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1.
Nat Commun ; 11(1): 4212, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839469

RESUMO

Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.


Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica/métodos , Genoma Fúngico/genética , Estudo de Associação Genômica Ampla/métodos , Monoéster Fosfórico Hidrolases/genética , Animais , Análise por Conglomerados , Criptococose/microbiologia , Cryptococcus neoformans/patogenicidade , Feminino , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Camundongos Endogâmicos , Monoéster Fosfórico Hidrolases/classificação , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/classificação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Transdução de Sinais/genética , Termotolerância/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genética
2.
Nat Commun ; 11(1): 4031, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788582

RESUMO

Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies.


Assuntos
Canais de Cálcio/genética , Evolução Molecular , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/química , Quitridiomicetos/genética , Proteínas Fúngicas/química , Células HeLa , Humanos , Funções Verossimilhança , Filogenia , Especificidade da Espécie
3.
Nat Commun ; 11(1): 3290, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620929

RESUMO

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Detergentes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sordariales/genética , Sordariales/metabolismo
4.
PLoS One ; 15(7): e0235746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678853

RESUMO

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.


Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Esterol 14-Desmetilase/genética
5.
Gene ; 759: 145002, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32726608

RESUMO

Recent evidence has shown that microRNAs are transferred from one species to another through cross-species transmission and exhibit biological activities in the receptor. However, the cross-kingdom regulation of pathogen virulence by plant-derived miRNAs is rarely reported. This study investigated the regulatory role of novel tomato miRNA miR1001 in the growth and development of Botrytis cinerea. Results showed that miR1001 inhibited the virulence of B. cinerea-infected plants, and the inhibitory effect of miR1001/miR1001* was stronger than that of miR1001. Moreover, miR1001 exerted a significant inhibitory effect on the conidiospore germination of B. cinerea. Degradome-seq experiment showed that miR1001 can directly target the Bcin03g02170.1 and Bcin10g01400.1 genes, which respectively encode the ATP-dependent metallopeptidase and cysteine-type endopeptidase, in B. cinerea. The interactions of both targets with miR1001 were further confirmed by using transient co-expression in tobacco. Real-time RT-PCR analysis showed that the expression levels of the two target genes were significantly downregulated in B. cinerea with miR1001 treatment. Our findings provide new evidence into the coevolution of pathogens and host plants, as well as new directions for the use of plant-derived miRNAs to control pathogens.


Assuntos
Botrytis/patogenicidade , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Lycopersicon esculentum/genética , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , Botrytis/fisiologia , Proteínas Fúngicas/metabolismo , Lycopersicon esculentum/microbiologia , MicroRNAs/genética , RNA de Plantas/genética , Esporos Fúngicos/fisiologia
6.
PLoS Genet ; 16(7): e1008908, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32639995

RESUMO

The human fungal pathogen Candida albicans is constantly exposed to environmental challenges impacting the cell wall. Signaling pathways coordinate stress adaptation and are essential for commensalism and virulence. The transcription factors Sko1, Cas5, and Rlm1 control the response to cell wall stress caused by the antifungal drug caspofungin. Here, we expand the Sko1 and Rlm1 transcriptional circuit and demonstrate that Rlm1 activates Sko1 cell wall stress signaling. Caspofungin-induced transcription of SKO1 and several Sko1-dependent cell wall integrity genes are attenuated in an rlm1Δ/Δ mutant strain when compared to the treated wild-type strain but not in a cas5Δ/Δ mutant strain. Genome-wide chromatin immunoprecipitation (ChIP-seq) results revealed numerous Sko1 and Rlm1 directly bound target genes in the presence of caspofungin that were undetected in previous gene expression studies. Notable targets include genes involved in cell wall integrity, osmolarity, and cellular aggregation, as well as several uncharacterized genes. Interestingly, we found that Rlm1 does not bind to the upstream intergenic region of SKO1 in the presence of caspofungin, indicating that Rlm1 indirectly controls caspofungin-induced SKO1 transcription. In addition, we discovered that caspofungin-induced SKO1 transcription occurs through self-activation. Based on our ChIP-seq data, we also discovered an Rlm1 consensus motif unique to C. albicans. For Sko1, we found a consensus motif similar to the known Sko1 motif for Saccharomyces cerevisiae. Growth assays showed that SKO1 overexpression suppressed caspofungin hypersensitivity in an rlm1Δ/Δ mutant strain. In addition, overexpression of the glycerol phosphatase, RHR2, suppressed caspofungin hypersensitivity specifically in a sko1Δ/Δ mutant strain. Our findings link the Sko1 and Rlm1 signaling pathways, identify new biological roles for Sko1 and Rlm1, and highlight the complex dynamics underlying cell wall signaling.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Candida albicans/efeitos dos fármacos , Caspofungina/farmacologia , Proteínas de Domínio MADS/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Antifúngicos/farmacologia , Candida albicans/genética , Candida albicans/patogenicidade , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Fosforilação/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética
7.
PLoS Genet ; 16(7): e1008611, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658892

RESUMO

Epimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and releases its control when a stressful environment challenges the fungus. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.


Assuntos
Mucorales/genética , Mucormicose/genética , Interferência de RNA , Ribonuclease III/genética , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Instabilidade Genômica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Mucorales/patogenicidade , Mucormicose/microbiologia , Mutação/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Virulência/genética
8.
PLoS One ; 15(7): e0227529, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730337

RESUMO

The pretreatment of biomass remains a critical requirement for bio-renewable fuel production from lignocellulose. Although current processes primarily involve chemical and physical approaches, the biological breakdown of lignin using enzymes and microorganisms is quickly becoming an interesting eco-friendly alternative to classical processes. As a result, bioprospection of wild fungi from naturally occurring lignin-rich sources remains a suitable method to uncover and isolate new species exhibiting ligninolytic activity. In this study, wild species of white rot fungi were collected from Colombian forests based on their natural wood decay ability and high capacity to secrete oxidoreductases with high affinity for phenolic polymers such as lignin. Based on high activity obtained from solid-state fermentation using a lignocellulose source from oil palm as matrix, we describe the isolation and whole-genome sequencing of Dictyopanus pusillus, a wild basidiomycete fungus exhibiting ABTS oxidation as an indication of laccase activity. Functional characterization of a crude enzymatic extract identified laccase activity as the main enzymatic contributor to fungal extracts, an observation supported by the identification of 13 putative genes encoding for homologous laccases in the genome. To the best of our knowledge, this represents the first report of an enzymatic extract exhibiting laccase activity in the Dictyopanus genera, offering means to exploit this species and its enzymes for the delignification process of lignocellulosic by-products from oil palm.


Assuntos
Agaricales/genética , Genoma Fúngico , Lignina/metabolismo , Óleo de Palmeira/metabolismo , Agaricales/classificação , Agaricales/enzimologia , Biomassa , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Lacase/genética , Lacase/metabolismo , Oxirredução , Filogenia , Temperatura , Sequenciamento Completo do Genoma
9.
PLoS Genet ; 16(6): e1008881, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32525871

RESUMO

Iron is an essential nutrient required as a cofactor for many biological processes. As a fungal commensal-pathogen of humans, Candida albicans encounters a range of bioavailable iron levels in the human host and maintains homeostasis with a conserved regulatory circuit. How C. albicans senses and responds to iron availability is unknown. In model yeasts, regulation of the iron homeostasis circuit requires monothiol glutaredoxins (Grxs), but their functions beyond the regulatory circuit are unclear. Here, we show Grx3 is required for virulence and growth on low iron for C. albicans. To explore the global roles of Grx3, we applied a proteomic approach and performed in vivo cross-linked tandem affinity purification coupled with mass spectrometry. We identified a large number of Grx3 interacting proteins that function in diverse biological processes. This included Fra1 and Bol2/Fra2, which function with Grxs in intracellular iron trafficking in other organisms. Grx3 interacts with and regulates the activity of Sfu1 and Hap43, components of the C. albicans iron regulatory circuit. Unlike the regulatory circuit, which determines expression or repression of target genes in response to iron availability, Grx3 amplifies levels of gene expression or repression. Consistent with the proteomic data, the grx3 mutant is sensitive to heat shock, oxidative, nitrosative, and genotoxic stresses, and shows growth dependence on histidine, leucine, and tryptophan. We suggest Grx3 is a conserved global regulator of iron-dependent processes occurring within the cell.


Assuntos
Candida albicans/fisiologia , Candidíase Invasiva/microbiologia , Proteínas Fúngicas/metabolismo , Glutarredoxinas/metabolismo , Ferro/metabolismo , Animais , Candida albicans/patogenicidade , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Homeostase , Humanos , Hifas , Masculino , Camundongos , Mutação , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Proteômica , Virulência/genética
10.
Gen Physiol Biophys ; 39(3): 205-218, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32525814

RESUMO

The GABA shunt is one of the metabolic pathways that is ubiquitous in prokaryotes and eukaryotes. γ-aminobutyric acid (GABA) in fungi is required in the stress responses, virulence and development. The number of genes encoding glutamate decarboxylase (gad), GABA transaminase (gta) and succinic semialdehyde dehydrogenase (ssadh) varies between fungal species. The genome-wide analysis in Neurospora crassa resulted in the identification of a gta and a ssadh. Disruption of either gta or ssadh decreased respiration rate and biomass accumulation, reduced growth on GABA and beta-alanine. The gta and ssadh mutants exhibited aberrant hyphal morphology and displayed differential transcription of the GABA shunt genes. In the gta mutant, protoperithecia and perithecia formation was almost completely suppressed in the presence of GABA and beta-alanine, indicating GTA requirement for the turnover of these amino acids. The strains displayed differential metabolic dysregulations in response to different nitrogen sources. The phenotypic differences between the gta and ssadh mutants could be contributed to accumulation of intermediates of the GABA shunt and/or GABA shunt-independent functions. Together, our data suggest that the GABA shunt could function as a moderate modulator of multiple biological events, including respiration, energy metabolism, carbon and nitrogen metabolism, growth, as well as sexual development in N. crassa.


Assuntos
4-Aminobutirato Transaminase/genética , Proteínas Fúngicas/genética , Neurospora crassa/enzimologia , Succinato-Semialdeído Desidrogenase/genética , Ácido gama-Aminobutírico/metabolismo , Aminoácidos/metabolismo , Metabolismo Energético
11.
PLoS Genet ; 16(6): e1008836, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479508

RESUMO

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.


Assuntos
Anticódon/genética , Uso do Códon , Elongação Traducional da Cadeia Peptídica/genética , Proteoma/genética , RNA de Transferência de Arginina/genética , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Anticódon/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Inosina/metabolismo , Neurospora crassa/genética , RNA de Transferência de Arginina/metabolismo , Ribossomos/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(27): 15884-15894, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576698

RESUMO

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn's disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.


Assuntos
Bactérias/genética , Hemeproteínas/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Malassezia/genética , Malassezia/metabolismo , Óxido Nítrico/metabolismo , Pele/microbiologia , Animais , Bactérias/metabolismo , Cristalografia por Raios X , Ergosterol/biossíntese , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Transferência Genética Horizontal , Hemeproteínas/química , Hemeproteínas/metabolismo , Humanos , Malassezia/classificação , Modelos Moleculares , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Filogenia , Pele/metabolismo , Simbiose
14.
PLoS Pathog ; 16(6): e1008652, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32574207

RESUMO

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.


Assuntos
Ascomicetos/química , Quitina/química , Proteínas Fúngicas/química , Hifas/química , Multimerização Proteica , Ascomicetos/genética , Ascomicetos/metabolismo , Quitina/genética , Quitina/metabolismo , Cladosporium/química , Cladosporium/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Estrutura Quaternária de Proteína , Triticum/genética , Triticum/metabolismo , Triticum/microbiologia
15.
Mol Genet Genomics ; 295(5): 1269-1279, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32561986

RESUMO

Aspergillus nidulans nrtA encodes a nitrate transporter that plays an important role in the [Formula: see text] assimilatory process. Many studies have focused on protein functions rather than gene regulation. The knowledge of nrtA[Formula: see text] uptake process, particularly in the regulation mechanism of transcription factors AreA and NirA on nrtA transcription, is very limited. Herein, we investigated the transcriptional regulation of nrtA in response to various N-sources in detail and characterized the promoter activity of nrtA. We confirmed that nrtA was induced by [Formula: see text] and repressed by preferred N-sources. Additionally, for the first time, we found that the transcription of nrtA increased under N-starvation conditions. AreA mediates nrtA transcription under both [Formula: see text] and N-starvation conditions, while NirA is effective only under [Formula: see text] conditions. All of the proposed AreA and NirA binding sites in the promoter region were capable of binding to their corresponding transcription factors in vitro. In vivo, all of the NirA binding sites showed regulation activities, but to AreA, only several of the initiation-codon-proximal binding sites participated in nrtA transcription. Moreover, the active binding sites contributed in different degrees of regulation strength to nrtA transcription, which is unrelated to the distance between the binding sites and initiation codon. These results provided an extensive map of nrtA promoter, defining the functional regulatory elements of A. nidulans nrtA.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Nitratos/metabolismo , Proteínas de Transporte de Ânions/química , Aspergillus nidulans/genética , Sítios de Ligação , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
16.
Mol Genet Genomics ; 295(5): 1295-1304, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32566991

RESUMO

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the - 973 base pair (bp) to - 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5'-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.


Assuntos
Metanol/metabolismo , Pichia/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aldeído Oxidase/metabolismo , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Deleção de Sequência , Fatores de Transcrição/química , Sítio de Iniciação de Transcrição
17.
Mol Cell ; 79(1): 127-139.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32437639

RESUMO

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.


Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , DNA Fúngico/metabolismo , Proteínas Fúngicas/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/genética , Hidrólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
J Med Microbiol ; 69(6): 844-849, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32459615

RESUMO

Introduction. Signal transducer and activator of transcription 3 (STAT3) deficiency is a rare primary immunodeficiency associated with increased susceptibility to bacterial and fungal infections, notably pulmonary aspergillosis.Aim. We describe the emergence of azole-resistant Aspergillus fumigatus infections in STAT3-deficient patients.Methodology. During a retrospective study of 13 pulmonary aspergillosis cases in STAT3-deficient patients conducted in France, we identified patients infected with azole-resistant A. fumigatus isolates.Results. Two out of the 13 STAT3-deficient patients with aspergillosis had azole-resistant A. fumigatus infection, indicating an unexpectedly high prevalence of resistance. The first patient with STAT3 deficiency presented several flares of allergic bronchopulmonary aspergillosis-like episodes. He was chronically infected with two azole-resistant A. fumigatus isolates (TR34/L98). Despite prolonged antifungal treatment, including caspofungin and amphotericin B, the patient was not able to clear the azole-resistant A. fumigatus. The second patient had chronic cavitary pulmonary aspergillosis (CCPA). The A. fumigatus isolate was initially azole susceptible but harboured three F46Y, M172V and E427K point mutations. Despite prolonged antifungal therapies, lesions worsened and the isolate became resistant to all azoles. Surgery and caspofungin treatments were then required to cure CCPA. Resistance was probably acquired from the environment (TR34/L98) in the first case whereas resistance developed under antifungal treatments in the second case. These infections required long-term antifungal treatments and surgery.Conclusions. The emergence of azole-resistant A. fumigatus infections in STAT3-deficiency dramatically impacts both curative and prophylactic antifungal strategies. Physicians following patients with primary immune-deficiencies should be aware of this emerging problem as it complicates management of the patient.


Assuntos
Antifúngicos/uso terapêutico , Aspergillus fumigatus/efeitos dos fármacos , Azóis/uso terapêutico , Farmacorresistência Fúngica/efeitos dos fármacos , Aspergilose Pulmonar/tratamento farmacológico , Aspergilose Pulmonar/genética , Fator de Transcrição STAT3/deficiência , Adulto , Anfotericina B/uso terapêutico , Caspofungina/uso terapêutico , Criança , Doenças Transmissíveis/tratamento farmacológico , Doenças Transmissíveis/genética , Doenças Transmissíveis/microbiologia , Farmacorresistência Fúngica/genética , França , Proteínas Fúngicas/genética , Genótipo , Humanos , Masculino , Testes de Sensibilidade Microbiana , Estudos Retrospectivos , Adulto Jovem
19.
J Ind Microbiol Biotechnol ; 47(4-5): 437-448, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32377991

RESUMO

Sophorolipids (SLs) are surface-active molecules produced by the non-pathogenic yeast Starmerella bombicola CGMCC 1576. Several genes involved in the synthesis of SLs have been identified. However, the regulation mechanism of the synthesis pathway for SLs has not been investigated. We recently discovered a protein in S. bombicola, which is structurally related to Yarrowia lipolytica YlBro1. To identify the function of the protein SbBro1 in S. bombicola, the deletion, overexpression, and complementary mutant strains were constructed. We found that the deletion mutant no longer produced SLs. Transcriptome analysis indicated that the expression levels of the key enzyme genes of SLs biosynthetic pathway were significantly down-regulated in the Δbro1, especially the expression level of cyp52m1 encoding the first rate-limiting enzyme in SL synthesis pathway was down-regulated 13-folds and the expression of fatty acid ß-oxidation-related enzymes was also down-regulated. This study can give insight into the regulation of SL synthesis.


Assuntos
Proteínas Fúngicas/metabolismo , Ácidos Oleicos/biossíntese , Saccharomycetales/metabolismo , Biologia Computacional , Proteínas Fúngicas/genética , Saccharomycetales/genética , Transcriptoma
20.
Nucleic Acids Res ; 48(12): e67, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32421771

RESUMO

We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.


Assuntos
Proteínas Fúngicas/metabolismo , Genes Reporter , Oxigenases/metabolismo , Ácidos Picolínicos/metabolismo , Análise de Célula Única/métodos , Absorção de Radiação , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas Fúngicas/genética , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Oxigenases/genética , Ácidos Picolínicos/efeitos da radiação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência/métodos , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , Raios Ultravioleta
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