RESUMO
Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.
Assuntos
Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Equinocandinas/farmacologia , Proteínas Fúngicas/metabolismo , Pulmão/efeitos dos fármacos , Proteínas Quinases/deficiência , Animais , Antifúngicos/farmacologia , Aspergilose/enzimologia , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus fumigatus/enzimologia , Feminino , Pulmão/microbiologia , Pulmão/patologia , CamundongosRESUMO
Caused by fungus, fungal keratitis is a kind of infections corneal disease with high rate of blindness, which patients are mainly farmers in developing countries. Interleukin, as important proinflammatory cytokines, involve in immune defense process against fungal infection of cornea. The expression of interleukin in the pathogenesis of fungal keratitis, especially the main source of its cells, is not clear and the cell signaling pathways which regulate the synthesis and modification of interleukin is still unknown. Caspase-11 was obtained and cultured. And the ELISA and Western-blot methods were used to explore the regulatory effect of Caspse-11 on Interleukin-1ß in the fungal keratitis. neutrophils were the main cell lineage of IL-1ß to take part in the innate anti-fungi immunity in the cornea; IL-1ß generation induced by fungal infection might not be through the pre-excitation in the classical signal pathway; TLR4/TRIF pathway was not involved in pro-IL-1ß generation; while Dectin-1/syk pathway was involved in IL-1ß generation in the fungal keratitis; Caspase-l participated in the modification of IL-1ß to change from the precursor into the mature body; but NLRP3 inflammasome and ASC inflammasome were not involved in IL-1ß generation; Caspase-11 was involved in IL-1ß generation through regulating the modified process of Caspase-l to turning from precursor into mature body. TLR4/TRIF pathway and NLRP3 inflammasome and ASC inflammasome are not involved in the pro-IL-1ß generation, while Caspase-l, Caspase-11 and Dectin-1/syk pathway are involved in the IL-1ß generation.
Assuntos
Aspergilose/enzimologia , Aspergillus fumigatus/patogenicidade , Caspase 1/metabolismo , Caspases/metabolismo , Córnea/enzimologia , Infecções Oculares Fúngicas/enzimologia , Interleucina-1beta/metabolismo , Ceratite/enzimologia , Neutrófilos/enzimologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Aspergilose/genética , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus/imunologia , Caspase 1/genética , Caspases Iniciadoras , Células Cultivadas , Córnea/imunologia , Córnea/microbiologia , Modelos Animais de Doenças , Infecções Oculares Fúngicas/genética , Infecções Oculares Fúngicas/imunologia , Infecções Oculares Fúngicas/microbiologia , Feminino , Genótipo , Interações Hospedeiro-Patógeno , Ceratite/genética , Ceratite/imunologia , Ceratite/microbiologia , Lectinas Tipo C/deficiência , Lectinas Tipo C/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/microbiologia , Fenótipo , Transdução de Sinais , Quinase Syk/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.
Assuntos
Aspergilose/enzimologia , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/patogenicidade , Parede Celular/enzimologia , Polissacarídeos Fúngicos/biossíntese , Proteínas Fúngicas/metabolismo , Glucosiltransferases/metabolismo , Animais , Aspergilose/genética , Aspergilose/patologia , Aspergillus fumigatus/genética , Parede Celular/genética , Polissacarídeos Fúngicos/genética , Proteínas Fúngicas/genética , Deleção de Genes , Glucosiltransferases/genética , Humanos , Camundongos Knockout , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genéticaRESUMO
The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.
Assuntos
Aspergillus fumigatus/enzimologia , Calcineurina/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/enzimologia , Modelos Biológicos , Motivos de Aminoácidos , Animais , Antifúngicos/química , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/enzimologia , Aspergilose/genética , Aspergillus fumigatus/genética , Calcineurina/química , Calcineurina/imunologia , Inibidores de Calcineurina , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Hifas/genética , Masculino , Camundongos , Fosforilação , Estrutura Terciária de ProteínaRESUMO
Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ß-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.
Assuntos
Aspergillus fumigatus/imunologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/imunologia , Monócitos/enzimologia , Monócitos/imunologia , NADPH Oxidases/fisiologia , Doença Aguda , Animais , Aspergilose/enzimologia , Aspergilose/imunologia , Aspergilose/patologia , Predisposição Genética para Doença , Inflamação/enzimologia , Inflamação/microbiologia , Inflamação/prevenção & controle , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monócitos/microbiologia , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Zimosan/farmacologiaRESUMO
Aspergillus spp. are widespread in nature and cause severe infections especially in immunocompromised patients. Aspergillus fumigatus complex is the most common species that causes infections in humans; however Aspergillus niger complex and Aspergillus flavus complex are the emerging agents that are isolated frequently from clinical specimens more recently. Besides the host factors such as immunosuppression, hematologic malignancy and corticosteroid use, fungal virulence factors such as elastase, acid protease and phospholipase enzymes are considered among the factors that affect the development of invasive aspergillosis. The aim of this study was to detect the acid proteinase and phospholipase enzyme activities in 30 A.fumigatus complex, nine A.flavus complex and four A.niger complex strains isolated from clinical specimens. Acid proteinase and phospholipase activities of the isolates were investigated by using bovine serum albumin agar (BSA), and egg yolk agar plates, respectively. Acid proteinase and phospholipase activity was detected in 76.7% (23/30) and 93.3% (28/30) of A.fumigatus complex isolates, respectively. None of the nine A.flavus complex isolates exhibited acid proteinase or phospholipase activity. Acid proteinase activity was not detected in any of the A.niger complex isolates (n= 4), however phospholipase activity was detected in one (25%) isolate. All of the acid proteinase positive A.fumigatus complex strains (n= 23) were also positive for phospholipase activity. In conclusion, further larger scale multicenter studies supported by clinical data, are needed to enlighten the roles of acid proteinase and phospholipase in the pathogenesis of infections due to Aspergillus spp.
Assuntos
Aspergilose/microbiologia , Aspergillus/enzimologia , Peptídeo Hidrolases/metabolismo , Fosfolipases/metabolismo , Fatores de Virulência/metabolismo , Aspergilose/enzimologia , Aspergillus/patogenicidade , Humanos , VirulênciaRESUMO
Invasive fungal infections are a major threat for patients suffering from chronic granulomatous disease (CGD), a primary immunodeficiency caused by a defect in the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase. Interestingly, Aspergillus (Emericella) nidulans is the second most encountered mold in CGD patients, causing almost exclusively invasive infections in this specific host, and is characterized by its aggressive behavior. A proper diagnosis is complicated by the often mild clinical presentation, the low sensitivity of the currently used diagnostic tools, and the difficulties in accurate identification of the Emericella species. According to the hitherto accepted view on the role of the NADPH-oxidase in the innate host-defense pathway, the pathogenesis of A. nidulans in CGD cannot be explained. This synopsis covers the current understanding of invasive infections caused by A. nidulans in the CGD patient and is intended to direct further research by indicating gaps in our knowledge and to guide optimal management strategies.
Assuntos
Aspergilose/microbiologia , Aspergillus nidulans/fisiologia , Doença Granulomatosa Crônica/complicações , Animais , Aspergilose/enzimologia , Aspergilose/imunologia , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Neutrófilos/microbiologiaRESUMO
BACKGROUND: Blood levels of voriconazole, a first line therapy for invasive aspergillosis, may correlate with adverse events and treatment response. However, no randomized controlled studies have been conducted to evaluate the clinical utility of routine therapeutic drug monitoring (TDM) of voriconazole. This study aimed to determine whether routine TDM of voriconazole reduces drug adverse events or improves treatment response in invasive fungal infections. METHODS: This was a randomized, assessor-blinded, controlled, single center trial. One hundred ten adult patients were randomly assigned to TDM or non-TDM groups. In the TDM group, voriconazole dosage was adjusted (target range, 1.0-5.5 mg/L) according to the serum trough level measured on the fourth day after initiation of voriconazole. The non-TDM group received a fixed, standard dosage. Voriconazole-related adverse events were monitored, and treatment response was assessed three months after the initiation of therapy. RESULTS: Baseline characteristics including the CYP2C19 genotype were comparable between the two groups. While the incidence of adverse events was not different between the TDM group and the non-TDM group (both 42%; P = .97), the proportion of voriconazole discontinuation due to adverse events was significantly lower in the TDM group than in the non-TDM group (4% vs 17%; P = .02). A complete or partial response was observed in 81% (30 of 37) of patients in the TDM group compared to 57% (20 of 34) in the non-TDM group (P = .04). CONCLUSIONS: Routine TDM of voriconazole may reduce drug discontinuation due to adverse events and improve the treatment response in invasive fungal infections. CLINICAL TRIAL REGISTRATION: NCT00890708.
Assuntos
Aspergilose/tratamento farmacológico , Monitoramento de Medicamentos/estatística & dados numéricos , Pirimidinas/efeitos adversos , Pirimidinas/uso terapêutico , Triazóis/efeitos adversos , Triazóis/uso terapêutico , Adulto , Idoso , Antifúngicos/efeitos adversos , Antifúngicos/uso terapêutico , Hidrocarboneto de Aril Hidroxilases/genética , Aspergilose/enzimologia , Aspergilose/genética , Distribuição de Qui-Quadrado , Citocromo P-450 CYP2C19 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Pirimidinas/sangue , Método Simples-Cego , Resultado do Tratamento , Triazóis/sangue , VoriconazolRESUMO
NADPH oxidase plays a critical role in antimicrobial host defense, as evident in chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal diseases. Invasive aspergillosis and other moulds are the major cause of mortality in CGD. We also learn from CGD patients that NADPH oxidase plays an important role in regulating inflammation; CGD patients are prone to developing inflammatory diseases such as inflammatory bowel disease, obstructive granulomata of the genitourinary tract, and hypersensitivity pneumonitis. Indeed, the NADPH oxidase plays an essential role in calibrating innate and T-cell responses to control the growth of inhaled fungi while protecting against excessive and injurious inflammation. Knowledge gained on the mechanisms by which NADPH oxidase kills fungi and regulates inflammation may lead to new therapeutics for CGD and will have broad relevance to understanding host-pathogen interactions between mammals and ubiquitous moulds to which we are continually exposed.
Assuntos
Aspergilose/enzimologia , Aspergilose/microbiologia , Aspergillus/patogenicidade , Doença Granulomatosa Crônica/enzimologia , Interações Hospedeiro-Patógeno , NADPH Oxidases/metabolismo , Animais , Aspergilose/imunologia , Aspergilose/mortalidade , Aspergillus/imunologia , Doença Granulomatosa Crônica/complicações , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Inflamação/enzimologia , Inflamação/etiologia , Inflamação/imunologia , Camundongos , Neutrófilos , Fenótipo , Isoformas de Proteínas/metabolismo , Linfócitos T/classificação , Linfócitos T/imunologiaRESUMO
Mammalian Ste20-like kinase 4 (MST4), a new member of the germinal-center kinase STE20 family, was recently demonstrated to be a negative regulator of inflammation. However, whether MST4 participates in the inflammatory response to fungal infection remains unknown. Our study investigated the role and molecular mechanisms of MST4 in mice cornea and corneal epithelial cells exposed to Aspergillus fumigatus (A. fumigatus). Protein level of MST4 was detected in mice corneas and human corneal epithelial cells (HCECs) by Western blot analysis. The MST4 protein level was significantly elevated in mice corneas infected with A. fumigatus and HCECs exposed to A. fumigatus. MST4 expression was also detected in mice corneas by immunofluorescence staining. Furthermore, we found recombinant MST4 inhibited proinflammatory cytokines expressions induced by A. fumigatus at both the mRNA and protein levels in mice corneas and HCECs. To further investigate the mechanism of MST4's anti-inflammatory effect in A. fumigatus keratitis, we verified recombinant MST4 can inhibit curdlan-mediated proinflammatory cytokines production in HCECs. Surprisingly, recombinant MST4 protein downregulated A. fumigatus-induced Dectin-1 expression in both mRNA and protein levels in mice corneas. Recombinant MST4 can inhibit the mRNA expression level of Dectin-1 which was induced by curdlan in HCECs. MST4 can also inhibit the expression of Dectin-1 in mRNA levels increased by Dectin-1 overexpression plasmid in HCECs. Moreover, A. fumigatus or curdlan significantly induced the phosphorylation of Syk, which was consequently suppressed by recombinant MST4. Finally, recombinant MST4 promotes HCECs proliferation, which contribute to cornea wound healing. Taken together, our results provide evidences that MST4 inhibits inflammatory signaling response in A. fumigatus keratitis by downregulating Dectin-1/p-Syk pathway and simultaneously promotes HCECs proliferation.
Assuntos
Aspergilose/enzimologia , Aspergillus fumigatus , Ceratite/microbiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.
Assuntos
Aspergilose/enzimologia , Aspergillus fumigatus/enzimologia , Parede Celular/metabolismo , Furanos/metabolismo , Galactose/metabolismo , Transferases Intramoleculares/metabolismo , Mananas/metabolismo , Animais , Aspergilose/genética , Aspergilose/fisiopatologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Temperatura Corporal/fisiologia , Proliferação de Células , Parede Celular/genética , Modelos Animais de Doenças , Farmacorresistência Fúngica/genética , Feminino , Regulação Fúngica da Expressão Gênica/genética , Hospedeiro Imunocomprometido/fisiologia , Transferases Intramoleculares/genética , Camundongos , Camundongos Endogâmicos BALB C , Infecções Oportunistas/genética , Infecções Oportunistas/metabolismo , Infecções Oportunistas/fisiopatologia , Virulência/genéticaRESUMO
Fungal lipases are efficient and environment-friendly biocatalysts for many industrially relevant processes. One of the most widely applied lipases in the manufacturing industry is Candida antarctica lipase B (CALB). Here, we report the biochemical and structural characterization of a novel CALB-like lipase from an important human pathogen-Aspergillus fumigatus (AFLB), which has high sn-1,3-specificity toward triolein. AFLB crystal structure displays a CALB-like catalytic domain and hosts a unique tightly closed 'lid' domain that contains a disulfide bridge, as well as an extra N-terminal subdomain composed of residues 1-128 (including the helix α1-α5 located above the active site). To gain insight into the function of this novel lid and N-terminal subdomain, we constructed and characterized a series of mutants in these two domains. Deleting the protruding bulk lid's residues, replacing the bulk and tight lid with a small and loose lid from CALB, or breaking the disulfide bridge increased the affinity of CALB for glyceride substrates and improved its catalytic activity, along with the loss of enzyme fold stability and thermostability. N-terminal truncation mutants revealed that the N-terminal peptide (residues 1-59) is a strong inhibitor of AFLB binding to lipid films. This peptide thus limits AFLB's penetration power and specific activity, revealing a unique enzyme activity regulatory mechanism. Our findings on the functional and structural properties of AFLB provide a better understanding of the functions of the CALB-like lipases and pave the way for its future protein engineering. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6IDY.
Assuntos
Aspergilose/enzimologia , Aspergillus fumigatus/enzimologia , Lipase/química , Conformação Proteica , Aspergilose/genética , Aspergilose/microbiologia , Aspergillus fumigatus/química , Domínio Catalítico/genética , Estabilidade Enzimática/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Lipase/genética , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Relação Estrutura-AtividadeRESUMO
Aspergillus is the causative agent of human diseases ranging from asthma to invasive infection. Genetic and environmental factors are crucial in regulating the interaction between the host and Aspergillus. The role played by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the first and rate-limiting step of tryptophan catabolism along the kynurenine pathway, is increasingly being recognized, but whether and how genetic variation of IDO1 influences the risk of aspergillosis in susceptible patients is incompletely understood. In addition, whether the closely related protein IDO2 plays a similar role remains unexplored. In the present study, we performed genetic association studies in two different cohorts of susceptible patients [cystic fibrosis (CF) patients and recipients of hematopoietic stem cell transplantation (HSCT)], and identified IDO1 polymorphisms that associate with the risk of infection in both cohorts. By using human bronchial epithelial cells and PBMC from CF and HSCT patients, respectively, we could show that the IDO1 polymorphisms appeared to down-modulate IDO1 expression and function in response to IFNγ or Aspergillus conidia, and to associate with an increased inflammatory response. In contrast, IDO2 polymorphisms, including variants known to profoundly affect protein expression and function, were differently associated with the risk of aspergillosis in the two cohorts of patients as no association was found in CF patients as opposed to recipients of HSCT. By resorting to a murine model of bone marrow transplantation, we could show that the absence of IDO2 more severely affected fungal burden and lung pathology upon infection with Aspergillus as compared to IDO1, and this effect appeared to be linked to a deficit in the antifungal effector phagocytic activity. Thus, our study confirms and extends the role of IDO1 in the response to Aspergillus, and shed light on the possible involvement of IDO2 in specific clinical settings.
Assuntos
Aspergilose/genética , Predisposição Genética para Doença/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Polimorfismo Genético , Adolescente , Adulto , Animais , Aspergilose/enzimologia , Aspergilose/microbiologia , Aspergillus/fisiologia , Criança , Pré-Escolar , Fibrose Cística/enzimologia , Fibrose Cística/genética , Fibrose Cística/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Interações Hospedeiro-Patógeno , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/microbiologia , Masculino , Camundongos , Adulto JovemRESUMO
The ubiquitous fungus Aspergillus flavus is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent Aspergillus pathogen after A. fumigatus infecting immunosuppressed patients. In many human fungal pathogens including A. fumigatus, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In A. fumigatus, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that A. flavus tolerates higher concentrations of Cu than A. fumigatus and other Aspergillus spp. associated with the presence of two homologs of A. fumigatus CrpA termed CrpA and CrpB. Both crpA and crpB are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of crpA or crpB alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor aceA. The ΔcrpAΔcrpB and ΔaceA strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed ΔcrpAΔcrpB and to a lesser degree ΔaceA to be significantly reduced in virulence, following the greater sensitivity of ΔcrpAΔcrpB to Cu than ΔaceA.
Assuntos
Aspergillus flavus/patogenicidade , ATPases Transportadoras de Cobre/metabolismo , Cobre/farmacologia , Proteínas Fúngicas/genética , Zea mays/microbiologia , Animais , Aspergilose/enzimologia , Aspergillus flavus/efeitos dos fármacos , Aspergillus fumigatus/efeitos dos fármacos , ATPases Transportadoras de Cobre/genética , Feminino , Deleção de Genes , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Fatores de Transcrição/genética , Virulência , Zea mays/enzimologiaRESUMO
In a previous work we demonstrated a clear link between elastase activity and pathogenicity using what we have named the Elastase Activity Index (EAI). In the present study we have evaluated the possible variability of this index as a consequence of successive inoculations in mice. Two strains of Aspergillus fumigatus isolated from the environment without elastase activity were used. These strains were inoculated into successive batches of ten mice. Our results showed that with each inoculation there was an increase in the number of mice on each batch from which the strain could be isolated and an increase in the number of strains with an EAI>1. This study suggests that A. fumigatus could adapt to the environment in which it is developed, increasing its pathogenic capabilities from host to host.
Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/análise , Pneumopatias Fúngicas/microbiologia , Elastase Pancreática/análise , Administração Intranasal , Animais , Aspergilose/enzimologia , Aspergillus fumigatus/citologia , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/patogenicidade , Ciclofosfamida/toxicidade , Suscetibilidade a Doenças , Microbiologia Ambiental , Imunossupressores/toxicidade , Pneumopatias Fúngicas/enzimologia , Masculino , Camundongos , VirulênciaRESUMO
OBJECTIVE: To investigate whether or not NADPH oxidase (NOX) participates in Aspergillus fumigatus induced macrophage autophagy in RAW264.7 cells and explore the possible mechanism. METHODS: RAW264.7 cells in the exponential growth phase were randomized into three groups: Aspergillus fumigatus conidia (MOI 5:1) treated group, Aspergillus fumigatus conidia plus NOX inhibitor diphenyleneiodonium chloride (DPI) treated group; negative control group (untreated). The levels of intracellular reactive oxygen species (ROS) were measured with dichiorodihydrofluorescein diacetate (DCFH-DA) staining by fluorescence microscope. The expression of microtubule-associated protein light chain-3 (LC3) protein in RAW264.7 cells was detected by Western blotting. Then the expression and positioning of LC-3 were observed by immunofluorescence staining. RESULTS: After stimulated with conidia of Aspergillus fumigatus, RAW264.7 cells showed significant increases of ROS and LC3BII. Interestingly, LC3 was recruited to phagosomes which contained Aspergillus fumigatus conidia. After the NOX inhibitor DPI blocked the ROS production, the expressions of ROS and LC3BII had no significant increases as compared with the negative control group, and the distribution of LC3 was diffuse in cytoplasm. CONCLUSION: NOX induces macrophage autophagy by producing ROS in Aspergillus fumigatus infection.
Assuntos
Aspergilose/enzimologia , Aspergillus fumigatus/fisiologia , Autofagia , Macrófagos/citologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergilose/fisiopatologia , Linhagem Celular , Humanos , Macrófagos/enzimologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , NADPH Oxidases/genéticaRESUMO
Clinical isolates of Aspergillus species were tested, both retrospectively and prospectively, for elastase activity using rose bengal-elastin agar plates. Patient records were then reviewed to determine the clinical diagnosis including the presence or absence of aspergillosis. All isolates that caused invasive aspergillosis produced elastase, but not all isolates producing elastase were associated with invasive disease.
Assuntos
Aspergillus/enzimologia , Elastase Pancreática/biossíntese , Aspergilose/enzimologia , Aspergilose/microbiologia , Aspergillus/isolamento & purificação , Humanos , Elastase Pancreática/metabolismoRESUMO
Aspergillus fumigatus produced a 33-kDa serine protease (ALP) in vitro and in vivo. In vitro, this alkaline protease was secreted when the fungus was cultivated in the absence of protein, if the pH of the medium remained close to neutrality. Western blotting and immunoelectronmicroscopy studies showed that ALP was localised in the wall of the fungus and was degraded after secretion in the culture medium under conditions of low pH. Although present in the lung during infection, ALP did not appear to be diagnostically useful and was different from the precipitating chymotrypsin antigen used in the diagnosis of aspergilloma.
Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Serina Endopeptidases/biossíntese , Animais , Aspergilose/enzimologia , Western Blotting , Meios de Cultura , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Soros Imunes/imunologia , Immunoblotting , Pulmão/enzimologia , Pulmão/microbiologia , Masculino , Camundongos , Coelhos , Serina Endopeptidases/imunologia , Serina Endopeptidases/isolamento & purificaçãoRESUMO
The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis. Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified. An A. fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe. Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli. Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified. These results indicate that A. fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB. The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A. fumigatus. Staining was observed heterogeneously throughout the fungal hyphae. This result indicates that catalase is produced by A. fumigatus during invasive aspergillosis.
Assuntos
Aspergillus fumigatus/enzimologia , Catalase/genética , Animais , Aspergilose/enzimologia , Aspergilose/microbiologia , Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Western Blotting , Catalase/química , Catalase/metabolismo , Clonagem Molecular , Éxons/genética , Feminino , Glicosilação , Íntrons/genética , Rim/enzimologia , Rim/microbiologia , Fígado/enzimologia , Fígado/microbiologia , Pulmão/enzimologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos , Neutropenia , Sinais Direcionadores de Proteínas , Homologia de Sequência de Aminoácidos , Coloração e RotulagemRESUMO
Cystine aminopeptidase (CAP) activity in tissues and in plasma from pregnant cows was measured at various stages of gestation by using the substrate S-benzyl-L-cysteine-4-nitroanilide. The CAP activity was as high or higher in hepatic and renal tissues as in cotyledonary tissue. Plasma CAP activity was low and did not change with length of gestation, nor did it change when placentation was disrupted by infection with Aspergillus fumigatus.