RESUMO
The filamentous mold Aspergillus fumigatus causes invasive aspergillosis, a potentially life-threatening infectious disease, in humans. The sidE gene encodes a bimodular peptide synthetase and was shown previously to be strongly upregulated during initiation of murine lung infection. In this study, we characterized the two adenylation domains of SidE with the ATP-[(32)P]pyrophosphate exchange assay in vitro, which identified fumarate and l-alanine, respectively, as the preferred substrates. Using full-length holo-SidE, fumarylalanine (FA) formation was observed in vitro. Furthermore, FA was identified in A. fumigatus culture supernatants under inducing conditions, unless sidE was genetically inactivated. As FA is structurally related to established pharmaceutical products exerting immunomodulatory activity, this work may contribute to our understanding of the virulence of A. fumigatus.
Assuntos
Alanina/biossíntese , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/patogenicidade , Fumaratos/metabolismo , Peptídeo Sintases/metabolismo , Filogenia , Alanina/metabolismo , Sequência de Bases , Northern Blotting , Escherichia coli , Teste de Complementação Genética , Dados de Sequência Molecular , Peptídeo Sintases/genética , Plasmídeos/genética , Alinhamento de Sequência , VirulênciaRESUMO
Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein-tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl-CoA ligase), SidH (mevalonyl-CoA hydratase) and SidF (anhydromevalonyl-CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH-targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen-type siderophore-producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus nidulans/enzimologia , Compostos Férricos/metabolismo , Ácidos Hidroxâmicos/metabolismo , Peroxissomos/metabolismo , Sideróforos/metabolismo , Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Sequência Conservada , Redes e Vias Metabólicas/genética , Neurospora crassa/enzimologia , Neurospora crassa/genética , Homologia de Sequência de AminoácidosRESUMO
Aspergillus fumigatus is the most common airborne fungal pathogen for humans. In this mold, iron starvation induces production of the siderophore triacetylfusarinine C (TAFC). Here we demonstrate a link between TAFC and ergosterol biosynthetic pathways, which are both critical for virulence and treatment of fungal infections. Consistent with mevalonate being a limiting prerequisite for TAFC biosynthesis, we observed increased expression of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (Hmg1) under iron starvation, reduced TAFC biosynthesis following lovastatin-mediated Hmg1 inhibition, and increased TAFC biosynthesis following Hmg1 overexpression. We identified enzymes, the acyl-CoA ligase SidI and the enoyl-CoA hydratase SidH, linking biosynthesis of mevalonate and TAFC, deficiency of which under iron starvation impaired TAFC biosynthesis, growth, oxidative stress resistance, and murine virulence. Moreover, inactivation of these enzymes alleviated TAFC-derived biosynthetic demand for mevalonate, as evidenced by increased resistance to lovastatin. Concordant with bilateral demand for mevalonate, iron starvation decreased the ergosterol content and composition, a phenotype that is mitigated in TAFC-lacking mutants.
Assuntos
Aspergillus fumigatus/metabolismo , Ergosterol/biossíntese , Ácido Mevalônico/metabolismo , Sideróforos/biossíntese , Anfotericina B/farmacologia , Animais , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Biomassa , Vias Biossintéticas/efeitos dos fármacos , Enoil-CoA Hidratase/metabolismo , Compostos Férricos/metabolismo , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Ácidos Hidroxâmicos/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Deficiências de Ferro , Ligases/metabolismo , Lovastatina/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Aspergilose Pulmonar/microbiologia , Pirimidinas/farmacologia , Triazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Virulência/efeitos dos fármacos , Virulência/genética , VoriconazolRESUMO
Zinc plays a critical role in a diverse array of biochemical processes. However, excess of zinc is deleterious to cells. Therefore, cells require finely tuned homeostatic mechanisms to balance uptake and storage of zinc. Here we show that iron starvation affects zinc metabolism by downregulating expression of the plasma membrane zinc importer encoding zrfB and upregulating the putative vacuolar zinc transporter-encoding zrcA in Aspergillus fumigatus. Nevertheless, the zinc content of iron-starved mycelia exceeded that of iron replete mycelia, possibly due to unspecific metal uptake induced by iron starvation. In agreement with increased zinc excess and zinc toxicity during iron starvation, deficiency in siderophore-mediated high-affinity iron uptake caused hypersensitivity to zinc. Moreover, an increase of zinc uptake by conditional overexpression of zrfB was more toxic under iron depleted compared to iron replete conditions. This deregulated zinc uptake under iron starvation caused a decrease in heme production and an increase in protoporphyrin IX accumulation. Furthermore, zinc excess impaired production of the extracellular siderophore triacetylfusarinine C but not the intracellular siderophore ferricrocin. Taken together, these data demonstrate a fine tuned coordination of zinc and iron metabolism in A. fumigatus.
