RESUMO
PURPOSE: To investigate the effect of Glabridin (GLD) in Aspergillus fumigatus keratitis and its associated mechanisms. METHODS: Aspergillus fumigatus (A. fumigatus) conidia was inoculated in 96-well plate, and minimal inhibitory concentration (MIC) and biofilm formation ability were evaluated after GLD treatment. Spore adhesion ability was evaluated in conidia infected human corneal epithelial cells (HCECs). Keratitis mouse model was created by corneal intrastromal injection with A. fumigatus conidia, and GLD treatment started at the day after infection. The number of fungal colonies was calculated by plate count, and degree of corneal inflammation was assessed by clinical score. Flow cytometry, myeloperoxidase (MPO), and immunofluorescence staining (IFS) experiments were used to assess neutrophil infiltrations. PCR, ELISA and Western blot were conducted to determine levels of TLR4, Dectin-1 as well as downstream inflammatory factors. RESULTS: GLD treatment suppressed the proliferation, biofilm formation abilities and adhesive capability of A. fumigatus. In mice upon A. fumigatus infection, treatment of GLD showed significantly decreased severity of corneal inflammation, reduced number of A. fumigatus in cornea, and suppressed neutrophil infiltration in cornea. GLD treatment obviously inhibited mRNA and protein levels of Dectin-1, TLR4 and proinflammatory mediators such as IL-1ß, HMGB1, and TNF-α in mice corneas compared to the control group. CONCLUSION: GLD has antifungal and anti-inflammatory effects in fungal keratitis through suppressing A. fumigatus proliferation and alleviating neutrophil infiltration, and repressing the expression of TLR4, Dectin-1 and proinflammatory mediators.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/fisiologia , Úlcera da Córnea/tratamento farmacológico , Infecções Oculares Fúngicas/tratamento farmacológico , Isoflavonas/uso terapêutico , Fenóis/uso terapêutico , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Western Blotting , Úlcera da Córnea/microbiologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Fúngicas/microbiologia , Feminino , Citometria de Fluxo , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Infiltração de Neutrófilos , Reação em Cadeia da Polimerase , Receptor 4 Toll-Like/metabolismoRESUMO
Plant immunity to pathogen infections is a dynamic response that involves multiple organelles and defence signalling systems such as hypersensitive response (HR) and systemic acquired resistance (SAR). The latter requires the function of Pathogenesis-related (PR) proteins, a common plant protein family with diverse roles in plant innate immunity. Our previous proteomics study showed that a PR gene (ITC1587_Bchr9_P26466_MUSBA) was differentially regulated during a compatible banana-M. incognita interaction, substantiating the isolation of this gene in the current study. Here, we successfully isolated and characterised Pathogenesis-related-10 (PR10) gene with ß-1,3-glucanase and ribonuclease (RNase) activities from two Musa acuminata cultivars (denoted as MaPR10) namely Berangan and Grand Naine (ITC1256). We found that MaPR10 cloned sequences possess glycine-rich loop domain and shared conserved motifs specific to PR10 gene group, confirming its identity as a member of this group. Interestingly, we also found a catalytic domain sequence for glycoside hydrolase family 16 (EXDXXE), unique only to MaPR10 cloned sequences. Two peptide variants closely related to the reference sequence ITC1587_Bchr9_P26466_MUSBA namely MaPR10-BeB5 and MaPR10-GNA5 were overexpressed and purified to test for their functionality. Here, we confirmed that both protein variants possess ß-1,3-glucanase and ribonuclease (RNase) activities, and inhibit the growth of Aspergillus fumigatus, a human opportunistic pathogen. To our knowledge, this is the first PR10 plant proteins with such properties to be reported thus far.
Assuntos
Musa/genética , Musa/parasitologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tylenchoidea/patogenicidade , Animais , Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus niger/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Parasita/genética , Cebolas/genética , Filogenia , Imunidade Vegetal/genética , Proteínas de Plantas/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , Plantas Geneticamente ModificadasRESUMO
Aspergillus fumigatus is an important fungal pathogen that causes allergic reactions but also life-threatening infections. One of the most abundant A. fumigatus proteins is Asp f3. This peroxiredoxin is a major fungal allergen and known for its role as a virulence factor, vaccine candidate, and scavenger of reactive oxygen species. Based on the hypothesis that Asp f3 protects A. fumigatus against killing by immune cells, we investigated the susceptibility of a conditional aspf3 mutant by employing a novel assay. Surprisingly, Asp f3-depleted hyphae were killed as efficiently as the wild type by human granulocytes. However, we identified an unexpected growth defect of mutants that lack Asp f3 under low-iron conditions, which explains the avirulence of the Δaspf3 deletion mutant in a murine infection model. A. fumigatus encodes two Asp f3 homologues which we named Af3l (Asp f3-like) 1 and Af3l2. Inactivation of Af3l1, but not of Af3l2, exacerbated the growth defect of the conditional aspf3 mutant under iron limitation, which ultimately led to death of the double mutant. Inactivation of the iron acquisition repressor SreA partially compensated for loss of Asp f3 and Af3l1. However, Asp f3 was not required for maintaining iron homeostasis or siderophore biosynthesis. Instead, we show that it compensates for a loss of iron-dependent antioxidant enzymes. Iron supplementation restored the virulence of the Δaspf3 deletion mutant in a murine infection model. Our results unveil the crucial importance of Asp f3 to overcome nutritional immunity and reveal a new biological role of peroxiredoxins in adaptation to iron limitation. IMPORTANCE Asp f3 is one of the most abundant proteins in the pathogenic mold Aspergillus fumigatus. It has an enigmatic multifaceted role as a fungal allergen, virulence factor, reactive oxygen species (ROS) scavenger, and vaccine candidate. Our study provides new insights into the cellular role of this conserved peroxiredoxin. We show that the avirulence of a Δaspf3 mutant in a murine infection model is linked to a low-iron growth defect of this mutant, which we describe for the first time. Our analyses indicated that Asp f3 is not required for maintaining iron homeostasis. Instead, we found that Asp f3 compensates for a loss of iron-dependent antioxidant enzymes. Furthermore, we identified an Asp f3-like protein which is partially functionally redundant with Asp f3. We highlight an unexpected key role of Asp f3 and its partially redundant homologue Af3l1 in overcoming the host's nutritional immunity. In addition, we uncovered a new biological role of peroxiredoxins.
Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/metabolismo , Ferro/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Feminino , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Homeostase , Humanos , Ferro/farmacologia , Estresse Oxidativo , Virulência , Fatores de Virulência/metabolismoRESUMO
Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Adenilato Quinase/antagonistas & inibidores , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Estudo de Prova de Conceito , Bibliotecas de Moléculas Pequenas/farmacologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/enzimologiaRESUMO
Invasive fungal infections (IFIs) are fatal infections, but treatment options are limited. The clinical efficacies of existing drugs are unsatisfactory because of side effects, drug-drug interaction, unfavorable pharmacokinetic profiles, and emerging drug-resistant fungi. Therefore, the development of antifungal drugs with a new mechanism is an urgent issue. Herein, we report novel aryl guanidine antifungal agents, which inhibit a novel target enzyme in the ergosterol biosynthesis pathway. Structure-activity relationship development and property optimization by reducing lipophilicity led to the discovery of 6h, which showed potent antifungal activity against Aspergillus fumigatus in the presence of serum, improved metabolic stability, and PK properties. In the murine systemic A. fumigatus infection model, 6h exhibited antifungal efficacy equivalent to voriconazole (1e). Furthermore, owing to the inhibition of a novel target in the ergosterol biosynthesis pathway, 6h showed antifungal activity against azole-resistant A. fumigatus.
Assuntos
Antifúngicos/farmacologia , Ergosterol/antagonistas & inibidores , Guanidina/farmacologia , Infecções Fúngicas Invasivas/tratamento farmacológico , Tiazóis/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Aspergillus fumigatus/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ergosterol/biossíntese , Guanidina/análogos & derivados , Guanidina/química , Humanos , Infecções Fúngicas Invasivas/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Purpose: To investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. Methods: The noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro. Results: Baicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1ß, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1ß, IL-6, and TNF-α were significantly lower in the TSLP siRNA-treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1ß, IL-6, and TNF-α. Conclusions: Baicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.
Assuntos
Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Citocinas/antagonistas & inibidores , Infecções Oculares Fúngicas/tratamento farmacológico , Flavanonas/uso terapêutico , Inflamação/tratamento farmacológico , Ceratite/tratamento farmacológico , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/ultraestrutura , Western Blotting , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Feminino , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase em Tempo Real , Linfopoietina do Estroma do TimoRESUMO
Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis. Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR. Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1ß, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro. Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.
Assuntos
Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Citocinas/antagonistas & inibidores , Infecções Oculares Fúngicas/tratamento farmacológico , Ceratite/tratamento farmacológico , Quercetina/análogos & derivados , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Western Blotting , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/ultraestrutura , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Feminino , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Quercetina/uso terapêutico , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
The prevalence of azole-resistant Aspergillus fumigatus (ARAF) among chronic pulmonary aspergillosis (CPA) patients treated with azoles in Japan is unknown. The aim of this study was to determine the detection rate of ARAF in isolates from CPA patients who were treated with azoles for varying durations. The potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104) between February 2012 and February 2019, at National Hospital Organization Tokyo National Hospital. The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF among all isolates was 8.3% (n = 10). Of the 10 resistant isolates, eight were ITCZ-resistant and five were VRCZ-resistant. Among 47 isolates obtained from 36 CPA patients who were treated with ITCZ (for an average of 256 days) and/or VRCZ (for an average of 29 days), the resistance rates were 17.0% and 10.6%, respectively. In addition, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients, particularly in those with ongoing long-term azole treatment, at the time of azole antifungal therapy failure.
Aspergillus fumigatus can acquire azole resistance during long-term treatment with azole drugs in patients with chronic pulmonary aspergillosis (CPA). The aim of this study was to determine the detection rate of azole-resistant A. fumigatus (ARAF) in isolates from CPA patients who had been treated with azoles. In addition, a potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104). The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF from all isolates was 8.3% (n = 10). Greater than 10% of the 47 isolates obtained from 36 CPA patients who had been treated with azoles exhibited resistance. Furthermore, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients undergoing long-term azole treatment at the time of antifungal therapy failure.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Azóis/farmacologia , Azóis/uso terapêutico , Farmacorresistência Fúngica/genética , Hospitais/estatística & dados numéricos , Aspergilose Pulmonar/tratamento farmacológico , Idoso , Aspergillus fumigatus/genética , Azóis/classificação , Doença Crônica/terapia , Feminino , Proteínas Fúngicas/genética , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Prevalência , Aspergilose Pulmonar/epidemiologia , Aspergilose Pulmonar/microbiologia , Estudos Retrospectivos , Tóquio/epidemiologiaRESUMO
Aspergillus spp. infections remain a global concern, with â¼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 µg*h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 µg*h/ml). When A. fumigatus isolates with in vitro susceptibilities of 0.25 and 1.0 µg/ml were used in a disseminated IA model, VT-1598 treatment produced no decrease in kidney fungal burden at QD 10 mg/kg, intermediate decreases at QD 20 mg/kg and maximum or near maximum decreases at 40 mg/kg QD and BID. The PK/PD relationships of AUCfree/MIC for 1-log killing for the two strains were 5.1 and 1.6 h, respectively, similar to values reported for approved CYP51 inhibitors. In a survival study where animals were observed for 12 days after the last treatment, survival was 100% at the doses tested (20 and 40 mg/kg QD), and fungal burden remained suppressed even though drug wash-out was complete. Similar dose-dependent reductions in lung fungal burden were observed in a pulmonary model of IA. These data strongly support further exploration of VT-1598 for the treatment of this lethal mold infection.
Assuntos
Inibidores de 14-alfa Desmetilase/uso terapêutico , Antifúngicos/uso terapêutico , Aspergillus fumigatus/efeitos dos fármacos , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Piridinas/uso terapêutico , Tetrazóis/uso terapêutico , Animais , Antifúngicos/farmacocinética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Neutropenia , Piridinas/farmacocinética , Tetrazóis/farmacocinéticaRESUMO
Aspergillus fumigatus can cause a variety of lung diseases in immunocompromised patients, including life-threatening invasive aspergillosis. There are only three main classes of antifungal drugs currently used to treat aspergillosis, and antifungal resistance is increasing. Experimental results in fungal biology research are usually obtained as average measurements across whole populations while ignoring what is happening at the single cell level. In this study, we show that conidia with the same genetic background in the same cell population at a similar developmental stage show heterogeneity in their cell wall labeling at the single cell level. We present a rigorous statistical method, newly applied to quantify the level of cell heterogeneity, which allows for direct comparison of the heterogeneity observed between treatments. We show the extent of cell wall labeling heterogeneity in dormant conidia and how the level of heterogeneity changes during germination. The degree of heterogeneity is influenced by deletions of cell wall synthesizing genes and environmental conditions, including medium composition, method of inoculation, age of conidia, and the presence of antifungals. This heterogeneity results in subpopulations of germinating conidia with heterogeneous fitness to the antifungal caspofungin, which targets cell wall synthesis and heterogeneous sensitivity of dormant conidia to phagocytosis by macrophages.IMPORTANCE The fungus Aspergillus fumigatus can cause invasive lung diseases in immunocompromised patients resulting in high mortality. Treatment using antifungal compounds is often unsuccessful. Average population measurements hide what is happening at the individual cell level. We set out to test what impact individual differences between the cell walls of fungal conidia have on their behavior. We show that a population of cells having the same genetic background gives rise to subpopulations of cells that exhibit distinct behavior (phenotypic heterogeneity). This cell heterogeneity is dependent on the strain type, gene deletions, cell age, and environmental conditions. By looking at the individual cell level, we discovered subpopulations of cells that show differential fitness during antifungal treatment and uptake by immune cells.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Parede Celular/química , Fagocitose/efeitos dos fármacos , Animais , Farmacorresistência Fúngica , Regulação Fúngica da Expressão Gênica , Camundongos , Células RAW 264.7 , Análise de Célula Única , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/genéticaRESUMO
Introduction. Signal transducer and activator of transcription 3 (STAT3) deficiency is a rare primary immunodeficiency associated with increased susceptibility to bacterial and fungal infections, notably pulmonary aspergillosis.Aim. We describe the emergence of azole-resistant Aspergillus fumigatus infections in STAT3-deficient patients.Methodology. During a retrospective study of 13 pulmonary aspergillosis cases in STAT3-deficient patients conducted in France, we identified patients infected with azole-resistant A. fumigatus isolates.Results. Two out of the 13 STAT3-deficient patients with aspergillosis had azole-resistant A. fumigatus infection, indicating an unexpectedly high prevalence of resistance. The first patient with STAT3 deficiency presented several flares of allergic bronchopulmonary aspergillosis-like episodes. He was chronically infected with two azole-resistant A. fumigatus isolates (TR34/L98). Despite prolonged antifungal treatment, including caspofungin and amphotericin B, the patient was not able to clear the azole-resistant A. fumigatus. The second patient had chronic cavitary pulmonary aspergillosis (CCPA). The A. fumigatus isolate was initially azole susceptible but harboured three F46Y, M172V and E427K point mutations. Despite prolonged antifungal therapies, lesions worsened and the isolate became resistant to all azoles. Surgery and caspofungin treatments were then required to cure CCPA. Resistance was probably acquired from the environment (TR34/L98) in the first case whereas resistance developed under antifungal treatments in the second case. These infections required long-term antifungal treatments and surgery.Conclusions. The emergence of azole-resistant A. fumigatus infections in STAT3-deficiency dramatically impacts both curative and prophylactic antifungal strategies. Physicians following patients with primary immune-deficiencies should be aware of this emerging problem as it complicates management of the patient.
Assuntos
Antifúngicos/uso terapêutico , Aspergillus fumigatus/efeitos dos fármacos , Azóis/uso terapêutico , Farmacorresistência Fúngica/efeitos dos fármacos , Aspergilose Pulmonar/tratamento farmacológico , Aspergilose Pulmonar/genética , Fator de Transcrição STAT3/deficiência , Adulto , Anfotericina B/uso terapêutico , Caspofungina/uso terapêutico , Criança , Doenças Transmissíveis/tratamento farmacológico , Doenças Transmissíveis/genética , Doenças Transmissíveis/microbiologia , Farmacorresistência Fúngica/genética , França , Proteínas Fúngicas/genética , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Aspergillus fumigatus, an opportunistic fungal pathogen is associated with a wide array of diseases. It produces 1, 8-dihydroxy naphthalene (DHN) melanin that imparts greenish grey color to conidia and is an important virulence factor. It masks various molecular patterns associated with A. fumigatus and protects the fungus from host immune system. Myristica fragrans, enriched with secondary metabolites has been traditionally used for the treatment of infectious and inflammatory diseases. The present study was aimed to explore the anti-melanogenic effect of M. fragrans extracts on A. fumigatus. METHODS: M. fragrans extracts (hexane, chloroform, methanol and ethanol) were prepared through polarity guided extraction. Phytochemical analysis was performed to detect the chemical constituents of the extracts. The minimum effective concentration (MEC) of the extracts against A. fumigatus melanin was determined by broth micro-dilution assay. Various virulence factors were assayed by spectrophotometric methods. Electron microscopic studies were performed to evaluate the effect of the hexane extract of M. fragrans on A. fumigatus cell surface morphology. The major active compounds of the extract were detected by gas chromatography-mass spectrometry (GC-MS). Docking was performed to study the interaction between the major identified compounds and the ketosynthase domain of polyketide synthase protein. RESULTS: The results indicated that the hexane extract of M. fragrans inhibited melanin production (76.09%), reduced ergosterol content (83.63%) and hydrophobicity of the cell (72.2%) at the MEC of 0.078 mg/mL. Altered conidial surface, disappearance of protrusions and absence of melanin layer on outer cell surface was observed in electron microscopy. Forty-two compounds were identified by GC-MS. The main constituents were identified as sabinene (12.2%), linoleic acid (11.7%), hexadecanoic acid (10.5%), safrole (8.1%) and elemicin (7.8%). Docking studies revealed that hexadecanoic acid, its derivative compound cis-9-hexadecenal and isoeugenol have lower binding energy forming proper hydrogen bond with ketosynthase domain of polyketide synthase protein. CONCLUSION: The study concludes that the extract of M. fragrans has potential antifungal properties that can be explored in combination with available antifungals. This combination approach may be helpful for large number of patients suffering with A. fumigatus infections.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Melaninas/antagonistas & inibidores , Myristica/química , Extratos Vegetais/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Índia , Testes de Sensibilidade Microbiana , Esporos Fúngicos/efeitos dos fármacosRESUMO
Aspergillus infections are increasingly recognized as a global health problem because of limited antifungal drugs and occurrence of azole resistance worldwide. More cyp51-mediated and non-cyp51-mediated mechanisms of azole resistance have been identified in clinical and laboratory studies in recent years with applications of molecular biotechnology including next-generation sequencing, reverse genetics and so on. In this review, current research on the molecular mechanisms of azole resistance in A. fumigatus were presented and summarized and meanwhile the putative clinical relevance of these findings from bench work were discussed. Important aims are to gain more insight to mechanism of azole resistance and provide some efficient lead for prevention strategy.
Assuntos
Antifúngicos/uso terapêutico , Aspergillus fumigatus/genética , Azóis/uso terapêutico , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Aspergilose/genética , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Mutação , Esterol 14-Desmetilase/genéticaRESUMO
PURPOSE: Nerolidol, a naturally occurring sesquiterpene has both anti-microbial and anti-inflammatory properties. The current study aims to investigate the antifungal and the anti-inflammatory effects of nerolidol against mouse Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration (MIC) and cytotoxicity tests were used to study the antifungal ability. For in vivo and in vitro studies, the mouse corneas and the human corneal epithelial cells (HCECs) infected with A. fumigatus spores were intervented with nerolidol or phosphate buffer saline (PBS). Thereafter, the effect of the nerolidol on the response against inflammation was analyzed using the following parameters: recruitment of the neutrophils or macrophages and the expression of the lectin-type oxidized low density lipoprotein receptor-1 (LOX-1) and interleukin 1ß (IL-1ß). Techniques used were the slit lamp, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. RESULTS: Nerolidol directly inhibits the growth of A. fumigatus. The administration of nerolidol reduced the severity of fungal keratitis with infiltration of fewer inflammatory cells and reduced levels of the LOX-1, as well the anti-inflammatory cytokines such as IL-1ß were reduced compared with the PBS group. Additionally, in vitro studies showed that treatment with nerolidol inhibited the production of the LOX-1 / IL-1ß levels in A. fumigatus stimulated HCECs. CONCLUSION: Nerolidol attenuated the A. fumigatus keratitis inflammatory response by inhibiting the growth of A. fumigatus, reducing the recruitment of the neutrophils and the macrophages, and inhibiting the LOX-1/ IL-1ß signaling.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Ceratite/tratamento farmacológico , Sesquiterpenos/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Antifúngicos/farmacologia , Aspergilose/imunologia , Aspergilose/patologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Córnea/efeitos dos fármacos , Córnea/imunologia , Córnea/patologia , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Ceratite/imunologia , Ceratite/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Receptores Depuradores Classe E/genética , Receptores Depuradores Classe E/imunologia , Sesquiterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Chronic pulmonary aspergillosis (CPA) is a progressive respiratory disease, caused most commonly by A fumigatus, with significant morbidity and mortality. Azole resistance in A fumigatus is a growing concern worldwide, with resistance to itraconazole reported in up to 50% of patients. AIM: The aim of this study was to determine whether a positive Aspergillus PCR (polymerase chain reaction) is a marker of resistance in CPA patients on azole therapy. METHODS: Patients were selected via a consecutive database search for the first 50 CPA patients with a positive Aspergillus PCR from January to September 2016. Data were collected regarding concurrent and subsequent culture results, current therapy and serum antifungal levels. PCR-positive patients not on therapy were included as the control group. RESULTS: Twenty-three patients were on therapy (15 itraconazole, 4 voriconazole and 4 posaconazole). Cycle threshold (Ct) values ranged from 20.8 to 37.9; no significant difference was found between each treatment and the control group (P = .47). In treated patients, concurrent azole-resistant A fumigatus was found in 75% of A fumigatus-positive cultures (6/8). All of the resistant isolates in the itraconazole group showed therapy resistance. Twenty per cent of all itraconazole levels were sub-therapeutic. No significant difference was found in serum itraconazole levels for patients on itraconazole with a positive PCR versus negative PCR (P = .44). CONCLUSION: Positive sputum, Aspergillus-specific PCR can be associated with azole resistance in CPA patients on therapy.
Assuntos
Aspergillus fumigatus/isolamento & purificação , Azóis/uso terapêutico , Farmacorresistência Fúngica , Aspergilose Pulmonar/tratamento farmacológico , Antifúngicos/uso terapêutico , Aspergillus/efeitos dos fármacos , Aspergillus/genética , Aspergillus/isolamento & purificação , Aspergillus fumigatus/efeitos dos fármacos , Feminino , Proteínas Fúngicas/genética , Humanos , Masculino , Reação em Cadeia da Polimerase , Estudos RetrospectivosRESUMO
Aspergillus fumigatus is the predominant etiological agent of invasive aspergillosis (IA), a difficult-to-manage fungal disease associated with a high case fatality rate. Azole antifungals, particularly voriconazole, have significantly improved the survival rate of patients with IA. However, the clinical advances made possible through the use of medical azoles could be threatened by the emergence of azole-resistant strains which has been reported in an ever-increasing number of countries over the last 10 years. The major resistance mechanism, that combines point mutation(s) in the coding sequence of cyp51A gene and an insertion of a tandem repeat in the promoter region of this gene which leads to its overexpression (TR34/L98H and TR46/Y121F/T289A), is presumed to be of environmental origin. However, the emergence of clinical and environmental azole-resistant strains without the cyp51A gene mutation suggests that other mechanisms could also be responsible for azole resistance (for example, overexpression of efflux pumps). The development of resistance may be linked to either long-term use of azole antifungals in patients with chronic aspergillosis (patient-acquired route) or selection pressure of the fungicides in the environment (environmental route). The fungicide-driven route could be responsible for resistance in azole-naive patients with IA. This literature review aims to summarize recent findings, focusing on the current situation of azole-resistance in A. fumigatus, and provides better understanding of the importance of the environmental route in resistance acquisition.
Assuntos
Aspergilose/tratamento farmacológico , Aspergillus fumigatus , Azóis/uso terapêutico , Farmacorresistência Fúngica , Antifúngicos/uso terapêutico , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/fisiologia , Azóis/química , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Voriconazol/uso terapêuticoRESUMO
Galactomannan (GM) detection in biological samples has been shown to predict therapeutic response by azoles and polyenes. In a murine invasive pulmonary aspergillosis model, fosmanogepix or posaconazole treatment resulted in an â¼6- to 7-log reduction in conidial equivalents (CE)/g lung tissue after 96 h versus placebo. Changes in GM levels in BAL fluid and serum mirrored reductions in lung CE, with significant decreases seen after 96 h or 72 h for fosmanogepix or posaconazole, respectively (P < 0.02).
Assuntos
Antifúngicos/uso terapêutico , Biomarcadores/metabolismo , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Aspergilose Pulmonar Invasiva/metabolismo , Mananas/metabolismo , Animais , Aspergilose/tratamento farmacológico , Aspergilose/metabolismo , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Galactose/análogos & derivados , Hospedeiro Imunocomprometido , Pulmão/microbiologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Triazóis/uso terapêuticoRESUMO
Rezafungin acetate is a novel echinocandin in clinical development for prevention and treatment of invasive fungal infections. Rezafungin is differentiated by a pharmacokinetic/pharmacodynamic (PK/PD) profile that includes a long half-life allowing once-weekly administration, front-loaded plasma drug exposures associated with antifungal efficacy, and penetration into deep-seated infections, such as intra-abdominal abscesses. In this series of in vivo studies, rezafungin demonstrated efficacy in the treatment of neutropenic mouse models of disseminated candidiasis, including infection caused by azole-resistant Candida albicans, and aspergillosis. These results contribute to a growing body of evidence demonstrating the antifungal efficacy and potential utility of rezafungin in the treatment of invasive fungal infections.
Assuntos
Antifúngicos/farmacocinética , Aspergilose/tratamento farmacológico , Candidíase Invasiva/tratamento farmacológico , Equinocandinas/farmacocinética , Administração Oral , Animais , Antifúngicos/administração & dosagem , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/isolamento & purificação , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Candidíase Invasiva/sangue , Candidíase Invasiva/imunologia , Candidíase Invasiva/microbiologia , Modelos Animais de Doenças , Esquema de Medicação , Equinocandinas/administração & dosagem , Feminino , Meia-Vida , Humanos , Hospedeiro Imunocomprometido , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Neutropenia/imunologiaRESUMO
We evaluated extended-interval dosing of the investigational echinocandin rezafungin (1, 4, and 16 mg/kg on days 1, 4, and 7 postinoculation) for the treatment of disseminated invasive aspergillosis caused by azole-resistant Aspergillus fumigatus Survival was significantly improved in mice treated with each dose of rezafungin and supratherapeutic posaconazole (20 mg/kg twice daily). Kidney fungal burden, as measured by quantitative real-time PCR, was also significantly reduced in mice treated with rezafungin although variability was observed.
Assuntos
Antifúngicos/uso terapêutico , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Azóis/uso terapêutico , Equinocandinas/uso terapêutico , Animais , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Azóis/efeitos adversos , Farmacorresistência Fúngica/genética , Equinocandinas/efeitos adversos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Rim/virologia , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Triazóis/uso terapêuticoRESUMO
The crude methanol extract of Sphenocentrum jollyanum root exhibited 98% and 80% antimicrobial activity against Aspergillus fumigatus Pinh and Vancomycin resistant enterococcus (VRE) at a concentration of 200⯵g/mL, with IC50 11.45 and 12.95⯵g/mL, respectively. The ethyl acetate fraction of methanol extract showed in-vitro antimicrobial activity against A. fumigatus Pinh at 83% with IC50 ofâ¯<8⯵g/mL. The phytochemical investigation of ethyl acetate fraction yielded six compounds, which were identified by their NMR, IR and MS spectral analyses as two new phytoecdysteroidal glycosides Sphenocentroside A (1), and Sphenocentroside B (2), and four known phytoecdysteroids: polypodoaurein (3), polypodine B (4), ecdysterone (5), and 20, 26-dihydroxyecdysone (6).