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1.
mSphere ; 8(5): e0031423, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37578262

RESUMO

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 Celular
2.
bioRxiv ; 2023 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-37131584

RESUMO

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.

3.
Antimicrob Agents Chemother ; 66(8): e0005222, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35862738

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/metabolismo
4.
J Fungi (Basel) ; 8(4)2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35448567

RESUMO

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.

5.
mBio ; 12(5): e0244621, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34579578

RESUMO

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 , Virulência
6.
mBio ; 11(1)2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019801

RESUMO

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.


Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/genética , Esporos Fúngicos/efeitos dos fármacos , Fatores de Transcrição/genética , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Doxiciclina/farmacologia , Feminino , Larva/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mariposas/microbiologia , Estudo de Prova de Conceito , Esporos Fúngicos/genética , Virulência , Fatores de Virulência
7.
Cell Microbiol ; 20(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29114981

RESUMO

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/metabolismo
8.
Proc Natl Acad Sci U S A ; 114(27): 7124-7129, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28634301

RESUMO

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 , Virulência
9.
mBio ; 7(2): e00252-16, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27048799

RESUMO

UNLABELLED: The mold Aspergillus fumigatus causes invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetylgalactosamine (GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(ß1-6)-N-acetyl-D-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster gene agd3 encodes a protein containing a deacetylase domain. Because deacetylation of N-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3 mutant in the presence of culture supernatants of the GAG-deficient Δuge3 mutant rescued the biofilm defect of the Δagd3 mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of the Pezizomycotina subphylum of the Ascomycota including a number of plant-pathogenic fungi and a single basidiomycete species,Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation. IMPORTANCE: This study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatus virulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.


Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/fisiologia , Biofilmes , Polissacarídeos/metabolismo , Acetilação , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos
10.
PLoS Pathog ; 11(10): e1005187, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26492565

RESUMO

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 , Virulência
11.
J Biol Chem ; 290(46): 27438-50, 2015 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-26342082

RESUMO

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 Sequência
12.
J Biol Chem ; 289(3): 1243-56, 2014 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-24257745

RESUMO

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/genética
13.
Curr Microbiol ; 68(1): 1-5, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23925951

RESUMO

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/química
14.
PLoS Pathog ; 9(8): e1003575, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23990787

RESUMO

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/genética
15.
PLoS One ; 7(11): e49959, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23185496

RESUMO

MedA is a developmental regulator that is conserved in the genome of most filamentous fungi. In the pathogenic fungus Aspergillus fumigatus MedA regulates conidiogenesis, adherence to host cells, and pathogenicity. The mechanism by which MedA governs these phenotypes remains unknown. Although the nuclear import of MedA orthologues has been reported in other fungi, no nuclear localization signal, DNA-binding domain or other conserved motifs have been identified within MedA. In this work, we performed a deletion analysis of MedA and identified a novel domain within the C-terminal region of the protein, designated MedA(346-557), that is necessary and sufficient for nuclear localization of MedA. We further demonstrate that MedA nuclear localization is required for the function of MedA. Surprisingly, expression of the minimal nuclear localization fragment MedA(346-557) alone was sufficient to restore conidogenesis, biofilm formation and virulence to the medA mutant strain. Collectively these results suggest that MedA functions in the regulation of transcription, and that the MedA(346-557) domain is both necessary and sufficient to mediate MedA function.


Assuntos
Aspergillus fumigatus , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas , Sinais de Localização Nuclear , Proteínas Nucleares/genética , Esporos Fúngicos , Transporte Ativo do Núcleo Celular/genética , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Estrutura Terciária de Proteína/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
16.
Appl Environ Microbiol ; 78(11): 3855-63, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22467499

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/metabolismo
17.
Methods Mol Biol ; 845: 119-30, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22328371

RESUMO

The construction of a fungal strain that lacks a specific gene product is often accomplished by replacing the gene of interest with a selection marker using site-specific recombination. Transformation of Aspergillus fumigatus, like many related fungal species, must overcome two major obstacles. First, the cell wall limits the entry of exogenous DNA, and second, a high rate of nonhomologous recombination leads to random ectopic integration of the marker. Here, we describe an experimental strategy that has been successfully used to overcome these challenges through protoplast transformation with split-marker cassettes. Each cassette is constructed to contain sequences flanking the gene of interest fused to an incomplete fragment of a dominant selection marker. The resistance marker is only functional if both fragments undergo recombination to regenerate an intact resistance cassette. This event is favored by the proximity of the DNA constructs that arises as a result of homologous recombination between the target-gene sequences in the deletion construct with the fungal chromosome. A similar strategy can be employed using a second resistance marker to complement the deletion mutant with an intact allele of the gene of interest.


Assuntos
Aspergillus fumigatus/genética , Farmacorresistência Fúngica/genética , Deleção de Genes , Marcadores Genéticos/genética , Mutagênese Insercional/métodos , Aspergillus fumigatus/efeitos dos fármacos , Cinamatos/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacos , Higromicina B/análogos & derivados , Higromicina B/farmacologia
18.
Mol Microbiol ; 78(4): 1038-54, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21062375

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 , Virulência
19.
Eukaryot Cell ; 9(10): 1432-40, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20675576

RESUMO

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 , Virulência
20.
Infect Immun ; 78(7): 3007-18, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20439478

RESUMO

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/biossíntese
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