RESUMO
Siderophores are specialized molecules produced by bacteria and fungi to scavenge iron, a crucial nutrient for growth and metabolism. Catecholate-type siderophores are mainly produced by bacteria, while hydroxamates are mostly from fungi. This study investigates the capacity of nine hydroxamate-type siderophores from fungi and Streptomyces to facilitate iron acquisition by the human pathogen Pseudomonas aeruginosa. Growth assays under iron limitation and 55Fe incorporation tests showed that all nine siderophores promoted bacterial growth and iron transport. The study also aimed to identify the TonB-dependent transporters (TBDTs) involved in iron import by these siderophores. Using mutant strains lacking specific TBDT genes, it was found that iron is imported into P. aeruginosa cells by FpvB for coprogen, triacetylfusarinine, fusigen, ferrirhodin, and ferrirubin. Iron complexed by desferioxamine G is transported by FpvB and FoxA, ferricrocin-Fe and ferrichrycin-Fe by FpvB and FiuA, and rhodotoluric acid-Fe by FpvB, FiuA, and another unidentified TBDT. These findings highlight the effectiveness of hydroxamate-type siderophores in iron transport into P. aeruginosa and provide insights into the complex molecular mechanisms involved, which are important for understanding microbial interactions and ecological balance.
Assuntos
Proteínas de Bactérias , Ácidos Hidroxâmicos , Ferro , Pseudomonas aeruginosa , Sideróforos , Sideróforos/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/genética , Ferro/metabolismo , Ácidos Hidroxâmicos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Ferricromo/metabolismo , Ferricromo/análogos & derivados , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas da Membrana Bacteriana Externa , Proteínas de Membrana , Receptores de Superfície CelularRESUMO
The putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.
Assuntos
Beauveria/crescimento & desenvolvimento , Beauveria/metabolismo , Ferricromo/análogos & derivados , Interações Hospedeiro-Patógeno , Insetos/microbiologia , Ferro/metabolismo , Animais , Beauveria/classificação , Beauveria/patogenicidade , Biologia Computacional , Ferricromo/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Homeostase , Mutação , Estresse Oxidativo , Filogenia , Virulência/genéticaRESUMO
Albomycin δ2 is a sulfur-containing sideromycin natural product that shows potent antibacterial activity against clinically important pathogens. The l-serine-thioheptose dipeptide partial structure, known as SB-217452, has been found to be the active seryl-tRNA synthetase inhibitor component of albomycin δ2 . Herein, it is demonstrated that AbmF catalyzes condensation between the 6'-amino-4'-thionucleoside with the d-ribo configuration and seryl-adenylate supplied by the serine adenylation activity of AbmK. Formation of the dipeptide is followed by C3'-epimerization to produce SB-217452 with the d-xylo configuration, which is catalyzed by the radical S-adenosyl-l-methionine enzyme AbmJ. Gene deletion suggests that AbmC is involved in peptide assembly linking SB-217452 with the siderophore moiety. This study establishes how the albomycin biosynthetic machinery generates its antimicrobial component SB-217452.
Assuntos
Antibacterianos/biossíntese , Ferricromo/análogos & derivados , Pirimidinonas/metabolismo , Serina-tRNA Ligase/metabolismo , Tiofenos/metabolismo , Antibacterianos/química , Biocatálise , Ferricromo/química , Ferricromo/metabolismo , Peptídeo Sintases/metabolismo , Pirimidinonas/química , Serina-tRNA Ligase/antagonistas & inibidores , Serina-tRNA Ligase/genética , Streptomyces/química , Streptomyces/metabolismo , Tiofenos/químicaRESUMO
Albomycins are peptidyl thionucleoside natural products that display antimicrobial activity against clinically important pathogens. Their structures are characterized by a thioheptose with atypical stereochemistry including a d-xylofuranose ring modified with a d-amino acid moiety. Herein it is demonstrated that AbmH is a pyridoxal 5'-phosphate (PLP)-dependent transaldolase that catalyzes a threo-selective aldol-type reaction to generate the thioheptose core with a d-ribofuranose ring and an l-amino acid moiety. The conversion of l-to d-amino acid configuration is catalyzed by the PLP-dependent epimerase AbmD. The d- ribo to d- xylo conversion of the thiofuranose ring appears according to gene deletion experiments to be mediated by AbmJ, which is annotated as a radical S-adenosyl-l-methionine (SAM) enzyme. These studies establish several key steps in the assembly of the thioheptose core during the biosynthesis of albomycins.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Ferricromo/análogos & derivados , Heptoses/química , Nucleosídeos/química , Biocatálise , Ferricromo/química , Ferricromo/metabolismo , Estereoisomerismo , Transaldolase/metabolismoRESUMO
Aureobasidium melanogenum HN6.2 is a unique yeast strain who can produce the siderophore of fusigen under iron starvation to guarantee its survival. However, a comprehensive understanding of mechanisms involved in iron acquisition and homeostasis for it is still vacant. In this study, genome sequencing and mining revealed that A. melanogenum HN6.2 strain was the first yeast species that exclusively possessed all the four known mechanisms for the iron acquisition: (i) the siderophore-mediated iron uptake; (ii) reductive iron assimilation; (iii) low-affinity ferrous uptake; and (iv) heme utilization, which suggested its stronger adaptability than Aspergillus fumigatus and Saccharomyces cerevisiae. This HN6.2 strain also employed the vacuolar iron storage for immobilizing the excessive iron to avoid its cellular toxicity. Specially, genome mining indicated that A. melanogenum HN6.2 strain could also synthesize ferricrocin siderophore. Further HPLC and Q-Tof-MS analysis confirmed that the siderophores synthesized by this strain consisted of cyclic fusigen, linear fusigen, ferricrocin, and hydroxyferricrocin and they played parallel roles as both intracellular and extracellular siderophores. Also, the heme utilization for this strain was experimentally verified by the knock-out of heme oxygenase gene. For iron homeostasis, the transcriptome analysis revealed that this strain mainly employed two central regulators of SreA/HapX to tune iron uptake and storage at the transcriptional level. It was also noted that mitogen-activated protein kinase C gene (MpkC) exhibited a transcriptional up-regulation under iron sufficiency, suggesting that it may serve as another factor involved in the repression of siderophore biosynthesis. This is the first genetic blueprint of iron acquisition and homeostasis for A. melanogenum.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Ferro/metabolismo , Saccharomycetales/metabolismo , Sideróforos/metabolismo , Transcriptoma , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Mineração de Dados , Compostos Férricos/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/metabolismo , Ontologia Genética , Heme/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Homeostase/genética , Ácidos Hidroxâmicos/metabolismo , Transporte de Íons , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Anotação de Sequência Molecular , Oxirredução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Vacúolos/metabolismoRESUMO
Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and ß-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2 in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.
Assuntos
Aspergillus fumigatus/metabolismo , Ergotioneína/biossíntese , Proteínas Fúngicas/metabolismo , Antioxidantes/química , Aspergillus fumigatus/genética , Carbono-Oxigênio Liases/metabolismo , Compostos Férricos/química , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Teste de Complementação Genética , Gliotoxina/química , Glutationa/metabolismo , Histidina/química , Peróxido de Hidrogênio/química , Ácidos Hidroxâmicos/química , Liases/metabolismo , Metais Pesados/química , Oxirredução , Estresse Oxidativo , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/química , Vitamina K 3/químicaRESUMO
Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels â¼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.
Assuntos
Aminoácidos Sulfúricos/metabolismo , Antibacterianos/biossíntese , Ferricromo/análogos & derivados , Streptomyces griseus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ferricromo/metabolismo , Família Multigênica , Streptomyces griseus/genética , Streptomyces griseus/crescimento & desenvolvimentoRESUMO
Iron is an essential element for life. However, the iron overload can be toxic. Here, we investigated the significant increase of tenellin and iron-tenellin complex production in ferricrocin-deficient mutants of Beauveria bassiana. Our chemical analysis indicated that the ferricrocin-deficient mutants T1, T3 and T5 nearly abolished ferricrocin production. In turn, these mutants had significant accumulation of iron-tenellin complex in their mycelia at 247-289 mg g(-1) cell dry weight under iron-replete condition. Both tenellin and iron-tenellin complex were not detected in the wild-type under such condition. Mass analysis of the mutants' crude extracts demonstrated that tenellin formed a 3:1 complex with iron in the absence of ferricrocin. The unexpected link between ferricrocin and tenellin biosynthesis in ferricrocin-deficient mutants could be a survival strategy during iron-mediated oxidative stress.
Assuntos
Beauveria/metabolismo , Ferricromo/análogos & derivados , Ferro/metabolismo , Piridonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/metabolismo , Beauveria/química , Beauveria/genética , Beauveria/ultraestrutura , Cromatografia Líquida de Alta Pressão , Ferricromo/química , Ferricromo/metabolismo , Espectrometria de Massas , Mutação , Piridonas/química , Interferência de RNARESUMO
Iron is an essential element for all eukaryotes but its excess has deleterious effects. Aspergillus fumigatus produces extracellular siderophores for iron uptake and the intracellular siderophore ferricrocin (FC) for distribution and storage of iron. Iron excess has previously been shown to increase the content of ferric FC and the expression of the putative vacuolar iron importer CccA (AFUA_4G12530), indicating a role of both the vacuole and FC in iron detoxification. In this study, we show that CccA-deficiency decreases iron resistance in particular in combination with derepressed iron uptake, while overproduction of CccA increases iron resistance. Green fluorescence protein-tagging confirmed localization of CccA in the vacuolar membrane. In contrast to CccA-deficiency, inactivation of FC biosynthesis did not affect iron resistance, which indicates that vacuolar rather than FC-mediated iron storage is the major iron detoxifying mechanism. After uptake, extracellular siderophore backbones are hydrolyzed and recycled. Lack of FC, CccA, and in particular lack of both increased the cellular content of iron chelated by siderophore breakdown products. These data indicate that the transfer of iron from extracellular siderophores to the metabolism, FC or the vacuole precedes recycling of siderophore breakdown products. Furthermore, this study indicates that CccA does not play an exclusive role in vacuolar iron storage for nutritional reuse.
Assuntos
Aspergillus fumigatus/metabolismo , Ferro/metabolismo , Sideróforos/metabolismo , Aspergillus fumigatus/genética , Transporte Biológico Ativo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Filogenia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Vacúolos/metabolismoRESUMO
Salmonella enterica subsp. enterica serovar Enteritidis is a leading causative agent of gastroenteritis in humans. This pathogen also colonizes the intestinal tracts of poultry and can spread systemically in chickens. Transfer to humans usually occurs through undercooked or improperly handled poultry meat or eggs. The bacterial twin-arginine transport (Tat) pathway is responsible for the translocation of folded proteins across the cytoplasmic membrane. In order to study the role of the Tat system in the infection and colonization of chickens by Salmonella Enteritidis, we constructed chromosomal deletion mutants of the tatB and tatC genes, which are essential components of the Tat translocon. We observed that the tat mutations affected bacterial cell morphology, motility, and sensitivity to albomycin, sodium dodecyl sulfate (SDS), and EDTA. In addition, the mutant strains showed reduced invasion of polarized Caco-2 cells. The wild-type phenotype was restored in all our Salmonella Enteritidis tat mutants by introducing episomal copies of the tatABC genes. When tested in chickens by use of a Salmonella Enteritidis Delta tatB strain, the Tat system inactivation did not substantially affect cecal colonization, but it delayed systemic infection. Taken together, our data demonstrated that the Tat system plays a role in Salmonella Enteritidis pathogenesis.
Assuntos
Proteínas de Bactérias/fisiologia , Células Epiteliais/microbiologia , Doenças das Aves Domésticas/microbiologia , Salmonelose Animal/microbiologia , Salmonella enteritidis/patogenicidade , Fatores de Virulência/fisiologia , Animais , Antibacterianos/toxicidade , Proteínas de Bactérias/genética , Células CACO-2 , Galinhas , Ácido Edético/toxicidade , Ferricromo/análogos & derivados , Ferricromo/toxicidade , Deleção de Genes , Teste de Complementação Genética , Humanos , Locomoção , Salmonella enteritidis/citologia , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/fisiologia , Dodecilsulfato de Sódio/toxicidade , Virulência , Fatores de Virulência/genéticaRESUMO
The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. McC is excreted from producing cells by the MccC transporter. The residual McC that remains in the producing cell can be processed by cellular aminopeptidases with the release of a non-hydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. Accumulation of processed McC inside producing cells should therefore lead to translation inhibition and cessation of growth. Here, we show that a product of another gene of the McC biosynthetic cluster, mccE, acetylates processed McC and converts it into a non-toxic compound. MccE also makes Escherichia coli resistant to albomycin, a Trojan horse inhibitor unrelated to McC that, upon processing, gives rise to a serine coupled to a thioxylofuranosyl pyrimidine, an inhibitor of seryl-tRNA synthetase. We speculate that MccE and related cellular acetyltransferases of the Rim family may detoxify various aminoacyl-nucleotides, either exogenous or those generated inside the cell.
Assuntos
Acetiltransferases/metabolismo , Bacteriocinas/farmacologia , Farmacorresistência Bacteriana/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Família Multigênica/fisiologia , Biossíntese de Proteínas/efeitos dos fármacos , Acetiltransferases/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Antibacterianos/farmacologia , Aspartato-tRNA Ligase/antagonistas & inibidores , Ácido Aspártico/análogos & derivados , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Bacteriocinas/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/farmacologia , Ferricromo/análogos & derivados , Ferricromo/farmacologia , Biossíntese de Proteínas/fisiologiaRESUMO
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.
Assuntos
Aspergillus fumigatus/metabolismo , Ferro/metabolismo , Zinco/metabolismo , Aspergillus fumigatus/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Compostos Férricos/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Heme/biossíntese , Homeostase , Ácidos Hidroxâmicos/metabolismo , Micélio/metabolismo , Protoporfirinas/metabolismo , RNA Fúngico/análise , RNA Fúngico/biossíntese , RNA Fúngico/genéticaRESUMO
Iron is an essential metal for virtually all organisms. Iron acquisition is well characterized for various organisms, whereas intracellular iron distribution is poorly understood. In contrast to bacteria, plants, and animals, most fungi lack ferritin-mediated iron storage but possess an intracellular siderophore shown to be involved in iron storage. Here we demonstrate that deficiency in the intracellular siderophore ferricrocin causes iron starvation in conidia of Aspergillus fumigatus, demonstrating that ferricrocin is also involved in intra- and transcellular iron distribution. Thus, ferricrocin represents the first intracellular iron transporter identified in any organism.
Assuntos
Aspergillus fumigatus/metabolismo , Ferricromo/análogos & derivados , Ferro/metabolismo , Sideróforos/metabolismo , Ferricromo/metabolismo , Esporos Fúngicos/metabolismoRESUMO
The saprophytic fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen, which is responsible for invasive aspergillosis in immunocompromised patients. Iron plays an essential role for the growth and proliferation of A. fumigatus. This fungus synthesizes three major siderophores. It excretes triacetylfusarinine C to capture iron, while it accumulates ferricrocin and hydroxyferricrocin for hyphal and conidial iron storage, respectively. Herein, we investigated the role of the siderophore system of A. fumigatus in the modulation of immune effector pathways and iron homeostasis in macrophages. We set up a co-culture system consisting of the murine macrophage cell line RAW264.7 and either A. fumigatus wild type or a siderophore-deficient mutant (DeltasidA). We used real-time quantitative RT-PCR and Western blot analyses to study the expression of macrophage iron metabolism and innate immune response genes in response to pathogen challenge. Infection of macrophages with A. fumigatus wild type, but not with the DeltasidA mutant, induced expression of TNF and phagocyte oxidase subunit 47 at the transcriptional level. Moreover, infection with A. fumigatus wild type, but not with the DeltasidA mutant, compromised macrophage iron homeostasis. Infection with wild-type A. fumigatus decreased expression of the two cellular iron importers, the divalent metal transporter-1 and the transferrin receptor, and the only known iron exporter ferroportin. At the same time, it increased macrophage iron retention and ferritin synthesis. These data indicate that A. fumigatus affects the regulation of macrophage iron homeostasis and innate immune effector pathways via its siderophore system. The changes in immune response may be a consequence of macrophage iron restriction.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/patogenicidade , Ferro/imunologia , Macrófagos/imunologia , Sideróforos/metabolismo , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/metabolismo , Linhagem Celular , Técnicas de Cocultura , Compostos Férricos/imunologia , Compostos Férricos/metabolismo , Ferricromo/análogos & derivados , Ferricromo/imunologia , Ferricromo/metabolismo , Ácidos Hidroxâmicos/imunologia , Ácidos Hidroxâmicos/metabolismo , Imunidade Inata , Ferro/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Fagocitose/imunologia , Receptores da Transferrina/biossíntese , Sideróforos/genéticaRESUMO
Aspergillus fumigatus, the most common airborne fungal pathogen of humans, employs two high-affinity iron uptake systems: iron uptake mediated by the extracellular siderophore triacetylfusarinine C and reductive iron assimilation. Furthermore, A. fumigatus utilizes two intracellular siderophores, ferricrocin and hydroxyferricrocin, to store iron. Siderophore biosynthesis, which is essential for virulence, is repressed by iron. Here we show that this control is mediated by the GATA factor SreA. During iron-replete conditions, SreA deficiency partially derepressed synthesis of triacetylfusarinine C and uptake of iron resulting in increased cellular accumulation of both iron and ferricrocin. Genome-wide DNA microarray analysis identified 49 genes that are repressed by iron in an SreA-dependent manner. This gene set, termed SreA regulon, includes all known genes involved in iron acquisition, putative novel siderophore biosynthetic genes, and also genes not directly linked to iron metabolism. SreA deficiency also caused upregulation of iron-dependent and antioxidative pathways, probably due to the increased iron content and iron-mediated oxidative stress. Consistently, the sreA disruption mutant displayed increased sensitivity to iron, menadion and phleomycin but retained wild-type virulence in a mouse model. As all detrimental effects of sreA disruption are restricted to iron-replete conditions these data underscore that A. fumigatus faces iron-depleted conditions during infection.
Assuntos
Aspergillus fumigatus/genética , Proteínas Fúngicas/genética , Fatores de Transcrição GATA/genética , Ferro/metabolismo , Proteínas Repressoras/genética , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , DNA Fúngico/genética , Compostos Férricos/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Proteínas Fúngicas/metabolismo , Fatores de Transcrição GATA/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Teste de Complementação Genética , Ácidos Hidroxâmicos/metabolismo , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo , Regiões Promotoras Genéticas , RNA Fúngico/genética , Regulon , Proteínas Repressoras/metabolismo , Sideróforos/biossíntese , Sideróforos/genética , VirulênciaRESUMO
Ferrichrysin (Fcy), which is produced by Aspergillus oryzae and is present in foods used for human consumption, belongs to a group of hydroxamate siderophore ferric iron chelators. Fcy (100 mg/mL) dissolves completely at both pH 2.0 and 7.0, being very stable at a wide range of pH, high temperatures and pressures, with little reactivity to dietary iron absorption inhibitors, phytic acid, tannic acid, and catechin. We studied the effect of Fcy in male Sprague-Dawley rats with iron-deficiency anemia, which were separated into three different dietary groups (n=5) and supplementing diets as follows: (i) ferric citrate, (ii) heme iron concentrate, and (iii) Fcy (35 mg Fe/kg diet) for three weeks. Fcy exhibited the same beneficial effect in improving iron deficiency anemia as ferric citrate, being significantly greater than the effect of heme iron. The iron concentration of liver in the Fcy group was 35% greater than that in the ferric citrate group. These findings indicate that Fcy could be an efficient oral iron supplement to prevent or treat iron deficiency.
Assuntos
Anemia Ferropriva/tratamento farmacológico , Ferricromo/análogos & derivados , Animais , Suplementos Nutricionais , Compostos Férricos/administração & dosagem , Ferricromo/administração & dosagem , Ferricromo/química , Heme/química , Concentração de Íons de Hidrogênio , Ferro/administração & dosagem , Ferro/análise , Ferro/sangue , Fígado/química , Masculino , Ratos , Ratos Sprague-Dawley , Solubilidade , Aumento de Peso/efeitos dos fármacosRESUMO
Iron is required by most organisms, but an excess of this metal is potentially toxic. Consequently, uptake and intracellular storage of iron are tightly controlled. The filamentous fungus A. nidulans lacks the iron storage compound ferritin but possesses an intracellular siderophore, which is accumulated in a highly regulated manner as iron-free desferri-ferricrocin or iron-containing ferricrocin via transcriptional regulation of the nonribosomal peptide synthetase SidC. Biosynthesis of desferri-ferricrocin was low during iron-replete conditions but up-regulated by both iron starvation and intracellular iron excess, the latter caused by either a shift from iron-depleted to high-iron conditions or deregulation of iron uptake. Consequently, ferricrocin constituted only about 5% of the total iron content under iron-replete conditions but up to 64% during conditions of intracellular excess. In contrast, during iron starvation, desferri-ferricrocin was accumulated, which appears to represent a proactive strategy to prevent iron toxicity. Accumulation of the intracellular siderophore was also up-regulated by oxidative stress, which underscores the intertwining of iron metabolism and oxidative stress. Lack of the intracellular siderophore causes pleiotropic effects, as SidC deficiency results in (i) less-efficient utilization of iron, indicated by reduced growth under iron-depleted conditions and a higher iron demand under iron-replete conditions, (ii) delayed germination under iron-depleted conditions, (iii) increased sensitivity of conidia to oxidative stress, and (iv) elimination of cleistothecia formation in homothallic conditions.
Assuntos
Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/fisiologia , Ferricromo/análogos & derivados , Ferro/metabolismo , Estresse Oxidativo , Sideróforos/metabolismo , Aspergillus nidulans/citologia , Aspergillus nidulans/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ferricromo/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Hifas/efeitos dos fármacos , Deficiências de Ferro , Peso Molecular , Mutação/genética , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sideróforos/análise , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The hydroxamate siderophore receptor FhuA is a TonB-dependent outer membrane protein of Escherichia coli composed of a C-terminal 22-stranded beta-barrel occluded by an N-terminal globular cork domain. During siderophore transport into the periplasm, the FhuA cork domain has been proposed to undergo conformational changes that allow transport through the barrel lumen; alternatively, the cork may be completely displaced from the barrel. To probe such changes, site-directed cysteine mutants in the cork domain (L109C and Q112C) and in the barrel domain (S356C and M383C) were created within the putative siderophore transport pathway. Molecular modeling predicted that the double cysteine mutants L109C/S356C and Q112C/M383C would form disulfide bonds, thereby tethering the cork and barrel domains. The double cysteine FhuA mutants were denatured under nonreducing conditions and fluorescently labeled with thiol-specific Oregon Green maleimide. Subsequent SDS-PAGE analysis revealed two distinct species: FhuA containing a disulfide bond and FhuA with free sulfhydryl groups. To address the role of the putative siderophore transport pathway and to evaluate possible rearrangements of the cork domain during ferricrocin transport, disulfide bond formation was enhanced by an oxidative catalyst. Cells containing double cysteine FhuA mutants that were subjected to oxidation during ferricrocin transport exhibited disulfide bond formation to near completion. After disulfide tethering of the cork to the barrel, ferricrocin transport was equivalent to transport by untreated cells. These results demonstrate that blocking the putative siderophore transport pathway does not abrogate ferricrocin uptake. We propose that, during siderophore transport through FhuA, the cork domain remains within the barrel rather than being displaced.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Receptores Virais/química , Proteínas da Membrana Bacteriana Externa/fisiologia , Transporte Biológico , Western Blotting , Reagentes de Ligações Cruzadas/farmacologia , Cisteína/química , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/fisiologia , Ferricromo/análogos & derivados , Ferricromo/farmacologia , Corantes Fluorescentes/farmacologia , Maleimidas/farmacologia , Microscopia de Fluorescência , Modelos Biológicos , Modelos Moleculares , Mutação , Oxigênio/química , Oxigênio/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Virais/fisiologia , Sideróforos/metabolismo , Compostos de Sulfidrila/químicaRESUMO
FhuA, an outer membrane receptor of Escherichia coli, facilitates transport of hydroxamate siderophores and siderophore-antibiotic conjugates. The cytoplasmic membrane complex TonB-ExbB-ExbD provides energy for transport via the proton motive force. This energy is transduced by protein-protein interactions between TonB and FhuA, but the molecular determinants of these interactions remain uncharacterized. Our analyses of FhuA and two recombinant TonB species by surface plasmon resonance revealed that TonB undergoes a kinetically limiting rearrangement upon initial interaction with FhuA: an intermediate TonB-FhuA complex of 1:1 stoichiometry was detected. The intermediate then recruits a second TonB protein. Addition of ferricrocin, a FhuA-specific ligand, enhanced amounts of the 2:1 complex but was not essential for its formation. To assess the role of the cork domain of FhuA in forming a 2:1 TonB-FhuA complex, we tested a FhuA deletion (residues 21-128) for its ability to interact with TonB. Analytical ultracentrifugation demonstrated that deletion of this region of the cork domain resulted in a 1:1 complex. Furthermore, the high-affinity 2:1 complex requires the N-terminal region of TonB. Together these in vitro experiments establish that TonB-FhuA interactions require sequential steps of kinetically limiting rearrangements. Additionally, domains that contribute to complex formation were identified in TonB and in FhuA.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferricromo/análogos & derivados , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Receptores Virais/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/metabolismo , Cisteína/genética , Citoplasma/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ferricromo/metabolismo , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Químicos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Receptores Virais/química , Receptores Virais/genética , Deleção de Sequência , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N'',N'''-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.