RESUMO
The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl. In this study, recombinant glycoside hydrolases (GHs) that degrade Pel and Psl were evaluated alone and in combination with antibiotics in a mouse model of P. aeruginosa infection. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that, although GHs have short half-lives, administration of two GHs in combination resulted in increased GH persistence. Combining GH prophylaxis and treatment with the antibiotic ciprofloxacin resulted in greater reduction in pulmonary bacterial burden than that with either agent alone. This study lays the foundation for further exploration of GH therapy in bacterial infections.
Assuntos
Infecções por Pseudomonas , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Glicosídeo Hidrolases/metabolismo , Pulmão/metabolismo , Camundongos , Polissacarídeos Bacterianos/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismoRESUMO
Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-d-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.
Assuntos
Aspergilose/terapia , Aspergillus fumigatus/enzimologia , Proteínas de Bactérias/química , Biofilmes , Glicosídeo Hidrolases/química , Pseudomonas aeruginosa/enzimologia , Células A549 , Animais , Anti-Infecciosos/química , Antifúngicos/química , Aspergilose/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Polissacarídeos/química , Especificidade da Espécie , VirulênciaRESUMO
The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. The developmental modifiers MedA, StuA, and SomA regulate GAG biosynthesis, but the mechanisms underlying this regulation are poorly understood. PtaB is a lim-domain binding protein that interacts with the transcription factor SomA and is required for normal conidiation and biofilm formation. Disruption of ptaB resulted in impaired GAG production and conidiation in association with a markedly reduced expression of GAG biosynthetic genes (uge3 and agd3), developmental regulators (medA and stuA), and genes involved in the core conidiation pathway. Overexpression of medA and dual overexpression of uge3 and agd3 in the ΔptaB mutant increased biofilm formation but not conidiation, whereas overexpression of core conidiation genes rescued conidiation but not biofilm formation. Overexpression of stuA modestly increased both conidiation and biofilm formation. Analysis of ptaB truncation mutants revealed that overexpression of the lim-domain binding region restored conidiation but not biofilm formation, suggesting that ptaB may govern these processes by interacting with different partners. These studies establish that PtaB governs GAG biosynthesis at the level of substrate availability and polymer deacetylation and that PtaB-mediated biofilm formation and conidiation are largely independent pathways.
Assuntos
Aspergillus fumigatus/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Polissacarídeos/biossíntese , Esporos Fúngicos/crescimento & desenvolvimento , Aspergilose/microbiologia , Aspergilose/patologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Adesão Celular/genética , Técnicas de Inativação de Genes , Polissacarídeos/genética , Fatores de Transcrição/metabolismoRESUMO
Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.
Assuntos
Aspergillus/patogenicidade , Armadilhas Extracelulares , Neutrófilos/imunologia , Polissacarídeos/fisiologia , Animais , Biofilmes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , VirulênciaRESUMO
Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (ß/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.
Assuntos
Aspergillus fumigatus/metabolismo , Coccidioidina/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Polissacarídeos/biossíntese , Sequência de Aminoácidos , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/patogenicidade , Catálise , Domínio Catalítico , Parede Celular/enzimologia , Coccidioidina/genética , Coccidioidina/fisiologia , Sequência Conservada , Cristalografia por Raios X , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/fisiologia , Hidrólise , Dados de Sequência Molecular , Mutação , Polissacarídeos/genética , Conformação Proteica , Alinhamento de SequênciaRESUMO
The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.
Assuntos
Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Polissacarídeos Fúngicos/biossíntese , Proteínas Fúngicas/metabolismo , Galactose/metabolismo , UDPglucose 4-Epimerase/metabolismo , Aspergillus fumigatus/genética , Parede Celular/genética , Polissacarídeos Fúngicos/genética , Proteínas Fúngicas/genética , Galactose/genética , UDPglucose 4-Epimerase/genéticaRESUMO
Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall ß-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Polissacarídeos Fúngicos/imunologia , Polissacarídeos/imunologia , Fatores de Virulência/imunologia , beta-Glucanas/imunologia , Animais , Aspergilose/genética , Aspergilose/patologia , Aspergillus fumigatus/genética , Carboidratos Epimerases/genética , Carboidratos Epimerases/imunologia , Linhagem Celular , Modelos Animais de Doenças , Polissacarídeos Fúngicos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Humanos , Hifas/genética , Hifas/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Camundongos , Polissacarídeos/genética , Fatores de Virulência/genéticaRESUMO
Production of ergot alkaloids in the opportunistic fungal pathogen Aspergillus fumigatus is restricted to conidiating cultures. These cultures typically accumulate several pathway intermediates at concentrations comparable to that of the pathway end product. We investigated the contribution of different cell types that constitute the multicellular conidiophore of A. fumigatus to the production of ergot alkaloid pathway intermediates versus the pathway end product, fumigaclavine C. A relatively minor share (11 %) of the ergot alkaloid yield on a molar basis was secreted into the medium, whereas the remainder was associated with the conidiating colonies. Entire conidiating cultures (containing hyphae, vesicle of conidiophore, phialides of conidiophore, and conidia) accumulated higher levels of the pathway intermediate festuclavine and lower levels of the pathway end product fumigaclavine C than did isolated, abscised conidia, indicating that conidiophores and/or hyphae have a quantitatively different ergot alkaloid profile compared to that of conidia. Differences in alkaloid accumulation among cell types also were indicated by studies with conidiophore development mutants. A ∆medA mutant, in which conidiophores are numerous but develop poorly, accumulated higher levels of pathway intermediates than did the wildtype or a complemented ∆medA mutant. A ∆stuA mutant, which grows mainly as hyphae and produces very few, abnormal conidiophores, produced no detectable ergot alkaloids. The data indicated heterogeneous spatial distribution of ergot alkaloid pathway intermediates versus pathway end product in conidiating cultures of A. fumigatus. This skewed distribution may reflect differences in abundance or activity of pathway enzymes among cell types of those conidiating cultures.
Assuntos
Alcaloides de Claviceps/análise , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Aspergillus fumigatus/química , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/metabolismo , Alcaloides de Claviceps/química , Alcaloides de Claviceps/metabolismo , Esporos Fúngicos/químicaRESUMO
During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro, little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. Using the HSAEC1-KT human small airway epithelial (HSAE) cell line, we developed an in vitro model to study the interaction of two strains of A. fumigatus with these cells. We then compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAE cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5ß1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5ß1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro. Importance During the initiation of invasive aspergillosis, Aspergillus fumigatus interacts with the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus-epithelial cell interactions in vitro used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line; the interactions of fungi with terminal bronchiolar epithelial cells were not investigated. Using the TERT-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line, we developed an in vitro model of the interactions of A. fumigatus with bronchiolar epithelial cells. We discovered that A. fumigatus invades and damages A549 and HSAE cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.
Assuntos
Aspergilose , Aspergillus fumigatus , Humanos , Aspergillus fumigatus/metabolismo , Integrina alfa5beta1/metabolismo , Células Epiteliais/microbiologia , Pulmão/microbiologia , Linhagem CelularRESUMO
During the initiation of invasive aspergillosis, inhaled Aspergillus fumigatus conidia are deposited on the epithelial cells lining the bronchi, terminal bronchioles, and alveoli. While the interactions of A. fumigatus with bronchial and type II alveolar cell lines have been investigated in vitro , little is known about the interactions of this fungus with terminal bronchiolar epithelial cells. We compared the interactions of A. fumigatus with the A549 type II alveolar epithelial cell line and the HSAEC1-KT human small airway epithelial (HSAE) cell line. We found that A. fumigatus conidia were poorly endocytosed by A549 cells, but avidly endocytosed by HSAE cells. A. fumigatus germlings invaded both cell types by induced endocytosis, but not by active penetration. A549 cell endocytosis of A. fumigatus was independent of fungal viability, more dependent on host microfilaments than microtubules, and induced by A. fumigatus CalA interacting with host cell integrin α5ß1. By contrast, HSAE cell endocytosis required fungal viability, was more dependent on microtubules than microfilaments, and did not require CalA or integrin α5ß1. HSAE cells were more susceptible than A549 cells to damage caused by direct contact with killed A. fumigatus germlings and by secreted fungal products. In response to A. fumigatus infection, A549 cells secreted a broader profile of cytokines and chemokines than HSAE cells. Taken together, these results demonstrate that studies of HSAE cells provide complementary data to A549 cells and thus represent a useful model for probing the interactions of A. fumigatus with bronchiolar epithelial cells in vitro . Importance: During the initiation of invasive aspergillosis, Aspergillus fumigatus invades, damages, and stimulates the epithelial cells that line the airways and alveoli. Previous studies of A. fumigatus - epithelial cell interactions in vitro have used either large airway epithelial cell lines or the A549 type II alveolar epithelial cell line. The interactions of fungi with terminal bronchiolar epithelial cells have not been investigated. Here, we compared the interactions of A. fumigatus with A549 cells and the Tert-immortalized human small airway epithelial HSAEC1-KT (HSAE) cell line. We discovered that A. fumigatus invades and damages these two cell lines by distinct mechanisms. Also, the proinflammatory responses of the cell lines to A. fumigatus are different. These results provide insight into how A. fumigatus interacts with different types of epithelial cells during invasive aspergillosis and demonstrate that HSAE cells are useful in vitro model for investigating the interactions of this fungus with bronchiolar epithelial cells.
RESUMO
Arsenic contamination of groundwater sources is a major issue worldwide, since exposure to high levels of arsenic has been linked to a variety of health problems. Effective methods of detection are thus greatly needed as preventive measures. In an effort to develop a fungal biosensor for arsenic, we first identified seven putative arsenic metabolism and transport genes in Aspergillus niger, a widely used industrial organism that is generally regarded as safe (GRAS). Among the genes tested for RNA expression in response to arsenate, acrA, encoding a putative plasma membrane arsenite efflux pump, displayed an over 200-fold increase in gene expression in response to arsenate. We characterized the function of this A. niger protein in arsenic efflux by gene knockout and confirmed that AcrA was located at the cell membrane using an enhanced green fluorescent protein (eGFP) fusion construct. Based on our observations, we developed a putative biosensor strain containing a construct of the native promoter of acrA fused with egfp. We analyzed the fluorescence of this biosensor strain in the presence of arsenic using confocal microscopy and spectrofluorimetry. The biosensor strain reliably detected both arsenite and arsenate in the range of 1.8 to 180 µg/liter, which encompasses the threshold concentrations for drinking water set by the World Health Organization (10 and 50 µg/liter).
Assuntos
Arsênio/metabolismo , Arsênio/farmacologia , Aspergillus niger/efeitos dos fármacos , Técnicas Biossensoriais/métodos , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Arsenitos/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Membrana Celular/metabolismo , Água Potável/química , Corantes Fluorescentes/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Água Subterrânea/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The mold Aspergillus fumigatus and bacterium Pseudomonas aeruginosa form biofilms in the airways of individuals with cystic fibrosis. Biofilm formation by A. fumigatus depends on the self-produced cationic exopolysaccharide galactosaminogalactan (GAG), while P. aeruginosa biofilms can contain the cationic exopolysaccharide Pel. GAG and Pel are rendered cationic by deacetylation mediated by either the secreted deacetylase Agd3 (A. fumigatus) or the periplasmic deacetylase PelA (P. aeruginosa). Given the similarities between these polymers, the potential for biofilm interactions between these organisms were investigated. P. aeruginosa were observed to adhere to A. fumigatus hyphae in a GAG-dependent manner and to GAG-coated coverslips of A. fumigatus biofilms. In biofilm adherence assays, incubation of P. aeruginosa with A. fumigatus culture supernatants containing de-N-acetylated GAG augmented the formation of adherent P. aeruginosa biofilms, increasing protection against killing by the antibiotic colistin. Fluorescence microscopy demonstrated incorporation of GAG within P. aeruginosa biofilms, suggesting that GAG can serve as an alternate biofilm exopolysaccharide for this bacterium. In contrast, Pel-containing bacterial culture supernatants only augmented the formation of adherent A. fumigatus biofilms when antifungal inhibitory molecules were removed. This study demonstrates biofilm interaction via exopolysaccharides as a potential mechanism of co-operation between these organisms in chronic lung disease.
RESUMO
Relatively few transcription factors that govern the virulence of Aspergillus fumigatus are known. We constructed 11 A. fumigatus transcription factor mutants and screened them for altered virulence in Galleria mellonella larvae. We discovered that the zinc cluster transcription factor, AcuM, is essential for maximal virulence in this model, as well as in murine models of haematogenously disseminated and invasive pulmonary aspergillosis. Transcriptional profiling experiments suggested that AcuM suppresses sreA and induces hapX to stimulate expression of genes involved in both reductive iron assimilation and siderophore-mediated iron uptake. Consistent with these results, a ΔacuM mutant had reduced iron incorporation, decreased extracellular siderophore production and impaired capacity to grow under iron-limited conditions. Interestingly, an Aspergillus nidulansΔacuM mutant had normal extracellular siderophore production and growth under iron-limited conditions, indicating that AcuM does not govern iron acquisition in this organism. A. fumigatus AcuM also regulated genes involved in gluconeogenesis, and the ΔacuM mutant had impaired growth on gluconeogenic carbon sources. Deletion of sreA in the ΔacuM mutant restored iron uptake, extracellular siderophore production and virulence, but not the defect in gluconeogenesis. Thus, AcuM represses SreA and thereby induces iron acquisition, a process that is essential for the maximal virulence of A. fumigatus.
Assuntos
Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Gluconeogênese , Ferro/metabolismo , Proteínas Repressoras/metabolismo , Animais , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Deleção de Genes , Perfilação da Expressão Gênica , Larva/microbiologia , Lepidópteros/microbiologia , Camundongos , Aspergilose Pulmonar/microbiologia , Aspergilose Pulmonar/patologia , Proteínas Repressoras/genética , VirulênciaRESUMO
In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusDeltamedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The DeltamedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusDeltamedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.
Assuntos
Aspergillus fumigatus/patogenicidade , Adesão Celular , Proteínas Fúngicas/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Virulência/fisiologia , Animais , Aspergilose/microbiologia , Aspergilose/mortalidade , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Células Endoteliais/microbiologia , Células Epiteliais/microbiologia , Fibronectinas/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Lepidópteros , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Esporos Fúngicos/crescimento & desenvolvimento , Análise de Sobrevida , Virulência , Fatores de Virulência/genéticaRESUMO
The transcription factors that regulate Aspergillus fumigatus interactions with host cells and virulence are incompletely defined. We investigated the role of the putative C2H2 transcription factor DvrA in governing these processes. Although DvrA was identified by its limited homology to Candida albicans Bcr1, a ΔdvrA mutant strain of A. fumigatus had wild-type adherence to host constituents in vitro. However, it had increased capacity to damage both endothelial cells and a pulmonary epithelial cell line compared to the ability of the wild-type strain and a ΔdvrA::dvrA-complemented strain. This increase in damage required direct contact between the mutant and host cells. The ΔdvrA mutant also stimulated greater CCL20, interleukin-8, and tumor necrosis factor mRNA expression in a pulmonary epithelial cell line compared to levels induced by the control strains. Also, it was resistant to nikkomycin Z, suggesting an altered cell wall composition. As predicted by these in vitro results, the ΔdvrA mutant had increased virulence and stimulated a greater pulmonary inflammatory response than the wild-type strain and ΔdvrA::dvrA-complemented strains in the nonneutropenic mouse model of invasive pulmonary aspergillosis. These results indicate that DvrA influences A. fumigatus virulence as well as its capacity to damage host cells and stimulate a proinflammatory response.
Assuntos
Aspergillus fumigatus/patogenicidade , Células Endoteliais/microbiologia , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Pulmão/microbiologia , Fatores de Transcrição/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Células Endoteliais/patologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Aspergilose Pulmonar Invasiva/microbiologia , Aspergilose Pulmonar Invasiva/patologia , Pulmão/citologia , Pulmão/patologia , Camundongos , Mariposas/microbiologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , VirulênciaRESUMO
Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.
Assuntos
Antifúngicos/administração & dosagem , Aspergillus fumigatus/patogenicidade , Biofilmes/efeitos dos fármacos , Glicosídeo Hidrolases/administração & dosagem , Glicosídeo Hidrolases/genética , Aspergilose Pulmonar Invasiva/prevenção & controle , Animais , Antifúngicos/farmacocinética , Biofilmes/crescimento & desenvolvimento , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Glicosídeo Hidrolases/farmacocinética , Aspergilose Pulmonar Invasiva/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Neutropenia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , VirulênciaRESUMO
Aspergillus fumigatus is a pathogenic mold which causes invasive, often fatal, pulmonary disease in immunocompromised individuals. Recently, proteins involved in the biosynthesis of trehalose have been linked with virulence in other pathogenic fungi. We found that the trehalose content increased during the developmental life cycle of A. fumigatus, throughout which putative trehalose synthase genes tpsA and tpsB were significantly expressed. The trehalose content of A. fumigatus hyphae also increased after heat shock but not in response to other stressors. This increase in trehalose directly correlated with an increase in expression of tpsB but not tpsA. However, deletion of both tpsA and tpsB was required to block trehalose accumulation during development and heat shock. The DeltatpsAB double mutant had delayed germination at 37 degrees C, suggesting a developmental defect. At 50 degrees C, the majority of DeltatpsAB spores were found to be nonviable, and those that were viable had severely delayed germination, growth, and subsequent sporulation. DeltatpsAB spores were also susceptible to oxidative stress. Surprisingly, the DeltatpsAB double mutant was hypervirulent in a murine model of invasive aspergillosis, and this increased virulence was associated with alterations in the cell wall and resistance to macrophage phagocytosis. Thus, while trehalose biosynthesis is required for a number of biological processes that both promote and inhibit virulence, in A. fumigatus the predominant effect is a reduction in pathogenicity. This finding contrasts sharply with those for other fungi, in which trehalose biosynthesis acts to enhance virulence.
Assuntos
Aspergillus fumigatus/patogenicidade , Trealose/fisiologia , Animais , Antifúngicos/farmacologia , Aspergillus fumigatus/química , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/fisiologia , Citometria de Fluxo , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos/fisiologia , Glucosiltransferases/genética , Aspergilose Pulmonar Invasiva/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Estresse Oxidativo/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trealose/análise , Trealose/biossínteseRESUMO
Aspergillus fumigatus causes serious and frequently fatal infections in immunocompromised patients. To investigate the regulation of virulence of this fungus, we constructed and analysed an A. fumigatus mutant that lacked the transcription factor Ace2, which influences virulence in other fungi. The Deltaace2 mutant had dysmorphic conidiophores, reduced conidia production and abnormal conidial cell wall architecture. This mutant produced an orange pigment when grown on solid media, although its conidia had normal pigmentation. Conidia of the Deltaace2 mutant were larger and had accelerated germination. The resulting germlings were resistant to hydrogen peroxide, but not other stressors. Non-neutropenic mice that were immunosuppressed with cortisone acetate and infected with the Deltaace2 mutant had accelerated mortality, greater pulmonary fungal burden, and increased pulmonary inflammatory responses compared with mice infected with the wild-type or Deltaace2::ace2-complemented strains. The Deltaace2 mutant had reduced ppoC, ecm33 and ags3 mRNA expression. It is known that A. fumigatus mutants with absent or reduced expression of these genes have increased virulence in mice, as well as other phenotypic similarities to the Deltaace2 mutant. Therefore, reduced expression of these genes likely contributes to the increased virulence of the Deltaace2 mutant.
Assuntos
Aspergillus fumigatus/genética , Proteínas Fúngicas/metabolismo , Pigmentos Biológicos/biossíntese , Fatores de Transcrição/metabolismo , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Parede Celular/metabolismo , Células Cultivadas , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Mutação , RNA Fúngico/genética , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genética , VirulênciaRESUMO
Conidiation (asexual sporulation) is a key developmental process in filamentous fungi. We examined the gene regulatory roles of the Aspergillus fumigatus developmental transcription factors StuAp and BrlAp during conidiation. Conidiation was completely abrogated in an A. fumigatus DeltabrlA mutant and was severely impaired in a DeltastuA mutant. We determined the full genome conidiation transcriptomes of wild-type and DeltabrlA and DeltastuA mutant A. fumigatus and found that BrlAp and StuAp governed overlapping but distinct transcriptional programs. Six secondary metabolite biosynthetic clusters were found to be regulated by StuAp, while only one cluster exhibited BrlAp-dependent expression. The DeltabrlA mutant, but not the DeltastuA mutant, had impaired downregulation of genes encoding ribosomal proteins under nitrogen-limiting, but not carbon-limiting, conditions. Interestingly, inhibition of the target of rapamycin (TOR) pathway also caused downregulation of ribosomal protein genes in both the wild-type strain and the DeltabrlA mutant. Downregulation of these genes by TOR inhibition was associated with conidiation in the wild-type strain but not in the DeltabrlA mutant. Therefore, BrlAp-mediated repression of ribosomal protein gene expression is not downstream of the TOR pathway. Furthermore, inhibition of ribosomal protein gene expression is not sufficient to induce conidiation in the absence of BrlAp.
Assuntos
Aspergillus fumigatus/genética , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Nitrogênio/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Aspergillus fumigatus/citologia , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Fatores de Transcrição/genéticaRESUMO
Inhalation of conidia of the opportunistic mold Aspergillus fumigatus by immunocompromised hosts can lead to invasive pulmonary disease. Inhaled conidia that escape immune defenses germinate to form filamentous hyphae that invade lung tissues. Conidiation rarely occurs during invasive infection of the human host, allowing the bulk of fungal energy to be directed toward vegetative growth. We hypothesized that forced induction of conidiation during infection can suppress A. fumigatus vegetative growth, impairing the ability of this organism to cause disease. To study the effects of conidiation pathway dysregulation on A. fumigatus virulence, a key transcriptional regulator of conidiation (brlA) was expressed under the control of a doxycycline-inducible promoter. Time- and dose-dependent brlA overexpression was observed in response to doxycycline both in vitro and in vivo. Exposure of the inducible brlA overexpression strain to low doses of doxycycline under vegetative growth conditions in vitro induced conidiation, whereas high doses arrested growth. Overexpression of brlA attenuated A. fumigatus virulence in both an invertebrate and mouse model of invasive aspergillosis. RNA sequencing studies and phenotypic analysis revealed that brlA overexpression results in altered cell signaling, amino acid, and carbohydrate metabolism, including a marked upregulation of trehalose biosynthesis and a downregulation in the biosynthesis of the polysaccharide virulence factor galactosaminogalactan. This proof of concept study demonstrates that activation of the conidiation pathway in A. fumigatus can reduce virulence and suggests that brlA-inducing small molecules may hold promise as a new class of therapeutics for A. fumigatus infection.IMPORTANCE The mold Aspergillus fumigatus reproduces by the production of airborne spores (conidia), a process termed conidiation. In immunocompromised individuals, inhaled A. fumigatus conidia can germinate and form filaments that penetrate and damage lung tissues; however, conidiation does not occur during invasive infection. In this study, we demonstrate that forced activation of conidiation in filaments of A. fumigatus can arrest their growth and impair the ability of this fungus to cause disease in both an insect and a mouse model of invasive infection. Activation of conidiation was linked to profound changes in A. fumigatus metabolism, including a shift away from the synthesis of polysaccharides required for cell wall structure and virulence in favor of carbohydrates used for energy storage and stress resistance. Collectively, these findings suggest that activation of the conidiation pathway may be a promising approach for the development of new agents to prevent or treat A. fumigatus infection.