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1.
Mycoses ; 64(11): 1354-1365, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34558115

RESUMO

BACKGROUND: Mutations in cyp51A gene are known as main mechanisms of azole resistance in Aspergillus fumigatus, whereas azole-susceptible strains also carry cyp51A mutations (polymorphisms). The polymorphisms found in Europe mainly consist of two combinations of mutations, that is combinations of five single-nucleotide polymorphisms (SNPs) of cyp51A, referred to as cyp51A-5SNPs, and combinations of three SNPs of cyp51A, referred to as cyp51A-3SNPs. Few studies have compared the distributions of cyp51A polymorphisms between different regions. OBJECTIVES: The aim of this study was to investigate the regional differences of cyp51A polymorphisms. METHODS: We compared the proportions of cyp51A polymorphisms in clinical and environmental strains isolated in various countries, and analysed the strains phylogenetically using short tandem repeats (STRs) and whole-genome sequence (WGS). RESULTS: Among the Japanese strains, 15 out of 98 (15.3%) clinical strains and 8 out of 95 (8.4%) environmental strains had cyp51A polymorphisms. A mutation of cyp51AN248K was the most prevalent polymorphism in both clinical (n = 14, 14.3%) and environmental strains (n = 3, 3.2%). Only one environmental strain harboured cyp51A-5SNPs, which was reported to be the most prevalent in Europe. For phylogenetic analyses using STRs and WGS, 183 and 134 strains, respectively, were employed. They showed that most of the strains with cyp51AN248K clustered in the clades different from those of the strains with cyp51A-5SNPs and cyp51A-3SNPs as well as from those with TR34 /L98H mutations. CONCLUSIONS: This study suggests that there are genetic differences between cyp51A polymorphisms of A. fumigatus in Japan and Europe.


Assuntos
Aspergillus fumigatus/genética , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Aspergilose Pulmonar Invasiva/microbiologia , Polimorfismo de Nucleotídeo Único , Aspergilose Pulmonar/microbiologia , Animais , Antifúngicos/farmacologia , Aspergillus fumigatus/classificação , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Bombyx/microbiologia , Doença Crônica , Microbiologia Ambiental , Europa (Continente) , Genótipo , Humanos , Japão , Testes de Sensibilidade Microbiana , Mutação , Filogenia , Virulência , Sequenciamento Completo do Genoma
2.
Anal Biochem ; 632: 114384, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34543643

RESUMO

Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production.


Assuntos
Anticorpos Monoclonais/imunologia , Aspergilose/diagnóstico , Compostos Férricos/imunologia , Ácidos Hidroxâmicos/imunologia , Imunoconjugados/química , Sideróforos/química , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/química , Aspergillus fumigatus/patogenicidade , Ensaio de Imunoadsorção Enzimática , Compostos Férricos/análise , Células HEK293 , Humanos , Ácidos Hidroxâmicos/análise , Camundongos , Proteínas Recombinantes/imunologia
3.
Medicine (Baltimore) ; 100(30): e26434, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34397685

RESUMO

ABSTRACT: This study to analyze the clinical characteristics of patients with invasive pulmonary aspergillosis (IPA) following influenza A (H1N1) infection.We retrospectively analyzed 10 cases with IPA following H1N1 infection. The clinical manifestations, laboratory examination results, chest computed tomography, and treatments were analyzed.Clinical manifestations: all 10 cases had typical flu-like symptoms at the onset of the disease, among which 7 patients developed dyspnea in the late stage, and 8 patients had hemoptysis. Laboratory examination: the absolute and percentage of peripheral blood lymphocytes in all 10 patients were declined, among which 5 cases were with decreased CD3+ CD4+ T cells/lymphocytes; 9 cases with increased bronchoalveolar lavage fluid galactomannan; 6 cases with increased serum galactomannan; 1 case with bronchoalveolar lavage fluid cultured aspergillus fumigatus; and 2 cases with aspergillus by second-generation sequencing. Chest computed tomography: all patients showed multiple diffused ground-glass opacities at the beginning, along with linear or reticular interstitial changes. Two cases had multiple subarachnoid nodules with halo signs, 3 cases had consolidation in multiple segments of both lungs, 2 cases had cavities, and 4 cases were with pleural effusion. Treatment: 10 patients were treated with antiviral and anti-Aspergillus drugs after admission. Four patients received respiratory support. All 10 cases were cured and discharged.Early diagnosis of IPA in influenza A (H1N1) patients is the key to successful treatment.


Assuntos
Influenza Humana/complicações , Aspergilose Pulmonar/etiologia , Adulto , Idoso , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Líquido da Lavagem Broncoalveolar , Distribuição de Qui-Quadrado , China , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Aspergilose Pulmonar/diagnóstico , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/métodos
4.
Front Immunol ; 12: 675294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34322116

RESUMO

Aspergillus fumigatus airway infections are associated with increased rates of hospitalizations and declining lung function in patients with chronic lung disease. While the pathogenesis of invasive A. fumigatus infections is well studied, little is known about the development and progression of airway infections. Previous studies have demonstrated a critical role for the IL-1 cytokines, IL-1α and IL-1ß in enhancing pulmonary neutrophil recruitment during invasive aspergillosis. Here we use a mouse model of A. fumigatus airway infection to study the role of these IL-1 cytokines in immunocompetent mice. In the absence of IL-1 receptor signaling, mice exhibited reduced numbers of viable pulmonary neutrophils and increased levels of neutrophil apoptosis during fungal airway infection. Impaired neutrophil viability in these mice was associated with reduced pulmonary and systemic levels of G-CSF, and treatment with G-CSF restored both neutrophil viability and resistance to A. fumigatus airway infection. Taken together, these data demonstrate that IL-1 dependent G-CSF production plays a key role for host resistance to A. fumigatus airway infection through suppressing neutrophil apoptosis at the site of infection.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/patogenicidade , Pulmão/imunologia , Neutrófilos/fisiologia , Aspergilose Pulmonar/imunologia , Receptores de Interleucina-1/fisiologia , Animais , Apoptose/imunologia , Quimiocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Interleucina-1alfa , Interleucina-1beta , Pulmão/patologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Neutrófilos/imunologia
5.
PLoS One ; 16(7): e0252948, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34242260

RESUMO

Conidia of Aspergillus fumigatus are inhaled by humans on daily basis. As a consequence, these conidia can cause infections that differ in severity ranging from allergic bronchopulmonary aspergillosis to invasive aspergillosis. In this study we compared virulence of five A. fumigatus isolates in four different infection models to address the predictive value of different model systems. Two of the A. fumigatus strains were isolated from dogs with a non-invasive sino-nasal aspergillosis (DTO271-B5 and DTO303-F3), while three strains were isolated from human patients with invasive aspergillosis (Af293, ATCC46645 and CEA10). Infection models used encompassed cultured type II A549 lung epithelial cells, Protostelium aurantium amoeba, Galleria melonella larvae and zebrafish embryos. No major differences in virulence between these five strains were observed in the lung epithelial cell model. In contrast, strain ATCC46645 was most virulent in the amoeba and zebrafish model, whereas it was much less virulent in the Galleria infection model. DTO303-F3 was most virulent in the latter model. In general, reference strain Af293 was less virulent as compared to the other strains. Genome sequence analysis showed that this latter strain differed from the other four strains in 136 SNPs in virulence-related genes. Together, our results show that virulence of individual A. fumigatus strains show significant differences between infection models. We conclude that the predictive value of different model systems varies since the relative virulence across fungal strains does not hold up across different infection model systems.


Assuntos
Aspergillus fumigatus/patogenicidade , Animais , Aspergillus fumigatus/genética , Cães , Mutação , Fenótipo , Virulência , Peixe-Zebra
6.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073107

RESUMO

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.


Assuntos
Células Epiteliais Alveolares , Aspergilose/microbiologia , Aspergillus fumigatus , Interações Hospedeiro-Patógeno , Células A549 , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/ultraestrutura , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Aspergillus fumigatus/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Imagem Óptica , Fagocitose , Esporos Fúngicos/metabolismo
7.
BMC Infect Dis ; 21(1): 573, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34126952

RESUMO

BACKGROUND: The recent increase in cases of azole-resistant Aspergillus fumigatus (ARAf) infections is a major clinical concern owing to its treatment limitations. Patient-derived ARAf occurs after prolonged azole treatment in patients with aspergillosis and involves various cyp51A point mutations or non-cyp51A mutations. The prognosis of patients with chronic pulmonary aspergillosis (CPA) with patient-derived ARAf infection remains unclear. In this study, we reported the case of a patient with ARAf due to HapE mutation, as well as the virulence of the isolate. CASE PRESENTATION: A 37-year-old male was presented with productive cough and low-grade fever. The patient was diagnosed with CPA based on the chronic course, presence of a fungus ball in the upper left lobe on chest computed tomography (CT), positivity for Aspergillus-precipitating antibody and denial of other diseases. The patient underwent left upper lobe and left S6 segment resection surgery because of repeated haemoptysis during voriconazole (VRC) treatment. The patient was postoperatively treated with VRC for 6 months. Since then, the patient was followed up without antifungal treatment but relapsed 4 years later, and VRC treatment was reinitiated. Although an azole-resistant isolate was isolated after VRC treatment, the patient did not show any disease progression in either respiratory symptoms or radiological findings. The ARAf isolated from this patient showed slow growth, decreased biomass and biofilm formation in vitro, and decreased virulence in the Galleria mellonella infection model compared with its parental strain. These phenotypes could be caused by the HapE splice site mutation. CONCLUSIONS: This is the first to report a case demonstrating the clinical manifestation of a CPA patient infected with ARAf with a HapE splice site mutation, which was consistent with the in vitro and in vivo attenuated virulence of the ARAf isolate. These results imply that not all the ARAf infections in immunocompetent patients require antifungal treatment. Further studies on the virulence of non-cyp51A mutations in ARAf are warranted.


Assuntos
Aspergillus fumigatus/genética , Azóis/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Proteínas Fúngicas/genética , Aspergilose Pulmonar/microbiologia , Adulto , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/patogenicidade , Azóis/uso terapêutico , Doença Crônica , Humanos , Masculino , Testes de Sensibilidade Microbiana , Mutação , Fenótipo , Aspergilose Pulmonar/tratamento farmacológico , Virulência/genética , Voriconazol/uso terapêutico
8.
PLoS Biol ; 19(6): e3001247, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34061822

RESUMO

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Sulfetos/metabolismo , Células A549 , Adulto , Animais , Aspergilose/epidemiologia , Aspergilose/genética , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Cistationina gama-Liase/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Incidência , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/microbiologia , Masculino , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Células THP-1 , Transplantados , Virulência/efeitos dos fármacos , Adulto Jovem
9.
Front Immunol ; 12: 675702, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34122436

RESUMO

Environmental factors, particularly fungi, influence the pathogenesis of allergic airway inflammation, but the mechanisms underlying these effects are still unclear. Melanin is one fungal component which is thought to modulate pulmonary inflammation. We recently identified a novel C-type lectin receptor, MelLec (Clec1a), which recognizes fungal 1,8-dihydroxynaphthalene (DHN)-melanin and is able to regulate inflammatory responses. Here we show that MelLec promotes pulmonary allergic inflammation and drives the development of Th17 T-cells in response to spores of Aspergillus fumigatus. Unexpectedly, we found that MelLec deficiency was protective, with MelLec-/- animals showing normal weight gain and significantly reduced pulmonary inflammation in our allergic model. The lungs of treated MelLec-/- mice displayed significantly reduced inflammatory foci and reduced bronchial wall thickening, which correlated with a reduced cellular influx (particularly neutrophils and inflammatory monocytes) and levels of inflammatory cytokines and chemokines. Notably, fungal burdens were increased in MelLec-/- animals, without apparent adverse effects, and there were no alterations in the survival of these mice. Characterization of the pulmonary T-cell populations, revealed a significant reduction in Th17 cells, and no alterations in Th2, Th1 or Treg cells. Thus, our data reveal that while MelLec is required to control pulmonary fungal burden, the inflammatory responses mediated by this receptor negatively impact the animal welfare in this allergic model.


Assuntos
Aspergillus fumigatus/patogenicidade , Asma/etiologia , Lectinas Tipo C/fisiologia , Animais , Asma/imunologia , Brônquios/patologia , Citocinas/biossíntese , Melaninas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/imunologia
10.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33972437

RESUMO

This paper presents a modular software design for the construction of computational modeling technology that will help implement precision medicine. In analogy to a common industrial strategy used for preventive maintenance of engineered products, medical digital twins are computational models of disease processes calibrated to individual patients using multiple heterogeneous data streams. They have the potential to help improve diagnosis, prognosis, and personalized treatment for a wide range of medical conditions. Their large-scale development relies on both mechanistic and data-driven techniques and requires the integration and ongoing update of multiple component models developed across many different laboratories. Distributed model building and integration requires an open-source modular software platform for the integration and simulation of models that is scalable and supports a decentralized, community-based model building process. This paper presents such a platform, including a case study in an animal model of a respiratory fungal infection.


Assuntos
Aspergilose/tratamento farmacológico , Biologia Computacional/métodos , Modelagem Computacional Específica para o Paciente , Medicina de Precisão/métodos , Software , Algoritmos , Animais , Antifúngicos/farmacologia , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Humanos , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/patogenicidade
11.
Pol J Microbiol ; 70(1): 3-11, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33815522

RESUMO

Aspergillus fumigatus is one of the ubiquitous fungi with airborne conidia, which accounts for most aspergillosis cases. In immunocompetent hosts, the inhaled conidia are rapidly eliminated. However, immunocompromised or immunodeficient hosts are particularly vulnerable to most Aspergillus infections and invasive aspergillosis (IA), with mortality from 50% to 95%. Despite the improvement of antifungal drugs over the last few decades, the therapeutic effect for IA patients is still limited and does not provide significant survival benefits. The drawbacks of antifungal drugs such as side effects, antifungal drug resistance, and the high cost of antifungal drugs highlight the importance of finding novel therapeutic and preventive approaches to fight against IA. In this article, we systemically addressed the pathogenic mechanisms, defense mechanisms against A. fumigatus, the immune response, molecular aspects of host evasion, and vaccines' current development against aspergillosis, particularly those based on AFMP4 protein, which might be a promising antigen for the development of anti-A. fumigatus vaccines.


Assuntos
Antígenos de Fungos/imunologia , Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Proteínas Fúngicas/imunologia , Vacinas Fúngicas/imunologia , Animais , Antígenos de Fungos/administração & dosagem , Antígenos de Fungos/genética , Aspergilose/microbiologia , Aspergilose/prevenção & controle , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/administração & dosagem , Proteínas Fúngicas/genética , Vacinas Fúngicas/administração & dosagem , Vacinas Fúngicas/genética , Humanos , Imunidade , Virulência
12.
Nat Commun ; 12(1): 1707, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731708

RESUMO

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA and its response to azole treatment is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo in a neutropenic mouse model and 3D light sheet fluorescence microscopy ex vivo in the infected mouse lungs to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antifúngicos/uso terapêutico , Aspergillus fumigatus/imunologia , Pulmão/efeitos dos fármacos , Pulmão/diagnóstico por imagem , Compostos Radiofarmacêuticos/imunologia , Animais , Anticorpos Antifúngicos/química , Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais Humanizados/química , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Azóis/uso terapêutico , Radioisótopos de Cobre/química , Monitoramento de Medicamentos , Corantes Fluorescentes/química , Humanos , Aspergilose Pulmonar Invasiva/diagnóstico por imagem , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Aspergilose Pulmonar Invasiva/microbiologia , Pulmão/microbiologia , Imageamento por Ressonância Magnética , Camundongos , Microscopia de Fluorescência , Tomografia por Emissão de Pósitrons , Radioimunodetecção , Compostos Radiofarmacêuticos/química
13.
Nat Commun ; 12(1): 1631, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712585

RESUMO

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , NAD+ Nucleosidase/química , NAD+ Nucleosidase/isolamento & purificação , NAD+ Nucleosidase/metabolismo , ADP-Ribosil Ciclase/metabolismo , Animais , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Cristalografia por Raios X , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Modelos Moleculares , NAD/metabolismo , NAD+ Nucleosidase/genética , Neurospora crassa/enzimologia , Neurospora crassa/genética , Neurospora crassa/metabolismo , Neurospora crassa/patogenicidade , Conformação Proteica , Células Sf9 , Transdução de Sinais
14.
PLoS Pathog ; 17(3): e1009235, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33780518

RESUMO

To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.


Assuntos
Aspergillus fumigatus/genética , Regulação Fúngica da Expressão Gênica/genética , Pulmão/virologia , Fatores de Transcrição/metabolismo , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica/métodos , Ferro/metabolismo , Pulmão/metabolismo , Camundongos , Virulência/genética
15.
Curr Genet ; 67(4): 583-593, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33760942

RESUMO

Iron acquisition is critical for pathogenic fungi to adapt to and survive within the host environment. However, to same extent, the fungi must also avoid the detrimental effects caused by excess iron. The importance of iron has been demonstrated for the physiology and virulence of major fungal pathogens of humans including Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans. In particular, numerous studies have revealed that aspects of iron acquisition, metabolism, and homeostasis in the fungal pathogens are tightly controlled by conserved transcriptional regulators including a GATA-type iron transcription factor and the CCAAT-binding complex (CBC)/HapX orthologous protein complex. However, the specific downstream regulatory networks are slightly different in each fungus. In addition, roles have been proposed or demonstrated for other factors including monothiol glutaredoxins, BolA-like proteins, and Fe-S cluster incorporation on the GATA-type iron transcription factor and the CBC/HapX orthologous protein complex, although limited information is available. Here we focus on recent work on C. neoformans in the context of an emerging framework for fungal regulation of iron acquisition, metabolism, and homeostasis. Our specific goal is to summarize recent findings on transcriptional networks governed by the iron regulators Cir1 and HapX in C. neoformans.


Assuntos
Proteínas Fúngicas/genética , Homeostase/genética , Ferro/metabolismo , Fatores de Transcrição/genética , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Candida albicans/genética , Candida albicans/patogenicidade , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Regulação Fúngica da Expressão Gênica/genética , Humanos , Virulência/genética
16.
Virulence ; 12(1): 818-834, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33682618

RESUMO

The invertebrate Galleria mellonella has increasingly and widely been used in the last few years to study complex host-microbe interactions. Aspergillus fumigatus is one of the most pathogenic fungi causing life-threatening diseases in humans and animals. Galleria mellonella larvae has been proven as a reliable model for the analysis of pathogenesis and virulence factors, enable to screen a large number of A. fumigatus strains. This review describes the different uses of G. mellonella to study A. fumigatus and provides a comparison of the different protocols to trace fungal pathogenicity. The review also includes a summary of the diverse mutants tested in G. mellonella, and their respective contribution to A. fumigatus virulence. Previous investigations indicated that G. mellonella should be considered as an interesting tool even though a mammalian model may be required to complete and verify initial data.


Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Larva/microbiologia , Mariposas/microbiologia , Fatores de Virulência/análise , Animais , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Fúngica , Interações Hospedeiro-Patógeno , Virulência
17.
Biomolecules ; 11(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669094

RESUMO

The ability to regulate the recruitment of immune cells makes chemokines and their receptors attractive drug targets in many inflammatory diseases. Based on its preferential expression on T helper type 2 (Th2) cells, C-C chemokine receptor type 4 (CCR4) has been widely studied in the context of allergic diseases, but recent evidence on the expression of CCR4 in other cell types has considerably expanded the potential applications of CCR4 antagonism. However, the current number of approved indications, as well as the portfolio of CCR4-targeting drugs, are still limited. In the present study, we have assessed the potential therapeutic efficacy of a CCR4 small molecule antagonist, SP50, discovered via an in silico-based approach, against a variety of pre-clinical settings of infection with the fungus Aspergillus fumigatus. We show that SP50 efficiently worked as prophylactic vaccine adjuvant in immunocompetent mice, protected against invasive aspergillosis in immunosuppressed mice. Further, the CCR4 antagonist prevented allergic bronchopulmonary aspergillosis in susceptible mice, and in a murine model of cystic fibrosis, a genetic disorder characterized by chronic pulmonary inflammation and recurrent infections. In conclusion, our results extend the potential applications of CCR4 antagonism and prompt for the development of novel compounds with the potential to progress to clinical trials.


Assuntos
Aspergilose Broncopulmonar Alérgica/tratamento farmacológico , Aspergilose Broncopulmonar Alérgica/imunologia , Aspergilose/tratamento farmacológico , Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Receptores CCR4/antagonistas & inibidores , Receptores CCR4/metabolismo , Animais , Aspergilose/prevenção & controle , Aspergilose Broncopulmonar Alérgica/prevenção & controle , Fibrose Cística/tratamento farmacológico , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Vacinação
18.
mSphere ; 6(1)2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597172

RESUMO

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease.IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.


Assuntos
Aspergilose Broncopulmonar Alérgica/classificação , Aspergilose Broncopulmonar Alérgica/microbiologia , Aspergillus fumigatus/patogenicidade , Pulmão/microbiologia , Fenótipo , Esporos Fúngicos/patogenicidade , Alérgenos/imunologia , Animais , Aspergilose Broncopulmonar Alérgica/imunologia , Aspergilose Broncopulmonar Alérgica/patologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/isolamento & purificação , Citocinas/imunologia , Feminino , Humanos , Inflamação/microbiologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Esporos Fúngicos/imunologia
19.
mSphere ; 6(1)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568450

RESUMO

Aspergillus fumigatus is the main cause of invasive aspergillosis (IA) with a high annual global incidence and mortality rate. Recent studies have indicated an increasing prevalence of azole-resistant A. fumigatus (ARAF) strains, with agricultural use of azole fungicides as a potential contributor. China has an extensive agricultural production system and uses a wide array of fungicides for crop production, including in modern growth facilities such as greenhouses. Soils in greenhouses are among the most intensively cultivated. However, little is known about the occurrence and distribution of ARAF in greenhouse soils. Here, we investigated genetic variation and triazole drug susceptibility in A. fumigatus from greenhouses around metropolitan Kunming in Yunnan, southwest China. Abundant allelic and genotypic variations were found among 233 A. fumigatus strains isolated from nine greenhouses in this region. Significantly, ∼80% of the strains were resistant to at least one medical triazole drug, with >30% showing cross-resistance to both itraconazole and voriconazole. Several previously reported mutations associated with triazole resistance in the triazole target gene cyp51A were also found in our strains, with a strong positive correlation between the frequency of mutations at the cyp51A promoter and that of voriconazole resistance. Phylogenetic analyses of cyp51A gene sequences showed evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in these greenhouses. Evidence for multiple origins of azole resistance and the widespread distributions of genetically very diverse triazole-resistant strains of A. fumigatus in greenhouses calls for significant attention from public health agencies.IMPORTANCE The origin and prevalence of azole-resistant Aspergillus fumigatus have been attracting increasing attention from biologists, clinicians, and public health agencies. Current evidence suggests agricultural fungicide use as a major cause. In southwest China, greenhouses are used to produce large amounts of fruits, flowers, and vegetables for consumers throughout China as well as those in other countries, primarily in southeast Asia. Here, we found a very high frequency (∼80%) of triazole-resistant A. fumigatus in our sample, the highest reported so far, with a significant proportion of these strains resistant to both tested agricultural fungicides and medical triazole drugs. In addition, we found novel allelic and genotypic diversities and evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in greenhouse populations in this region. Our study calls for a systematic evaluation of the effects of azole fungicide usage in greenhouses on human health.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Azóis/farmacologia , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Variação Genética , Agricultura , Aspergillus fumigatus/classificação , Aspergillus fumigatus/patogenicidade , China , Fungicidas Industriais/efeitos adversos , Fungicidas Industriais/farmacologia , Genótipo , Humanos , Mutação , Filogenia , Saúde Pública , Solo/química , Microbiologia do Solo
20.
Methods Mol Biol ; 2260: 83-109, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33405032

RESUMO

The respiratory epithelium is the initial point of host contact for inhaled particles, leading to orchestrated, but highly heterogeneous, responses. Human airway epithelial cells (AECs) play a crucial role in host defense by promoting uptake and killing of inhaled microorganisms and concomitant cytokine production in order to recruit professional phagocytes to the site of infection. However, inhaled pathogens can also reside and replicate intracellularly to evade host immune defenses or circulating antimicrobial drugs, ultimately causing apoptosis or cell death of the infected AECs. Imaging flow cytometry (IFC) combines flow cytometry, fluorescent microscopy, and advanced data-processing algorithms to dissect the heterogeneity of the interaction of AECs and inhaled microorganisms and its outcomes at the single-cell level. Here, we describe a novel single-cell approach based on differential fluorescent staining and state-of-the-art IFC to identify, quantify, and analyze individual host-pathogen complexes from cultured AECs infected with spores of the major human fungal pathogen Aspergillus fumigatus.


Assuntos
Células Epiteliais Alveolares/microbiologia , Aspergillus fumigatus/patogenicidade , Citometria de Fluxo , Corantes Fluorescentes/química , Microscopia de Fluorescência , Análise de Célula Única , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Apoptose , Interações Hospedeiro-Patógeno , Humanos , Processamento de Imagem Assistida por Computador , Necrose , Software
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