RT-PCR was performed to quantify the expression of the transporter-related genes MDR1, CDR1, and CDR2 as well as ERG11, a gene encoding an enzyme targeted by antifungal drugs. Fluconazole resistance was shown to increase in biofilm in a time-dependent manner. No significant differences were observed between different strains of C. albicans. Genes encoding efflux pumps were overexpressed in early stages of biofilm formation and could also be induced by fluconazole. While ERG11 was not upregulated in biofilm, it was overexpressed upon the addition of fluconazole to biofilm and planktonic cells. Gene expression also appeared to be related to the original genotype of the strain. The upregulation of genes encoding efflux pumps demonstrates their role in the development of fluconazole resistance during the early stages of C. albicans biofilm formation.


Assuntos
Antifúngicos/farmacologia , Biofilmes , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Farmacorresistência Fúngica , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Biofilmes/efeitos dos fármacos , Candida albicans/isolamento & purificação , Candida albicans/fisiologia , China , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana
9.
Infect Genet Evol ; 44: 418-424, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27456280

RESUMO

To explore the putative correlation between the multilocus sequence types (MLST) and antifungal susceptibility of clinical Candida tropicalis isolates in Mainland China. Eighty-two clinical C. tropicalis isolates were collected from sixty-nine patients at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, from July 2012 to February 2015, and antifungal susceptibility tests were performed. Genetic profiles of those 82 isolates (30 azole-resistant and 52 azole-susceptible) were characterised by multilocus sequence typing. Phylogenetic analysis of the data was conducted with the clustering method, using UPGMA (unweighted pair group method with arithmetic averages) and the minimal spanning tree algorithm. MLST clonal clusters were analysed using the eBURST V3 package. Of the six gene fragments identified in multilocus sequence typing, SAPT4 presented the highest typing efficiency, whereas SAPT2 was the least efficient. Of the 44 diploid sequence types (DSTs) differentiated, 32 DSTs and 12 genotypes were identified as new to the C. tropicalis DST database. Twenty (45.45%) of the 44 DSTs were assigned to seven major groups based on eBURST analysis. Of these, Group 6, which contained DST 376, DST 505, DST 506 and DST 507, accounted for 76.7% of the 30 azole-resistant isolates. However, the genetic relationships among the azole-susceptible isolates were relatively decentralised. This MLST analysis of the putative correlation between the MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Mainland China shows that DSTs 376, 505, 506 and 507 are closely related azole-resistant C. tropicalis clones.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/genética , Candidíase/microbiologia , Farmacorresistência Fúngica , Alelos , Candida tropicalis/classificação , Candidíase/epidemiologia , China , Análise por Conglomerados , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia
10.
Int J Antimicrob Agents ; 46(5): 552-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26404130

RESUMO

The Candida albicans zinc cluster proteins are a family of transcription factors (TFs) that play essential roles in the development of antifungal drug resistance. Gain-of-function mutations in several TFs, such as Tac1p, Mrr1p and Upc2p, have been previously well documented in azole-resistant clinical C. albicans isolates. Mrr2p (multidrug resistance regulator 2) is a novel TF controlling expression of the ABC transporter gene CDR1 and mediating fluconazole resistance. In this study, the relationship between naturally occurring mutations in MRR2 and fluconazole resistance in clinical C. albicans isolates was investigated. Among a group of 20 fluconazole-resistant clinical C. albicans and 10 fluconazole-susceptible C. albicans, 12 fluconazole-resistant isolates overexpressed CDR1 by at least two-fold compared with the fluconazole-susceptible isolates. Of these 12 resistant isolates, three (C7, C9, C15) contained 11 identical missense mutations, 6 of which occurred only in the azole-resistant isolates. The contribution of these mutations to CDR1 overexpression and therefore to fluconazole resistance was further verified by generating recombinant strains containing the mutated MRR2 gene. The mutated MRR2 alleles from isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance; the missense mutations S466L and T470N resulted in an increase in CDR1 expression of more than two-fold and a four-fold increase in fluconazole resistance. In contrast, the other four missense mutations conferred only two- to four-fold increases in fluconazole resistance, with no significant increase in CDR1 expression. These findings provide some insight into the mechanism by which MRR2 regulates C. albicans multidrug resistance.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Farmacorresistência Fúngica , Fluconazol/farmacologia , Mutação de Sentido Incorreto , Fatores de Transcrição/genética , Candida albicans/isolamento & purificação , Candidíase/microbiologia , Proteínas Fúngicas/biossíntese , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras/biossíntese , Regulação para Cima
11.
Microbiol Res ; 178: 1-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26302841

RESUMO

The ability of Candida albicans to switch between multiple morphological states, including yeast (blastospores), fliamentous, pseudohyphal and hyphal forms, has been shown to be important for its pathogenicity and virulence. The transcription factor Flo8, which contains the LisH domain, is a downstream regulator of the cAMP/PKA pathway. Four clinical strains from adult women with recurrent vaginitis were isolated, and their morphology was observed. The results showed that two strains presented longer hyphal threads, stronger adherence to plastic and invasion into agar medium, and one strain was defective in filament and biofilm growth. Interestingly, mutations in the FLO8 gene were identified in these strains. We analyzed the contribution of these mutants to filamentous growth by constructing mutant strains and investigating their morphological and ultrastructural characteristics, including putative virulence traits, in vitro and in vivo. The results showed that the G723R and T751D Flo8 mutants enhanced activation of the Flo8C terminus, thereby promoting filamentous growth and increasing virulence.


Assuntos
Candida albicans/fisiologia , Hifas/crescimento & desenvolvimento , Mutação de Sentido Incorreto , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Adulto , Biofilmes/crescimento & desenvolvimento , Candida albicans/citologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Candidíase Vulvovaginal/microbiologia , Adesão Celular , Feminino , Humanos , Hifas/citologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fatores de Transcrição/genética , Virulência , Fatores de Virulência/genética
12.
Mycopathologia ; 180(3-4): 203-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25977140

RESUMO

The rate of Candida spp. infection is increasing, and resistance to azole antifungals is becoming increasingly common. Therefore, there is a need for discovery of new antifungal agents and for development of new modes of treatment using existing agents. In this in vitro study, the antifungal activity of two new imidazole derivatives was tested against a set of 20 Candida isolates, which included five different species. Treatment was carried out with the compound alone and in combination with fluconazole. Overall, we found that one of the new compounds, 31, was similar to fluconazole (FLC) in its efficacy against the Candida isolates and that compound 42 was superior to FLC. Furthermore, when combined with FLC, both compounds showed synergistic effects against 17 of the 20 tested isolates. No antagonistic interactions were observed. This study shows that our two new imidazole-derived compounds have good potential as general treatments for Candida infection and as a means to improve the current treatments with FLC.


Assuntos
Azóis/farmacologia , Candida/efeitos dos fármacos , Interações Medicamentosas , Azóis/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular
13.
Res Microbiol ; 166(3): 153-61, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25748216

RESUMO

This study was undertaken to characterize the mechanism(s) of azole resistance in clinical isolates of Candida albicans collected in Shanghai, China, focusing on the role of efflux pumps, target enzymes of fluconazole (Erg11), respiratory status and the ergosterol biosynthetic pathway. Clinical isolates of C. albicans (n = 30) were collected from 30 different non-HIV-infected patients in four hospitals in Shanghai. All 30 C. albicans isolates were susceptible to amphotericin B and 5-fluorocytosine. Twelve C. albicans isolates showed resistance to at least one type of triazole antifungal. Flow cytometry analysis of rhodamine 6G efflux showed that azole-resistant isolates had greater efflux pump activity, which was consistent with elevated levels of CDR1 and CDR2 genes that code for ABC efflux pumps. However, we did not observe increased expression of ERG11 and MDR1 or respiratory deficiency. Several mutations of ERG11 and TAC1 genes were detected. The F964Y mutation in the TAC1 gene was identified for the first time. Two main sterols, ergosterol and lanosterol, were identified by GC-MS chromatogram, and no missense mutations were found in ERG3. Furthermore, seven amino acid substitutions in ERG11, A114S, Y132H, Y132F, K143Q, K143R, Y257H and G448E were found, by Type II spectral quantitative analysis, to contribute to low affinity binding between Erg11 and fluconazole.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacocinética , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Farmacorresistência Fúngica , Fluconazol/farmacologia , Substituição de Aminoácidos , China , Farmacorresistência Fúngica/genética , Ergosterol/análise , Ergosterol/isolamento & purificação , Citometria de Fluxo , Fluconazol/metabolismo , Genes Fúngicos , Genes MDR , Humanos , Lanosterol/análise , Lanosterol/isolamento & purificação , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
14.
Thromb Haemost ; 113(3): 585-92, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25503412

RESUMO

Mutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %, 14.2 %, 5.2 %, 19.2 %, 1.8 % and 2.5 % of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.


Assuntos
Coagulação Sanguínea/genética , Fator VIII/genética , Hemofilia A/genética , Mutação , Sítios de Splice de RNA , RNA Mensageiro/genética , Simulação por Computador , Análise Mutacional de DNA , Éxons , Regulação da Expressão Gênica , Predisposição Genética para Doença , Hemofilia A/sangue , Humanos , Linhagem , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Molecules ; 19(10): 15653-72, 2014 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-25268720

RESUMO

A compound containing an imidazole moiety and a 2,4-dienone motif with significant activity toward several fungi was discovered in a screen for new antifungal compounds. Then, a total of 26 derivatives of this compound were designed, synthesized and evaluated through in vitro and in vivo antifungal activity assays. Several compounds exhibited improved antifungal activities compared to the lead compound. Of the derivatives, compounds 31 and 42 exhibited strong, broad-spectrum inhibitory effects toward Candida spp. In particular, the two derivatives exhibited potent antifungal activities toward the fluconazole-resistant isolate C. albicans 64110, with both having MIC values of 8 µg/mL. In addition, they had significant inhibitory effects toward two Gram-positive bacteria, Staphylococcus aureus UA1758 (compound 31: MIC = 8 µg/mL; compound 42: MIC = 4 µg/mL) and Staphylococcus epidermidis UF843 (compound 31: MIC = 8 µg/mL; compound 42: MIC = 8 µg/mL). The results of an animal experiment indicated that both compounds could improve the survival rate of model mice infected with ATCC 90028 (fluconazole-susceptible isolate). More importantly, the two compounds exhibited notable in vivo effects toward the fluconazole-resistant C. albicans isolate, which is promising with regard to the clinical problem posed by fluconazole-resistant Candida species.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Imidazóis/química , Imidazóis/farmacologia , Animais , Antibacterianos/síntese química , Antifúngicos/síntese química , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Candidíase/mortalidade , Técnicas de Química Sintética , Feminino , Imidazóis/síntese química , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/mortalidade , Staphylococcus/efeitos dos fármacos , Relação Estrutura-Atividade
16.
FEMS Yeast Res ; 13(4): 386-93, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23480635

RESUMO

The widespread use of azoles has led to increasing azole resistance among Candida albicans strains. One mechanism of azole resistance involves point mutations in the ERG11 gene, which encodes the target enzyme (cytochrome P450 lanosterol 14α-demethylase). In the present study, we amplified and sequenced the ERG11 gene of 23 C. albicans clinical isolates. Seventeen mutations encoding distinct amino acid substitutions were found, of which seven (K143Q, Y205E, A255V, E260V, N435V, G472R, and D502E) were novel. We further verified the contribution of the amino acid substitutions to azole resistance using site-directed mutagenesis of the ERG11 gene to recreate these mutations for heterologous expression in Saccharomyces cerevisiae. We observed that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significant increases (≧fourfold) in fluconazole and voriconazole resistance; changes in itraconazole resistance were not significant (≦twofold).


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Candida albicans/isolamento & purificação , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Análise Mutacional de DNA , DNA Fúngico/química , DNA Fúngico/genética , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Análise de Sequência de DNA
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