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1.
Medicine (Baltimore) ; 98(41): e17535, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31593129

RESUMO

Scedosporium genus as a significant emerging opportunist causes a broad spectrum of disease in not only immunosuppressed but also immunocompetent patients. The lung is one of the most commonly encountered sites of Scedosporium infection. Due to its very high levels of antifungal resistance, surgery has been recommended as an important part in the treatment of pulmonary Scedosporium spp infection, even in immunocompetent cases. However, whether lung surgery could help to reduce the risk of death in immunocompetent patients is not clear.We retrospectively retrieved the records of pulmonary infections with Scedosporium species in immunocompetent patients through a comprehensive literature search. The association of surgery on all-cause mortality was explored using binary logistic regression (BLR). Receiver operating characteristic (ROC) curve analysis was carried out to evaluate the capability of the model.The comprehensive searching strategy yielded 33 case reports and 3 case series in total, with 40 individual patients being included. The overall mortality was 12.50%. The fatality rate was 9.09% (2/22) in cases with surgery and 16.67% (3/18) in cases without surgery (odds ratio, 0.50; 95% confidence interval, 0.07-3.38; P = .48). Consistently, BLR analysis identified no statistical association between surgery and reduced mortality (odds ratio, 1.19; 95% confidence interval, 0.09-15.64; P = .89), after adjusting for age, gender, and antifungal chemotherapy. The area under the ROC curve was 0.88.For immunocompetent patients with pulmonary Scedosporium spp infection, surgical therapy may not be associated with reduced mortality. Surgical excision could be considered but is not imperative in this group of patients.


Assuntos
Pneumopatias Fúngicas/tratamento farmacológico , Pneumopatias Fúngicas/cirurgia , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/cirurgia , Scedosporium/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antifúngicos/uso terapêutico , Farmacorresistência Fúngica/fisiologia , Feminino , Humanos , Hospedeiro Imunocomprometido/efeitos dos fármacos , Pneumopatias Fúngicas/microbiologia , Pneumopatias Fúngicas/mortalidade , Masculino , Pessoa de Meia-Idade , Mortalidade/tendências , Micoses/tratamento farmacológico , Micoses/epidemiologia , Micoses/microbiologia , Micoses/mortalidade , Estudos Observacionais como Assunto , Cuidados Pós-Operatórios , Infecções Respiratórias/microbiologia , Infecções Respiratórias/mortalidade , Estudos Retrospectivos , Scedosporium/isolamento & purificação , Voriconazol/administração & dosagem , Voriconazol/uso terapêutico
2.
Res Microbiol ; 170(8): 417-425, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31562919

RESUMO

ABC transporters of the Pleiotropic Drug Resistance (PDR) family are the main actors of antifungal resistance in pathogenic fungi. While their involvement in clinical resistant strains has been proven, their transport mechanism remains unclear. Notably, one hallmark of PDR transporters is their asymmetry, with one canonical nucleotide-binding site capable of ATP hydrolysis while the other site is not. Recent publications reviewed here show that the so-called "deviant" site is of crucial importance for drug transport and is a step towards alleviating the mystery around the existence of non-catalytic binding sites.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/metabolismo , Candida albicans/metabolismo , Farmacorresistência Fúngica/fisiologia , Antifúngicos/farmacologia , Sítios de Ligação/fisiologia , Transporte Biológico/fisiologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candidíase/patologia , Humanos
3.
Sci Total Environ ; 681: 516-523, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31121401

RESUMO

Agrochemicals such as the non-azoles, used to improve crop productivity, poses severe undesirable effects on the environment and human health. In addition, they induce cross-resistance (CR) with clinical drugs in pathogenic fungi. However, till date emphasis has been given to the role of azoles on the induction of CR. Herein, we analyzed the effect of a non-azole agrochemical, pyraclostrobin (PCT), on the antifungal susceptibility and virulence of the human and animal pathogens Cryptococcus gattii and C. neoformans. We determined the minimum inhibitory concentration (MIC) of fluconazole (FLC), itraconazole, ravuconazole, amphotericin B, and PCT on colonies: (i) that were not exposed to PCT (non-adapted-NA-cultures), (ii) were exposed at the maximum concentration of PCT (adapted-A-cultures) and (iii) the adapted colonies after cultivation 10 times in PCT-free media (10 passages-10p-cultures). Our results showed that exposure to PCT induced both temporary and permanent CR to clinical azoles in a temperature-dependent manner. With the objective to understand the mechanism of induction of CR through non-azoles, the transcriptomes of NA and 10p cells from C. gattii R265 were analyzed. The transcriptomic analysis showed that expression of the efflux-pump genes (AFR1 and MDR1) and PCT target was higher in resistant 10p cells than that in NA. Moreover, the virulence of 10p cells was reduced as compared to NA cells in mice, as observed by the differential gene expression analysis of genes related to ion-metabolism. Additionally, we observed that FLC could not increase the survival rate of mice infected with 10p cells, confirming the occurrence of permanent CR in vivo. The findings of the present study demonstrate that the non-azole agrochemical PCT can induce permanent CR to clinical antifungals through increased expression of efflux pump genes in resistant cells and that such phenomenon also manifests in vivo.


Assuntos
Agroquímicos , Antifúngicos , Cryptococcus gattii/fisiologia , Farmacorresistência Fúngica/fisiologia , Estrobilurinas/toxicidade , Animais , Cryptococcus neoformans , Humanos , Camundongos , Testes de Sensibilidade Microbiana
4.
PLoS One ; 14(3): e0199484, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30856175

RESUMO

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate.


Assuntos
Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio/farmacologia , Triptofano/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Detergentes/farmacologia , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/fisiologia , Genes Fúngicos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos
6.
PLoS One ; 14(1): e0210883, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30673768

RESUMO

Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes. It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata. Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore, the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress), exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata Δvph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains. These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies.


Assuntos
Candida glabrata/efeitos dos fármacos , Candida glabrata/patogenicidade , Farmacorresistência Fúngica/fisiologia , Farmacorresistência Fúngica Múltipla/fisiologia , Proteínas Fúngicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Virulência/fisiologia , Animais , Antifúngicos/farmacologia , Candida glabrata/enzimologia , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Feminino , Fluconazol/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos , Humanos , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/metabolismo , Virulência/efeitos dos fármacos , Virulência/genética , Voriconazol/farmacologia
7.
Drug Dev Res ; 80(1): 24-27, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30370576

RESUMO

Natural products and derivatives thereof are of considerable importance in the discovery of new pharmaceuticals, for example, for the treatment of cancer, diabetes, inflammation diseases, and infection diseases caused by bacteria, fungi, viruses, or parasites. The great biodiversity of marine microorganisms is reflected in their huge chemical diversity, which provides a rich source of biologically active compounds. An increasing interest in marine microorganisms as promising producers of new compounds with potential medical applications has raised increasing interest in the sustainable exploration of marine microbial resources for the discovery of new antibiotics, which is highlighted. The bottlenecks in the development of drugs using the large marine natural product pipeline are also discussed.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Organismos Aquáticos , Produtos Biológicos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Organismos Aquáticos/isolamento & purificação , Organismos Aquáticos/microbiologia , Bactérias/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/fisiologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Fungos/isolamento & purificação , Humanos
8.
Biomed Pharmacother ; 110: 857-868, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30557835

RESUMO

Invasive candidiasis (IC) can affect individuals with various underlying diseases hospitalized in different parts of hospitals. In recent decades, IC has caused 27-55% mortality in general population. Although Candida albicans (C. albicans) is still the most common cause of IC, non-albicans infections such as C. krusei, C. glabrata, C. lusitaniae, C. tropicalis, and C. parapsilosis have been increased in recent years. Treatment of invasive fungal infections is challenging as the number of existing antifungals is limited and more problems include: toxicity, drug interactions, and drug resistance. These problems provide a clear rationale for the development of new immunotherapies to increase outcomes in patients with invasive fungal infections. Thus, the purpose of this paper is to complete review of the current and modern antifungal drugs in IC therapy and focus on the role of immunotherapy in preventing and controlling the disease. Therefore, we review the features of current research efforts directed towards devising safe and effective immunotherapeutic options for fungal infections, including work on antifungal vaccines, engineered T-cells, cytokines, monoclonal antibodies, and other agents.


Assuntos
Antifúngicos/uso terapêutico , Farmacorresistência Fúngica/efeitos dos fármacos , Imunoterapia/métodos , Micoses/tratamento farmacológico , Micoses/imunologia , Animais , Antifúngicos/farmacologia , Candidíase Invasiva/diagnóstico , Candidíase Invasiva/tratamento farmacológico , Candidíase Invasiva/imunologia , Farmacorresistência Fúngica/fisiologia , Humanos , Imunoterapia/tendências , Micoses/diagnóstico , Resultado do Tratamento
9.
Future Microbiol ; 13: 1175-1191, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30113223

RESUMO

The incidence of serious fungal infections is increasing rapidly, and yet the rate of new drugs becoming available to treat them is slow. The limited therapeutic armamentarium is a challenge for clinicians, because the available drugs are often toxic, expensive, difficult to administer, ineffective or a combination of all four. Given this setting, the emergence of resistance is especially concerning, and a review of the topic is timely. Here we discuss antifungal drug resistance in Candida spp. and Aspergillus spp. with reference to the most commonly used first-line antifungal agents - azoles and echinocandins. We review the resistance mechanisms of the leading pathogens, how resistance can be identified in the diagnostic lab and the clinical implications of resistance once detected.


Assuntos
Antifúngicos/farmacologia , Aspergillus/fisiologia , Candida/fisiologia , Farmacorresistência Fúngica , Aspergillus/classificação , Aspergillus/efeitos dos fármacos , Aspergillus/genética , Azóis/farmacologia , Candida/classificação , Candida/efeitos dos fármacos , Candida/genética , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/fisiologia , Equinocandinas/farmacologia , Humanos , Técnicas Microbiológicas , Modelos Biológicos , Micoses/diagnóstico , Micoses/tratamento farmacológico , Micoses/epidemiologia
10.
Int J Mol Sci ; 19(8)2018 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-30103374

RESUMO

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.


Assuntos
Farmacorresistência Fúngica/fisiologia , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Micélio/crescimento & desenvolvimento , Doenças das Plantas/microbiologia , Ácido Salicílico/metabolismo , Receptores Sulfonilureia/metabolismo , Triticum/microbiologia , Antifúngicos/farmacologia , Arabidopsis/microbiologia , Proteínas Fúngicas/genética , Fusarium/genética , Micélio/genética , Receptores Sulfonilureia/genética
11.
PLoS One ; 13(8): e0203079, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30157240

RESUMO

Among the several mechanisms of multidrug resistance (MDR), overexpression of drug efflux pumps CaCdr1p and CaMdr1p belonging to ATP binding cassette (ABC) and major facilitator superfamily (MFS) respectively remain the predominant mechanisms of candidal infections. Therefore inhibiting or modulating the function of these transporters continues to draw attention as effective strategy to combat MDR. We have previously reported the antifungal potential of Geraniol (Ger), a natural monoterpenoid from Palmarosa oil, against Candida albicans. Herein, we explored the fungicidal nature of Ger. The Rhodamine 6G (R6G) and Nile red accumulation confirms the specific effect on CaCdr1p. Mechanistic insights with Candida cells overexpressing CaCdr1p and CaMdr1p revealed that Ger specifically modulates CaCdr1p activity. Kinetic studies further unraveled the competitive inhibition of Ger for R6G efflux as evident from increased apparent Km without affecting Vmax value. The effect of Ger on CaCdr1p was substantiated by molecular docking analyses, which depicted in-silico binding affinity of Ger with CaCdr1p and explored that Ger binds to the active site of CaCdr1p with higher binding energy. Although RT-PCR and western blot revealed no change in expressions of CDR1 and CaCdr1p, confocal microscopy images however depicted CaCdr1p mislocalization in presence of Ger. Interestingly, Ger was synergistic (FICI<0.5) with fluconazole (FLC) which is a well known antifungal drug. Furthermore, Ger sensitizes the FLC sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated R6G efflux and depleted ergosterol. Furthermore, Rhodamine B labeling demonstrates altered mitochondrial potential with Ger which suggest possible linkage of dysfunctional mitochondria with CaCdr1p activity. We also estimated phenotypic virulence marker extracellular phospholipase activity which was considerably diminished along with inhibited cell adherence and biofilm biomass. Lastly, antifungal efficacy of Ger was demonstrated by enhanced survival of Caenorhabditis elegans model and negligible hemolytic activity (20%). Together, modulation of efflux pump activity by Ger and FLC synergism represent a promising approach for combinatorial treatment of candidiasis.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Terpenos/farmacologia , Animais , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans , Candida albicans/metabolismo , Adesão Celular/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Sinergismo Farmacológico , Células Epiteliais/metabolismo , Ergosterol/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Simulação de Acoplamento Molecular , Fosfolipases/metabolismo , Ligação Proteica
12.
Biosci Biotechnol Biochem ; 82(10): 1840-1848, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30011258

RESUMO

For strain improvement of Aspergillus oryzae, development of the transformation system is essential, wherein dominant selectable markers, including drug-resistant genes, are available. However, A. oryzae generally has a relatively high resistance to many antifungal drugs effective against yeasts and other filamentous fungi. In the course of the study, while investigating azole drug resistance in A. oryzae, we isolated a spontaneous mutant that exhibited high resistance to azole fungicides and found that pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter genes were upregulated in the mutant; their overexpression in the wild-type strain increased azole drug resistance. While deletion of the gene designated atrG resulted in increased azole susceptibility, double deletion of atrG and another gene (atrA) resulted in further azole hypersensitivity. Overall, these results indicate that the ABC transporters AtrA and AtrG are involved in azole drug resistance in A. oryzae.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Aspergillus oryzae/efeitos dos fármacos , Azóis/farmacologia , Farmacorresistência Fúngica/fisiologia
13.
Anticancer Res ; 38(7): 4281-4288, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29970562

RESUMO

BACKGROUND/AIM: Survivin expression has been shown to be associated with cancer progression, poor prognosis, and drug resistance. The aim of this study was to examine whether survivin knock-down could enhance paclitaxel-induced apoptosis in breast cancer cells in vitro. MATERIALS AND METHODS: MCF-7 cells were infected with an siRNA-expressing adenovirus vector against survivin (Adv-siSurv) or Renilla luciferase as a control (Adv-siRL). After treatment with paclitaxel, cells were analyzed by apoptotic, cell cycle and immunoblotting assays. RESULTS: Of cells treated with paclitaxel alone, only 20.2±2.08% showed apoptotic features. An increase in the paclitaxel dose was associated with increased survivin expression. In contrast, Adv-siSurv infection resulted in a marked increase in apoptotic cell death in paclitaxel-treated MCF-7 cells (49.9±7.70%). The percentage of cells in the G2M phase was lower (23.9±1.64%) in Adv-siSurv-infected cells than that of Adv-siRL-treated cells (40.0±2.43%). Adv-siSurv infection reduced survivin, procaspase-9, and procaspase-3 levels in paclitaxel-treated MCF-7 cells. CONCLUSION: Loss of survivin expression enhanced paclitaxel-induced apoptosis in MCF-7 breast cancer cells in vitro.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Terapia Genética/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Adenoviridae , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Farmacorresistência Fúngica/fisiologia , Feminino , Técnicas de Silenciamento de Genes/métodos , Vetores Genéticos , Humanos , Células MCF-7 , Paclitaxel/farmacologia , Survivina
14.
Artigo em Inglês | MEDLINE | ID: mdl-29038263

RESUMO

In vitro combinations of isavuconazole with echinocandins were evaluated against 30 Aspergillus strains with a two-dimensional checkerboard microdilution method and an agar-based diffusion method. With the checkerboard method, the three combinations showed indifferent interactions for all strains. With the agar-based method, indifferent interactions were found for all strains for isavuconazole-micafungin and isavuconazole-anidulafungin. For the isavuconazole-caspofungin combination, indifference was found in 24/30 strains, synergism in 4/30 strains, and antagonism in 2/30 strains.


Assuntos
Anidulafungina/farmacologia , Antifúngicos/farmacologia , Aspergillus flavus/efeitos dos fármacos , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus nidulans/efeitos dos fármacos , Aspergillus niger/efeitos dos fármacos , Micafungina/farmacologia , Nitrilos/farmacologia , Piridinas/farmacologia , Triazóis/farmacologia , Aspergillus flavus/isolamento & purificação , Aspergillus fumigatus/isolamento & purificação , Aspergillus nidulans/isolamento & purificação , Aspergillus niger/isolamento & purificação , Combinação de Medicamentos , Farmacorresistência Fúngica/fisiologia , Humanos , Testes de Sensibilidade Microbiana
16.
Microb Pathog ; 117: 32-42, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29229505

RESUMO

The increased incidence of candidemia in terciary hospitals worldwide and the cross-resistance frequency require the new therapeutic strategies development. Recently, our research group demonstrated three semi-synthetic naphthofuranquinones (NFQs) with a significant antifungal activity in a fluconazole-resistant (FLC) C. tropicalis strain. The current study aimed to investigate the action's preliminary mechanisms of NFQs by several standardized methods such as proteomic and flow cytometry analyzes, comet assay, immunohistochemistry and confocal microscopy evaluation. Our data showed C. tropicalis 24 h treated with all NFQs induced an expression's increase of proteins involved in the metabolic response to stress, energy metabolism, glycolysis, nucleosome assembly and translation process. Some aspects of proteomic analysis are in consonance with our flow cytometry analysis which indicated an augmentation of intracellular ROS, mitochondrial dysfunction and DNA strand breaks (neutral comet assay and γ-H2AX detection). In conclusion, our data highlights the great contribution of ROS as a key event, probably not the one, associated to anti-candida properties of studied NFQs.


Assuntos
Antifúngicos/farmacologia , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Naftoquinonas/farmacologia , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Antifúngicos/síntese química , Antifúngicos/química , Candida tropicalis/genética , Candidemia/microbiologia , Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Fúngico/genética , Metabolismo Energético/efeitos dos fármacos , Fluconazol/farmacologia , Glicólise/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Naftoquinonas/síntese química , Naftoquinonas/química , Estresse Psicológico
17.
Microbiology ; 163(11): 1540-1556, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29043954

RESUMO

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother-daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.


Assuntos
Acetilglucosamina/metabolismo , Parede Celular/metabolismo , Cryptococcus neoformans/metabolismo , Melaninas/metabolismo , Parede Celular/química , Parede Celular/ultraestrutura , Quitina/metabolismo , Quitosana/metabolismo , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/ultraestrutura , Farmacorresistência Fúngica/fisiologia , Ensaios Enzimáticos , Lacase/metabolismo , Melaninas/biossíntese , Testes de Sensibilidade Microbiana , Fenótipo
18.
Artigo em Inglês | MEDLINE | ID: mdl-28993330

RESUMO

Morphological heterogeneity of Aspergillus terreus cultures was observed during continued cultivation of amphotericin B (AMB)-resistant isolates on drug-free medium. Outgrowth leads to the emergence of multiple sectors that might result from increased growth rates at drug-free conditions. We evaluated the differences in AMB susceptibility and virulence between sector subcultures (ATSec), AMB-resistant (ATR) strains, and AMB-susceptible (ATS) strains. By comparing A. terreus AMB-resistant (ATR) strains and A. terreus sector (ATSec) cultures we observed a highly significant reduction of AMB MICs in ATSec (ATR MIC, 2 to 32 µg/ml; ATSec MIC, 0.12 to 5 µg/ml). Furthermore, Galleria mellonella survival studies revealed an enhanced virulence of ATSec, which was comparable with that of AMB-sensitive Aspergillus terreus strains (median survival rates for ATS isolates, 72 h; for ATSec isolate ATSecG1, 84 h; for ATR isolates, 144 h). Our findings clearly demonstrate that spontaneous culture degeneration occurs in A. terreus and, most importantly, crucially impacts drug efficacy and virulence.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergillus/patogenicidade , Farmacorresistência Fúngica/fisiologia , Mariposas/microbiologia , Animais , Aspergillus/crescimento & desenvolvimento , Larva/microbiologia , Testes de Sensibilidade Microbiana , Triazóis/farmacologia , Voriconazol/farmacologia
19.
Environ Microbiol ; 19(12): 5040-5059, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29076607

RESUMO

Hsp70 proteins play important roles in protein folding in the budding yeast, but their functions in pathogenic fungi are largely unknown. Here, we found that Fusarium graminearum Hsp70 proteins FgSsb, FgSsz and their cochaperone FgZuo formed a complex. This complex was required for microtubule morphology, vacuole fusion and endocytosis. More importantly, the ß2-tubulin FgTub2 and SNARE protein FgVam7 were identified as targeting proteins of this complex. We further found that the complex FgSsb-FgZuo-FgSsz controlled sensitivity of F. graminearum to the antimicrotubule drug carbendazim and cold stress via regulating the folding of FgTub2. Moreover, this complex assisted the folding of FgVam7, subsequently modulated vacuole fusion and responses to heavy metal, osmotic and oxidative stresses. In addition, the deletion of this complex led to dramatically decreased deoxynivalenol biosynthesis. This study uncovers a novel regulating mechanism of Hsp70 in multiple stress responses in a filamentous fungus.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Dobramento de Proteína , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Tubulina (Proteína)/metabolismo , Benzimidazóis/farmacologia , Carbamatos/farmacologia , Farmacorresistência Fúngica/fisiologia , Fungicidas Industriais/farmacologia , Fusarium/efeitos dos fármacos , Fusão de Membrana/fisiologia , Microtúbulos/efeitos dos fármacos , Micotoxinas/metabolismo , Pressão Osmótica/fisiologia , Estresse Oxidativo/fisiologia , Ligação Proteica , Tricotecenos/biossíntese
20.
Artigo em Inglês | MEDLINE | ID: mdl-28807920

RESUMO

The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor Tac1 and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate Tac1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C terminus of Tac1. GOF mutations within the Tac1 TAD, outside the context of full-length Tac1, generally do not enhance its absolute potential as a transcriptional activator. Negative regulation of the Tac1 TAD by the Tac1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length Tac1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator coactivator complex to the CDR1 promoter. Azole resistance and the activation of Tac1 target genes, such as CDR1, are dependent on the Tac1 TAD and subunits of the Mediator tail module. The dependence of different Tac1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of Tac1 is correlated with its Mediator-dependent phosphorylation, a potentially useful biomarker for Tac1 hyperactivation. The role of Mediator in events downstream of Tac1 hyperactivation in fluconazole-resistant clinical isolates is complex and provides opportunities and challenges for therapeutic intervention.


Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica/fisiologia , Proteínas Fúngicas/metabolismo , Candida albicans/patogenicidade , Eletroforese em Gel de Poliacrilamida , Fluconazol/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mutação , Regiões Promotoras Genéticas , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Xenobióticos/farmacologia
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