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1.
Mar Drugs ; 20(3)2022 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-35323462

RESUMO

The COVID-19 pandemic and its continuing emerging variants emphasize the need to discover appropriate treatment, where vaccines alone have failed to show complete protection against the new variants of the virus. Therefore, treatment of the infected cases is critical. This paper discusses the bio-guided isolation of three indole diketopiperazine alkaloids, neoechinulin A (1), echinulin (2), and eurocristatine (3), from the Red Sea-derived Aspergillus fumigatus MR2012. Neoechinulin A (1) exhibited a potent inhibitory effect against SARS-CoV-2 Mpro with IC50 value of 0.47 µM, which is comparable to the reference standard GC376. Despite the structural similarity between the three compounds, only 1 showed a promising effect. The mechanism of inhibition is discussed in light of a series of extensive molecular docking, classical and steered molecular dynamics simulation experiments. This paper sheds light on indole diketopiperazine alkaloids as a potential structural motif against SARS-CoV-2 Mpro. Additionally, it highlights the potential of different molecular docking and molecular dynamics simulation approaches in the discrimination between active and inactive structurally related Mpro inhibitors.


Assuntos
Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Alcaloides Indólicos/química , Piperazinas/química , SARS-CoV-2/enzimologia , Alcaloides/química , Alcaloides/isolamento & purificação , Antivirais/isolamento & purificação , Aspergillus fumigatus/química , Inibidores de Cisteína Proteinase/isolamento & purificação , Alcaloides Indólicos/isolamento & purificação , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Piperazinas/isolamento & purificação
2.
Appl Biochem Biotechnol ; 193(11): 3570-3585, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34313919

RESUMO

The current study was conducted to evaluate the antiproliferative and oxidative damage protection potential of endophytic fungi Aspergillus fumigatus and Chaetomium globosum isolated from Moringa oleifera. The chloroformic extract (CE) of both the fungi showed dose dependent antiproliferative activity against human prostate adenocarcinoma (PC-3) cell line with (IC50) value of 0.055 mg/ml and 0.008 mg/ml, respectively. Further, CE of both the fungi was studied for their ability to induce apoptosis in PC-3 cell line. Various deformities in the cancerous cells treated with CE of both the fungi have been observed by confocal microscopy which indicates the cell death by apoptosis. Further apoptosis inducing ability of CE of both the fungi was observed using various flow cytometric studies. The chloroformic extract of both the fungi showed slight increase in the level of reactive oxygen species to induce apoptosis. It also showed arrest of cancerous cells at G0/G1 phase of cell cycle to induce apoptosis. The externalization of phosphatidylserine (PS) to induce apoptosis was also observed when analysed using Annexin V-FITC/PI double staining assay where the CE of A. fumigatus and C. globosum showed the total apoptosis of 94.2% and 90.3%, respectively, at the highest tested concentration of GI70. The CE of both the fungi further showed the protective behaviour for plasmid DNA pBR322, when tested for their effect against the oxidative stress caused by the Fenton's reagent. Thus, the studies demonstrated a good antiproliferative and oxidative damage protection potential of the endophytic fungi.


Assuntos
Antioxidantes , Aspergillus fumigatus/química , Proliferação de Células/efeitos dos fármacos , Chaetomium/química , Misturas Complexas , Endófitos/química , Moringa oleifera/microbiologia , Neoplasias da Próstata , Antioxidantes/química , Antioxidantes/farmacologia , Misturas Complexas/química , Misturas Complexas/farmacologia , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo
3.
Arch Microbiol ; 203(7): 4635-4640, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34169338

RESUMO

The fungus is a great source of secondary metabolites which may possess various biological activities for the treatment of human diseases and disorders. The present study aimed to identify the secondary metabolites from Aspergillus fumigatus strain MF-1 using analytical techniques (chromatographic and spectroscopic). The instruments includes 1H-NMR, 13C-NMR, DEPT, COSY, HMQC, FTIR and UV-Vis spectrophotometer were employed to study the extracted compound and the produced compound is 2,5-dioxocyclopentylamino-7-oxohepta-1,3,5-trienyl-2,5-dihydroxy-3-chlorophenyl-2,4,6-trimethyldeca-2,4-dienamide. Furthermore, the four fractions were tested over HeLa cell lines for their cytotoxicity performance and found that fraction 4 exhibited potent cytotoxic effect with IC50 value of 74.38 ± 0.31 µg/mL on HeLa cell lines than other fractions.


Assuntos
Antineoplásicos , Aspergillus fumigatus , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Aspergillus fumigatus/química , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos
5.
J Microbiol ; 59(1): 64-75, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33201436

RESUMO

Aspergillus fumigatus is a well-known opportunistic pathogen that causes invasive aspergillosis (IA) infections with high mortality in immunosuppressed individuals. Morphogenesis, including hyphal growth, conidiation, and cell wall biosynthesis is crucial in A. fumigatus pathogenesis. Based on a previous random insertional mutagenesis library, we identified the putative polysaccharide synthase gene Afcps1 and its para-log Afcps2. Homologs of the cps gene are commonly found in the genomes of most fungal and some bacterial pathogens. Afcps1/cpsA is important in sporulation, cell wall composition, and virulence. However, the precise regulation patterns of cell wall integrity by Afcps1/cpsA and further effects on the immune response are poorly understood. Specifically, our in-depth study revealed that Afcps1 affects cell-wall stability, showing an increased resistance of ΔAfcps1 to the chitinmicrofibril destabilizing compound calcofluor white (CFW) and susceptibility of ΔAfcps1 to the ß-(1,3)-glucan synthase inhibitor echinocandin caspofungin (CS). Additionally, deletion of Afcps2 had a normal sporulation phenotype but caused hypersensitivity to Na+ stress, CFW, and Congo red (CR). Specifically, quantitative analysis of cell wall composition using high-performance anion exchange chromatography-pulsed amperometric detector (HPAEC-PAD) analysis revealed that depletion of Afcps1 reduced cell wall glucan and chitin contents, which was consistent with the down-regulation of expression of the corresponding biosynthesis genes. Moreover, an elevated immune response stimulated by conidia of the ΔAfcps1 mutant in marrow-derived macrophages (BMMs) during phagocytosis was observed. Thus, our study provided new insights into the function of polysaccharide synthase Cps1, which is necessary for the maintenance of cell wall stability and the adaptation of conidia to the immune response of macrophages in A. fumigatus.


Assuntos
Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/metabolismo , Macrófagos/imunologia , Esporos Fúngicos/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Parede Celular/genética , Parede Celular/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Imunidade , Macrófagos/microbiologia , Masculino , Camundongos , Alinhamento de Sequência , Esporos Fúngicos/química , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética
6.
mBio ; 11(5)2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051372

RESUMO

G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis.IMPORTANCEA. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.


Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Receptores Acoplados a Proteínas G/genética , Metabolismo Secundário , Animais , Aspergillus fumigatus/química , Regulação Fúngica da Expressão Gênica , Larva/microbiologia , Macrófagos/microbiologia , Masculino , Melaninas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Fagocitose , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Infect Immun ; 88(9)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32571987

RESUMO

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.


Assuntos
Aspergillus fumigatus/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Complemento C3/imunologia , Polissacarídeos Fúngicos/farmacologia , Macrófagos/efeitos dos fármacos , Soro/imunologia , Esporos Fúngicos/imunologia , Aspergilose/genética , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/química , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Parede Celular/química , Parede Celular/imunologia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/genética , Citocinas/biossíntese , Citocinas/imunologia , Polissacarídeos Fúngicos/imunologia , Polissacarídeos Fúngicos/isolamento & purificação , Galactose/análogos & derivados , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/imunologia , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Mananas/imunologia , Mananas/isolamento & purificação , Mananas/farmacologia , Proteínas Opsonizantes/farmacologia , Fagocitose/efeitos dos fármacos , Cultura Primária de Células , Ligação Proteica , Espécies Reativas de Oxigênio , Soro/química , Soro/microbiologia , Esporos Fúngicos/química , beta-Glucanas/imunologia , beta-Glucanas/isolamento & purificação , beta-Glucanas/farmacologia
8.
Toxins (Basel) ; 12(1)2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861936

RESUMO

Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology.


Assuntos
Aspergillus fumigatus/química , Cicloexanos/toxicidade , Ácidos Graxos Insaturados/toxicidade , Micotoxinas/toxicidade , Animais , Abelhas , Cicloexanos/química , Ácidos Graxos Insaturados/biossíntese , Ácidos Graxos Insaturados/química , Humanos , Micotoxinas/biossíntese , Micotoxinas/química , Sesquiterpenos/química , Sesquiterpenos/toxicidade
9.
Appl Microbiol Biotechnol ; 103(21-22): 8923-8935, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520132

RESUMO

UV and gamma irradiation mutagenesis was applied on Aspergillus fumigatus and Alternaria tenuissima in order to improve their producing ability of paclitaxel. Among the screened mutants, two stable strains (designated TXD105-GM6 and TER995-GM3) showed the maximum paclitaxel production. Paclitaxel titers of the two respective mutants were dramatically intensified to 1.22- and 1.24-fold, as compared by their respective parents. Immobilization using five different entrapment carriers of calcium alginate, agar-agar, Na-CMC, gelatin, and Arabic gum was successfully applied for production enhancement of paclitaxel by the two mutants. The immobilized cultures were superior to free-cell cultures and paclitaxel production by the immobilized mycelia was much higher than that of the immobilized spores using all the tried carriers. Moreover, calcium alginate gel beads were found the most conductive and proper entrapment carrier for maximum production of paclitaxel. The feasibility of the paclitaxel production by the immobilized mycelia as affected by incubation period, medium volume, and number of beads per flask was adopted. Under the favorable immobilization conditions, the paclitaxel titers were significantly intensified to 1.31- and 1.88-fold by the respective mutants, as compared by their free cultures. The obtained paclitaxel titers by the immobilized mycelia of the respective mutants (694.67 and 388.65 µg L-1) were found promising in terms of fungal production of paclitaxel. Hence, these findings indicate the future possibility to reduce the cost of producing paclitaxel and suggest application of the immobilization technique for the biotechnological production of paclitaxel at an industrial scale.


Assuntos
Alternaria/metabolismo , Antineoplásicos/metabolismo , Aspergillus fumigatus/metabolismo , Paclitaxel/biossíntese , Alginatos/química , Alternaria/química , Alternaria/genética , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Fermentação , Microbiologia Industrial , Micélio/química , Micélio/genética , Micélio/metabolismo
10.
Mar Drugs ; 16(7)2018 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-29966281

RESUMO

As part of research to search for antitumor agents in fungi originating from marine organisms, cephalimysins E⁻L were isolated from a culture broth of Aspergillus fumigatus that was separated from the marine fish Mugil cephalus. One- and two-dimensional nuclear magnetic resonance spectra revealed their planar structures, which are diastereomers of each other. Their absolute stereostructures were established by epimerization at C-8 with acidic methanol, nuclear Overhauser effect spectroscopy (NOESY), and circular dichroism (CD) spectroscopy. These demonstrated the detailed relationships between absolute configuration and CD Cotton effects. Additionally, in the growth inhibition assay against P388, HL-60, L1210, and KB cell lines, some of the fungal metabolites or reaction products exhibited moderate activities.


Assuntos
Lactamas/química , Animais , Antineoplásicos/química , Organismos Aquáticos/química , Aspergillus fumigatus/química , Linhagem Celular Tumoral , Dicroísmo Circular/métodos , Peixes/metabolismo , Células HL-60 , Humanos , Células KB , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Estereoisomerismo
11.
Nature ; 555(7696): 382-386, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29489751

RESUMO

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.


Assuntos
Aspergillus fumigatus/imunologia , Lectinas Tipo C/imunologia , Melaninas/imunologia , Naftóis/imunologia , Animais , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergilose/prevenção & controle , Aspergillus fumigatus/química , Aspergillus fumigatus/patogenicidade , Parede Celular/química , Parede Celular/imunologia , Feminino , Humanos , Macrófagos/imunologia , Melaninas/química , Camundongos , Camundongos Endogâmicos C57BL , Naftóis/química , Ratos , Ratos Sprague-Dawley , Esporos Fúngicos/química , Esporos Fúngicos/imunologia , Especificidade por Substrato
12.
Indoor Air ; 28(1): 28-39, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28922584

RESUMO

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.


Assuntos
Aspergillus fumigatus/imunologia , Hifas/imunologia , Penicillium chrysogenum/imunologia , Esporos Fúngicos/imunologia , Stachybotrys/imunologia , Aspergillus fumigatus/química , Citocinas/análise , Humanos , Hifas/química , Macrófagos/enzimologia , Monócitos/enzimologia , Micotoxinas/análise , Tamanho da Partícula , Penicillium chrysogenum/química , Peptídeo Hidrolases/análise , Esporos Fúngicos/química , Stachybotrys/química , Células THP-1 , beta-Glucanas/análise
13.
J Med Microbiol ; 66(7): 898-904, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28693685

RESUMO

PURPOSE: The detection of galactomannan (GM) in bronchoalveolar lavage (BAL) fluid is an important surrogate marker for the early diagnosis and therapeutic monitoring of invasive aspergillosis (IA), regardless of the involved species of Aspergillus. Here, we utilized the Platelia Aspergillus GM enzyme immunoassay (Bio-Rad) to evaluate the GM index in BAL fluid samples from patients with proven, probable or putative IA due to Aspergillusflavus versus Aspergillusfumigatus. METHODOLOGY: In a prospective study between 2009 and 2015, 116 BAL samples were collected from suspected IA patients referred to two university hospitals in Tehran, Iran. KEY FINDINGS: According to European Organization for Research and Treatment of Cancer and Mycoses Study Group and Blot criteria, 35 patients were classified as IA patients, of which 33 cases tested positive for GM above 0.5 and, among these patients, 22 had a GM index ≥1. Twenty-eight were culture positive for A. flavus and seven for A. fumigatus. The GM index for A. flavus cases was between 0.5-6.5 and those of A. fumigatus ranged from 1 to 6.5. The sensitivity and specificity of a GM index ≥0.5 in cases with A. flavus were 86 and 88 % and for A. fumigatus patients were 100 and 73 %, respectively. CONCLUSION: Overall, the mean GM index in patients with A. fumigatus (3.1) was significantly higher than those of A. flavus (1.6; P-value=0.031) and the sensitivity of GM lower for A. flavus when compared to A. fumigatus. This finding has implications for diagnosis in hospitals and countries with a high proportion of A. flavus infections.


Assuntos
Aspergillus flavus/química , Aspergillus fumigatus/química , Líquido da Lavagem Broncoalveolar/química , Testes Diagnósticos de Rotina/métodos , Aspergilose Pulmonar Invasiva/patologia , Mananas/análise , Adolescente , Adulto , Idoso , Aspergillus flavus/isolamento & purificação , Aspergillus fumigatus/isolamento & purificação , Criança , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Galactose/análogos & derivados , Humanos , Aspergilose Pulmonar Invasiva/diagnóstico , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Adulto Jovem
14.
Chem Biol Interact ; 269: 18-24, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28359723

RESUMO

Demethoxyfumitremorgin C, a secondary metabolite of the marine fungus, Aspergillus fumigatus, had been reported to demonstrate cytotoxic effect on mouse tsFT210 cells. However, no information is available regarding its functional mechanism and the chemo-sensitization effects on different kinds of human cancer cells. We found that treatment of demethoxyfumitremorgin C inhibited the cell viability of PC3 human advanced prostate cancer cells, induced apoptosis as determined by Annexin V/propidium iodide double staining, and decreased mitochondrial membrane potential. Demethoxyfumitremorgin C induced apoptosis was associated with downregulation of anti-apoptotic proteins: Ras, PI3K, Akt, Bcl-xL, and Bcl-2, and upregulation of pro-apoptotic Bax. Demethoxyfumitremorgin C activated caspase-3, -8, and -9, leading to PARP cleavage. Additionally, caspase inhibitors blocked demethoxyfumitremorgin C-induced apoptosis of PC3 cells. These results suggest that demethoxyfumitremorgin C from Aspergillus fumigatus inhibits the proliferation of PC3 human prostate cancer cells via the intrinsic (mitochondrial) and extrinsic pathway, followed by downstream events leading to apoptotic cell death. Demethoxyfumitremorgin C could therefore, serve as a useful agent to treat human advanced prostate cancer.


Assuntos
Apoptose/efeitos dos fármacos , Aspergillus fumigatus/química , Piperazinas/farmacologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Aspergillus fumigatus/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Piperazinas/química , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas ras/metabolismo
15.
J Proteomics ; 151: 83-96, 2017 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-27321585

RESUMO

Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. SIGNIFICANCE: Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus.


Assuntos
Aspergillus fumigatus/química , Células Endoteliais da Veia Umbilical Humana/química , Células Endoteliais da Veia Umbilical Humana/microbiologia , Hifas/química , Inflamação , Estresse Fisiológico , Aspergillus fumigatus/genética , Células Endoteliais/metabolismo , Proteínas Fúngicas/fisiologia , Deleção de Genes , Interações Hospedeiro-Patógeno , Humanos , Polissacarídeos/biossíntese , Trombose/etiologia , Trombose/microbiologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Virulence ; 7(8): 950-966, 2016 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-27687755

RESUMO

Invasive aspergillosis is a life-threatening infection caused by the opportunistic filamentous fungus Aspergillus fumigatus. Patients undergoing haematopoietic stem cell transplant (HSCT) for the treatment of hematological malignancy are at particularly high risk of developing this fatal infection. The susceptibility of HSCT patients to infection with A. fumigatus is a consequence of a complex interplay of both fungal and host factors. Here we review our understanding of the host-pathogen interactions underlying the susceptibility of the immunocompromised host to infection with A. fumigatus with a focus on the experimental validation of fungal and host factors relevant to HSCT patients. These include fungal factors such as secondary metabolites, cell wall constituents, and metabolic adaptations that facilitate immune evasion and survival within the host microenvironment, as well as the innate and adaptive immune responses involved in host defense against A. fumigatus.


Assuntos
Aspergillus fumigatus/patogenicidade , Interações Hospedeiro-Patógeno , Aspergilose Pulmonar Invasiva/fisiopatologia , Transplante de Células-Tronco/efeitos adversos , Aspergillus fumigatus/química , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/fisiologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/microbiologia , Neoplasias Hematológicas/terapia , Humanos , Evasão da Resposta Imune , Imunidade Inata , Hospedeiro Imunocomprometido , Aspergilose Pulmonar Invasiva/imunologia , Aspergilose Pulmonar Invasiva/microbiologia , Virulência
17.
Chembiochem ; 17(19): 1813-1817, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27442960

RESUMO

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.


Assuntos
Amidas/metabolismo , Aspergillus fumigatus/metabolismo , Fumaratos/metabolismo , Metiltransferases/metabolismo , Amidas/química , Aspergillus fumigatus/química , Biocatálise , Fumaratos/química , Metilação , Metiltransferases/genética , Estrutura Molecular
18.
mBio ; 7(3)2016 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-27247234

RESUMO

UNLABELLED: Chitin is an important cell wall component of Aspergillus fumigatus conidia, of which hundreds are inhaled on a daily basis. Previous studies have shown that chitin has both anti- and proinflammatory properties; however the exact mechanisms determining the inflammatory signature of chitin are poorly understood, especially in human immune cells. Human peripheral blood mononuclear cells were isolated from healthy volunteers and stimulated with chitin from Aspergillus fumigatus Transcription and production of the proinflammatory cytokine interleukin-1ß (IL-1ß) and the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) were measured from the cell culture supernatant by quantitative PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA), respectively. Chitin induced an anti-inflammatory signature characterized by the production of IL-1Ra in the presence of human serum, which was abrogated in immunoglobulin-depleted serum. Fc-γ-receptor-dependent recognition and phagocytosis of IgG-opsonized chitin was identified as a novel IL-1Ra-inducing mechanism by chitin. IL-1Ra production induced by chitin was dependent on Syk kinase and phosphatidylinositol 3-kinase (PI3K) activation. In contrast, costimulation of chitin with the pattern recognition receptor (PRR) ligands lipopolysaccharide, Pam3Cys, or muramyl dipeptide, but not ß-glucan, had synergistic effects on the induction of proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs). In conclusion, chitin can have both pro- and anti-inflammatory properties, depending on the presence of pathogen-associated molecular patterns and immunoglobulins, thus explaining the various inflammatory signatures reported for chitin. IMPORTANCE: Invasive aspergillosis and allergic aspergillosis are increasing health care problems. Patients get infected by inhalation of the airborne spores of Aspergillus fumigatus A profound knowledge of how Aspergillus and its cell wall components are recognized by the host cell and which type of immune response it induces is necessary to develop target-specific treatment options with less severe side effects than the treatment options to date. There is controversy in the literature about the receptor for chitin in human cells. We identified the Fc-γ receptor and Syk/PI3K pathway via which chitin can induce anti-inflammatory immune responses by inducing IL-1 receptor antagonist in the presence of human immunoglobulins but also proinflammatory responses in the presence of bacterial components. This explains why Aspergillus does not induce strong inflammation just by inhalation and rather fulfills an immune-dampening function. While in a lung coinfected with bacteria, Aspergillus augments immune responses by shifting toward a proinflammatory reaction.


Assuntos
Aspergillus fumigatus/imunologia , Parede Celular/química , Quitina/imunologia , Citocinas/imunologia , Leucócitos Mononucleares/imunologia , Transdução de Sinais , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Aspergillus fumigatus/química , Aspergillus fumigatus/metabolismo , Quitina/farmacologia , Citocinas/biossíntese , Humanos , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipoproteínas/farmacologia , Moléculas com Motivos Associados a Patógenos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores de IgG/imunologia , Quinase Syk/imunologia
19.
mBio ; 7(2): e00492-16, 2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27118592

RESUMO

UNLABELLED: Invasive fungal infections remain difficult to treat and require novel targeting strategies. The 12-kDa FK506-binding protein (FKBP12) is a ubiquitously expressed peptidyl-prolyl isomerase with considerable homology between fungal pathogens and is thus a prime candidate for future targeting efforts to generate a panfungal strategy. Despite decades of research on FKBPs, their substrates and mechanisms of action remain unclear. Here we describe structural, biochemical, and in vivo analyses of FKBP12s from the pathogenic fungi Candida albicans, Candida glabrata, and Aspergillus fumigatus Strikingly, multiple apo A. fumigatus and C. albicans FKBP12 crystal structures revealed a symmetric, intermolecular interaction involving the deep insertion of an active-site loop proline into the active-site pocket of an adjacent subunit. Such interactions have not been observed in previous FKBP structures. This finding indicates the possibility that this is a self-substrate interaction unique to the A. fumigatus and C. albicans fungal proteins that contain this central proline. Structures obtained with the proline in the cis and trans states provide more data in support of self-catalysis. Moreover, cysteine cross-linking experiments captured the interacting dimer, supporting the idea that it forms in solution. Finally, genetic studies exploring the impact of mutations altering the central proline and an adjacent residue provide evidence that any dimeric state formed in vivo, where FKBP12 concentrations are low, is transient. Taken together, these findings suggest a unique mechanism of self-substrate regulation by fungal FKBP12s, lending further novel understanding of this protein for future drug-targeting efforts. IMPORTANCE: FKBP12 is a cis-trans peptidyl-prolyl isomerase that plays key roles in cellular protein homeostasis. FKBP12s also bind the immunosuppressive drug FK506 to inhibit the phosphatase calcineurin (CaN). CaN is required for virulence of A. fumigatus, C. albicans, C. glabrata, and other deadly fungal pathogens, marking FKBP12 and CaN as potential broad-spectrum drug targets. Here we describe structures of fungal FKBP12s. Multiple apo A. fumigatus and C. albicans FKBP12 structures reveal the insertion of a proline, conspicuously conserved in these proteins, into the active sites of adjacent molecules. This suggests that these proteins might serve as their own substrates. Cysteine disulfide trapping experiments provide support for this self-interaction and hence possible intermolecular catalysis by these enzymes.


Assuntos
Aspergillus fumigatus/metabolismo , Candida albicans/metabolismo , Candida glabrata/metabolismo , Proteínas Fúngicas/química , Proteína 1A de Ligação a Tacrolimo/química , Sequência de Aminoácidos , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Candida albicans/química , Candida albicans/genética , Candida glabrata/química , Candida glabrata/genética , Domínio Catalítico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo
20.
Nat Commun ; 7: 10940, 2016 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-26956772

RESUMO

Fluorescent antimicrobial peptides are promising structures for in situ, real-time imaging of fungal infection. Here we report a fluorogenic probe to image Aspergillus fumigatus directly in human pulmonary tissue. We have developed a fluorogenic Trp-BODIPY amino acid with a spacer-free C-C linkage between Trp and a BODIPY fluorogen, which shows remarkable fluorescence enhancement in hydrophobic microenvironments. The incorporation of our fluorogenic amino acid in short antimicrobial peptides does not impair their selectivity for fungal cells, and enables rapid and direct fungal imaging without any washing steps. We have optimized the stability of our probes in human samples to perform multi-photon imaging of A. fumigatus in ex vivo human tissue. The incorporation of our unique BODIPY fluorogen in biologically relevant peptides will accelerate the development of novel imaging probes with high sensitivity and specificity.


Assuntos
Antifúngicos/química , Aspergilose/microbiologia , Aspergillus fumigatus/química , Compostos de Boro/química , Diagnóstico por Imagem/instrumentação , Corantes Fluorescentes/química , Peptídeos/química , Antifúngicos/farmacologia , Aspergilose/diagnóstico , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/fisiologia , Diagnóstico por Imagem/métodos , Humanos , Pulmão/microbiologia , Peptídeos/farmacologia
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