Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
Antimicrob Agents Chemother ; 66(8): e0045822, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35856665

RESUMO

Invasive aspergillosis (IA), caused predominantly by Aspergillus fumigatus, is the most common opportunistic mold infection in immunocompromised patients. Resistance of A. fumigatus to triazoles has been increasingly reported, leading to poor outcomes of IA to the front-line azoles. Triazole resistance is in part driven by exposure to agricultural azoles through mechanisms that are poorly understood beyond mutations in ergosterol biosynthetic genes. Priming is defined as a process in which prior exposures to sublethal stressful stimuli, such as antimicrobial drugs, can enhance the ability of pathogens to withstand reexposure to the same or other stressors. Here, we describe, for the first time, triazole priming, where exposure of conidia of three A. fumigatus strains to subinhibitory concentrations of either agricultural (tebuconazole difenoconazole, epoxiconazole) or medical triazoles (voriconazole) increases germination and growth during subsequent reexposure to subinhibitory triazole challenge. We demonstrate that priming in A. fumigatus is class specific to triazoles, is not confined to a particular isolate, and is retained for extended periods in primed dormant conidia, but is not transferred to subsequent generations. Furthermore, azole priming at subinhibitory triazole concentrations increased the frequency of development of stable resistance development at inhibitory triazole exposures. Triazole priming could have far-reaching clinical implications in generating resistance due to the widespread use of agricultural triazoles or breakthrough IA in patients with subtherapeutic serum levels of azoles.


Assuntos
Aspergilose , Aspergillus fumigatus , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Azóis/farmacologia , Azóis/uso terapêutico , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Humanos , Testes de Sensibilidade Microbiana , Triazóis/farmacologia , Triazóis/uso terapêutico
2.
J Antimicrob Chemother ; 72(8): 2263-2272, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28475687

RESUMO

Objectives: Over the last 30 years, the number of invasive fungal infections among immunosuppressed patients has increased significantly, while the number of effective systemic antifungal drugs remains low. The aim of this study was to identify and characterize antifungal compounds that inhibit fungus-specific metabolic pathways not conserved in humans. Methods: We screened a diverse compound library for antifungal activity in the pathogenic mould Aspergillus fumigatus . We determined the in vitro activity of bromoquinol by MIC determination against a panel of fungi, bacteria and cell lines. The mode of action of bromoquinol was determined by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring genes and by RNAseq analysis in A. fumigatus . In vivo efficacy was tested in Galleria mellonella and murine models of A. fumigatus infection. Results: Screening of a diverse chemical library identified three compounds interfering with fungal iron utilization. The most potent, bromoquinol, shows potent wide-spectrum antifungal activity that was blocked in the presence of exogenous iron. Mode-of-action analysis revealed that overexpression of the dba secondary metabolite cluster gene dbaD , encoding a metabolite transporter, confers bromoquinol resistance in A. nidulans , possibly by efflux. RNAseq analysis and subsequent experimental validation revealed that bromoquinol induces oxidative stress and apoptosis in A. fumigatus . Bromoquinol significantly reduced mortality rates of G. mellonella infected with A. fumigatus , but was ineffective in a murine model of infection. Conclusions: Bromoquinol is a promising antifungal candidate with a unique mode of action. Its activity is potentiated by iron starvation, as occurs during in vivo growth.


Assuntos
Antifúngicos/farmacologia , Apoptose , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus nidulans/efeitos dos fármacos , Estresse Oxidativo , Quinolinas/farmacologia , Animais , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Modelos Animais de Doenças , Lepidópteros , Testes de Sensibilidade Microbiana , Análise de Sobrevida
3.
Appl Environ Microbiol ; 82(9): 2585-94, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26896140

RESUMO

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well.S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol.


Assuntos
Biofilmes/crescimento & desenvolvimento , Fungos/fisiologia , Serratia marcescens/fisiologia , Antibiose/fisiologia , Aderência Bacteriana/fisiologia , Quitinases/genética , Quitinases/metabolismo , Fímbrias Bacterianas , Flagelos/genética , Flagelos/fisiologia , Fungos/citologia , Interações Hospedeiro-Patógeno , Hifas/citologia , Hifas/fisiologia , Viabilidade Microbiana , Mutação , Micélio/citologia , Micélio/fisiologia , Controle Biológico de Vetores , Rhizopus/citologia , Rhizopus/fisiologia , Serratia marcescens/citologia
4.
Fungal Genet Biol ; 63: 55-64, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24361821

RESUMO

Fungal cell-wall proteins containing the conserved fungal CFEM domain have been implicated in host-pathogen interactions and virulence. To determine the role of these proteins in the mold pathogen Aspergillus fumigatus, we deleted the entire family of three CFEM-containing genes (CfmA-C), singly and in all combinations. We found an additive increase in the susceptibility of the single, double and triple ΔCfm mutants towards the chitin/ß-glucan-microfibril destabilizing compounds Congo Red (CR) and Calcofluor White (CFW), indicating that the A. fumigatus CFEM proteins are involved in stabilizing the cell wall. No defects in growth or germination were observed, indicating that CFEM proteins do not have an essential role in the morphogenesis of A. fumigatus. Unlike in Candida albicans, the A. fumigatus CFEM proteins were not implicated in heme uptake or biofilm formation. The ΔTriple-Cfm deletion strain did not exhibit altered virulence in either insect or murine models of infection, suggesting that cell-wall proteins containing the conserved fungal CFEM domain are not a significant virulence factor in A. fumigatus.


Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Sequência de Aminoácidos , Animais , Aspergilose/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Virulência
5.
Antimicrob Agents Chemother ; 56(1): 1-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22006001

RESUMO

Many natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active against Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans but not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, for C. albicans, selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with ß-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was tested in vitro and in vivo using toxicity assays on erythrocytes, macrophages, and mice. In vitro cytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and no in vivo murine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity.


Assuntos
Anfotericina B/farmacologia , Peptídeos Catiônicos Antimicrobianos/síntese química , Colesterol/química , Vitamina E/química , beta-Ciclodextrinas/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/crescimento & desenvolvimento , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Micoses/tratamento farmacológico , Micoses/microbiologia , Especificidade da Espécie , Eletricidade Estática , Vitamina E/farmacologia
6.
mBio ; 13(5): e0219222, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36125294

RESUMO

Siderophores play an important role in fungal virulence, serving as trackers for in vivo imaging and as biomarkers of fungal infections. However, siderophore uptake is only partially characterized. As the major cause of aspergillosis, Aspergillus fumigatus is one of the most common airborne fungal pathogens of humans. Here, we demonstrate that this mold species mediates the uptake of iron chelated by the secreted siderophores triacetylfusarinine C (TAFC) and fusarinine C by the major facilitator-type transporters MirB and MirD, respectively. In a murine aspergillosis model, MirB but not MirD was found to be crucial for virulence, indicating that TAFC-mediated uptake plays a dominant role during infection. In the absence of MirB, TAFC becomes inhibitory by decreasing iron availability because the mutant is not able to recognize iron that is chelated by TAFC. MirB-mediated transport was found to tolerate the conjugation of fluorescein isothiocyanate to triacetylfusarinine C, which might aid in the development of siderophore-based antifungals in a Trojan horse approach, particularly as the role of MirB in pathogenicity restrains its mutational inactivation. Taken together, this study identified the first eukaryotic siderophore transporter that is crucial for virulence and elucidated its translational potential as well as its evolutionary conservation. IMPORTANCE Aspergillus fumigatus is responsible for thousands of cases of invasive fungal disease annually. For iron uptake, A. fumigatus secretes so-called siderophores, which are taken up after the binding of environmental iron. Moreover, A. fumigatus can utilize siderophore types that are produced by other fungi or bacteria. Fungal siderophores raised considerable interest due to their role in virulence and their potential for the diagnosis and treatment of fungal infections. Here, we demonstrate that the siderophore transporter MirB is crucial for the virulence of A. fumigatus, which reveals that its substrate, triacetylfusarinine C, is the most important siderophore during infection. We found that in the absence of MirB, TAFC becomes inhibitory by decreasing the availability of environmental iron and that MirB-mediated transport tolerates the derivatization of its substrate, which might aid in the development of siderophore-based antifungals. This study significantly improved the understanding of fungal iron homeostasis and the role of siderophores in interactions with the host.


Assuntos
Aspergilose , Micoses , Humanos , Animais , Camundongos , Sideróforos/metabolismo , Aspergillus fumigatus/metabolismo , Virulência , Antifúngicos/metabolismo , Aspergilose/microbiologia , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Isotiocianatos/metabolismo , Fluoresceínas
7.
Eukaryot Cell ; 9(9): 1403-15, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20656913

RESUMO

cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus. The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus. We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA-encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2. Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and beta-glucan. This is correlated with increasing susceptibility of the DeltacspA, DeltaECM33, and DeltacspA DeltaECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.


Assuntos
Aspergilose/microbiologia , Aspergillus fumigatus/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Esporos Fúngicos/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/patogenicidade , Linhagem Celular , Parede Celular/genética , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos ICR , Ligação Proteica , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/patogenicidade , Virulência
8.
Virulence ; 12(1): 2186-2200, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34468270

RESUMO

Aspergillus fumigatus is the leading cause of life-threatening invasive mold infections in immunocompromised individuals. This ubiquitous saprophyte possesses several natural attributes allowing it to evade the immune system, including the ability to withstand high toxic Cu concentrations within the phagosomes of macrophages and neutrophils. We previously established that at high levels, Cu binds and activates the A. fumigatus transcription factor AceA, which upregulates the expression of the Cu exporter CrpA to expel excess Cu. Deletion of aceA or crpA result in extreme Cu sensitivity and attenuated virulence.To identify other elements participating in resistance to Cu, we performed a genome-wide analysis of the transcriptome by RNAseq to analyze the AceA-dependent response of A. fumigatus to excess Cu. We deleted key genes whose transcription was strongly upregulated by high Cu, including those encoding homologs of the three Cu chaperones cox17, atx1 and ccs1. Detailed analysis of these genes indicates that in A. fumigatus, cox17 is an essential gene with a possible role in respiration, the atxA gene product participates in reductive iron uptake and ccsA encodes the Cu chaperone activating A. fumigatus Sod1. Interestingly, although the ccsA-null strain was extremely sensitive to high Cu and oxidative stress, it was not attenuated in virulence in a mouse model of invasive pulmonary aspergillosis.Our work provides (i) a detailed view of the genome-wide transcriptional response of A. fumigatus to excess Cu, (ii) identification of the AceA-dependent transcriptome and (iii) analysis of the roles of the three Cu chaperones cox17, atxA and ccsA.


Assuntos
Aspergillus fumigatus , Cobre , Proteínas Fúngicas , Chaperonas Moleculares , Animais , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Camundongos , Chaperonas Moleculares/genética , Fatores de Transcrição/genética , Virulência
9.
Front Microbiol ; 11: 1383, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32670238

RESUMO

The identification of cellular targets for antifungal compounds is a cornerstone for the development of novel antimycotics, for which a significant need exists due to increasing numbers of susceptible patients, emerging pathogens, and evolving resistance. For the human pathogenic mold Aspergillus fumigatus, the causative agent of the opportunistic disease aspergillosis, only a limited number of established targets and corresponding drugs are available. Among several targets that were postulated from a variety of experimental approaches, the conserved thioredoxin reductase (TrxR) activity encoded by the trxR gene was assessed in this study. Its essentiality could be confirmed following a conditional TetOFF promoter replacement strategy. Relevance of the trxR gene product for oxidative stress resistance was revealed and, most importantly, its requirement for full virulence of A. fumigatus in two different models of infection resembling invasive aspergillosis. Our findings complement the idea of targeting the reductase component of the fungal thioredoxin system for antifungal therapy.

10.
Genes (Basel) ; 11(4)2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32326414

RESUMO

Aspergillus fumigatus is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of A. fumigatus via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (argJ/AFUA_5G08120) and the ornithine carbamoyltransferase (argB/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in A. fumigatus. ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of A. fumigatus in the insect model Galleria mellonella and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.


Assuntos
Arginina/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/fisiologia , Proteínas de Bactérias/metabolismo , Mutação , Sideróforos/fisiologia , Virulência , Animais , Arginase/genética , Arginase/metabolismo , Aspergilose/metabolismo , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Mariposas/metabolismo , Mariposas/microbiologia
11.
Virulence ; 10(1): 925-934, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31694453

RESUMO

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.


Assuntos
Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/genética , Ferro/metabolismo , Leucina/biossíntese , Virulência , Adaptação Fisiológica , Animais , Aspergillus fumigatus/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Feminino , Regulação Fúngica da Expressão Gênica , Homeostase , Hidroliases/genética , Larva/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Mariposas/microbiologia , Aspergilose Pulmonar/microbiologia
12.
Front Microbiol ; 10: 2773, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31866965

RESUMO

Current suboptimal treatment options of invasive fungal infections and emerging resistance of the corresponding pathogens urge the need for alternative therapy strategies and require the identification of novel antifungal targets. Aspergillus fumigatus is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel in vivo conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of A. fumigatus in a murine model for pulmonary aspergillosis. The xylP promoter used has previously been shown to allow xylose-induced gene expression in different molds. Here, we demonstrate for the first time that this promoter also allows in vivo tuning of A. fumigatus gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an A. fumigatus strain expressing rpdA under control of the xylP promoter, rpdA xylP , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, rpdA xylP displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused rpdA induction in vivo. These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the xylP expression system is a powerful new tool for in vivo gene silencing in A. fumigatus.

13.
Fungal Genet Biol ; 45(3): 232-42, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17703972

RESUMO

The Aspergillus nidulans genes cetA (AN3079.2) and calA (AN7619.2) encode a novel class of fungal thaumatin-like proteins of unknown function. Deletion of cetA does not result in an observable phenotype [Greenstein, S., Shadkchan, Y., Jadoun, J., Sharon, C., Markovich, S., Osherov, N., 2006. Analysis of the Aspergillus nidulans thaumatin-like cetA gene and evidence for transcriptional repression of pyr4 expression in the cetA-disrupted strain. Fungal Genet. Biol. 43, 42-53]. We prepared knockout calA and calA/cetA A. nidulans strains. The calA mutants were phenotypically identical to the wild-type. In contrast, the cetA/calA double mutant showed a synthetic lethal phenotype suggesting that the two genes affect a single function or pathway: most of its conidia were completely inhibited in germination. Many collapsed and underwent lysis. A few showed abnormal germination characterized by short swollen hyphae and abnormal hyphal branching. Nongerminated conidia contained a single condensed nucleus suggesting a block in early germination. This is the first functional analysis of the novel cetA/calA family of thaumatin-like genes and their role in A. nidulans conidial germination. We show that CETA and CALA are secreted proteins that together play an essential role in early conidial germination.


Assuntos
Aspergillus nidulans/genética , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Esporos Fúngicos/genética , Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Northern Blotting , Western Blotting , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Genéticos , Morfogênese , Mutação , Fenótipo , Esporos Fúngicos/crescimento & desenvolvimento
14.
Front Microbiol ; 9: 2615, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30483221

RESUMO

The saprophytic mold Aspergillus fumigatus is the most common airborne fungal pathogen causing severe invasive fungal infections in immunocompromised patients. Siroheme is a heme-like prosthetic group used by plants and microorganisms for sulfate and nitrate assimilation but is absent in higher eukaryotes. Here, we investigated the role of siroheme in A. fumigatus by deletion of the gene encoding the bifunctional dehydrogenase/ferrochelatase enzyme Met8. Met8-deficiency resulted in the inability to utilize sulfate and nitrate as sulfur and nitrogen sources, respectively. These results match previous data demonstrating that siroheme is an essential cofactor for nitrite and sulfite reductases. Moreover, Met8-deficiency caused significantly decreased resistance against nitric oxide (NO) underlining the importance of nitrite reductase in NO detoxification. Met8-deficiency did not affect virulence in murine models for invasive aspergillosis indicating that neither NO-detoxification nor assimilation of sulfate and nitrate play major roles in virulence in this host. Interestingly, Met8-deficiency resulted in mild virulence attenuation in the Galleria mellonella infection model revealing differences in interaction of A. fumigatus with G. mellonella and mouse.

15.
Virulence ; 9(1): 1273-1286, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30027796

RESUMO

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/enzimologia
16.
Front Microbiol ; 8: 2490, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29312198

RESUMO

Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia) are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs) to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases) and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy.

17.
Cell Rep ; 19(5): 1008-1021, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28467895

RESUMO

The Fenton-chemistry-generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an Aspergillus fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages (AMΦs), and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses.


Assuntos
Aspergillus/metabolismo , Cobre/metabolismo , Interações Hospedeiro-Patógeno , Espécies Reativas de Oxigênio/metabolismo , Animais , Aspergillus/genética , Aspergillus/patogenicidade , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , ATPases do Tipo-P/genética , ATPases do Tipo-P/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
18.
J Med Chem ; 59(3): 1197-206, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26816333

RESUMO

Systemic fungal infections are an increasingly prevalent health problem, especially among immunocompromised patients. Antifungal drug development lags far behind in comparison to other types of antimicrobial drugs. Current commercially available antifungals are limited by their insufficient potency, side effects, drug-drug interactions, developing drug-resistance, and narrow formulation options. Here, we report the preparation and evaluation of two novel PEG amide conjugates of amphotericin B (AMB (1)): AB1 (4) and AM2 (5). These compounds are nonlabile, they are prepared in only two and three synthetic steps, respectively, and they show antifungal activity against a wide range of clinical fungal isolates. Their toxicity is significantly lower, and their water solubility is up to 5000-fold higher than that of AMB (1). In vivo efficacy studies in a mouse model of systemic candidiasis showed that AM2 (5) successfully cured all the mice at concentrations above 3.5 mg/kg body weight. In conclusion, these properties make AB1 (4) and AM2 (5) promising candidates for clinical use.


Assuntos
Anfotericina B/efeitos adversos , Anfotericina B/farmacologia , Antifúngicos/efeitos adversos , Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Polietilenoglicóis , Anfotericina B/química , Anfotericina B/uso terapêutico , Animais , Antifúngicos/química , Antifúngicos/uso terapêutico , Aspergillus/efeitos dos fármacos , Candida/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Estrutura Molecular , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Rhizopus/efeitos dos fármacos , Solubilidade , Relação Estrutura-Atividade , Água/química
19.
J Med Chem ; 55(3): 1296-302, 2012 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-22243686

RESUMO

The activity of antimicrobial peptides (AMPs) that contain a large proportion of histidine residues (pK(a) ∼ 6) depends on the physiological pH environment. Advantages of these AMPs include high activity in slightly acidic areas of the human body and relatively low toxicity in other areas. Also, many AMPs are highly active in a multivalent form, but this often increases toxicity. Here we designed pH dependent amphiphilic compounds consisting of multiple ultrashort histidine lipopeptides on a triazacyclophane scaffold, which showed high activity toward Aspergillus fumigatus and Cryptococcus neoformans at acidic pH, yet remained nontoxic. In vivo, treatment with a myristic acid conjugated trivalent histidine-histidine dipeptide resulted in 55% survival of mice (n = 9) in an otherwise lethal murine lung Aspergillus infection model. Fungal burden was assessed and showed completely sterile lungs in 80% of the mice (n = 5). At pH 5.5 and 7.5, differing peptide-membrane interactions and peptide nanostructures were observed. This study underscores the potential of unique AMPs to become the next generation of clinical antimicrobial therapy.


Assuntos
Antifúngicos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Lipopeptídeos/síntese química , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Linhagem Celular , Cryptococcus neoformans/efeitos dos fármacos , Feminino , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Nanoestruturas , Polímeros , Aspergilose Pulmonar/tratamento farmacológico , Aspergilose Pulmonar/microbiologia , Relação Estrutura-Atividade
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa