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1.
Phys Chem Chem Phys ; 23(32): 17656-17662, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34373871

RESUMO

In this manuscript the ability of selenium carbohydrates to undergo chalcogen bonding (ChB) interactions with protein residues has been studied at the RI-MP2/def2-TZVP level of theory. An inspection of the Protein Data Bank (PDB) revealed SeA (A = O, C and S) intermolecular contacts involving Se-pyranose ligands and ASP, TYR, SER and MET residues. Theoretical models were built to analyse the strength and directionality of the interaction together with "Atoms in Molecules" (AIM), Natural Bonding Orbital (NBO) and Non Covalent Interactions plot (NCIplot) analyses, which further assisted in the characterization of the ChBs described herein. We expect that the results from this study will be useful to expand the current knowledge regarding biological ChBs as well as to increase the visibility of the interaction among the carbohydrate chemistry community.


Assuntos
Lectinas/metabolismo , Monossacarídeos/metabolismo , Compostos Organosselênicos/metabolismo , Agaricales/química , Aspergillus oryzae/química , Bases de Dados de Proteínas , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ligação de Hidrogênio , Lectinas/química , Modelos Moleculares , Monossacarídeos/química , Compostos Organosselênicos/química , Ligação Proteica , Selênio/química , Eletricidade Estática , Termodinâmica
3.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445203

RESUMO

Wheat is a major staple food crop worldwide, due to its total yield and unique processing quality. Its grain yield and quality are threatened by Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum. Salicylic acid (SA) has a strong and toxic effect on F. graminearum and is a hopeful target for sustainable control of FHB. F. graminearum is capable of efficientdealing with SA stress. However, the underlying mechanisms remain unclear. Here, we characterized FgMFS1 (FGSG_03725), a major facilitator superfamily (MFS) transporter gene in F. graminearum. FgMFS1 was highly expressed during infection and was upregulated by SA. The predicted three-dimensional structure of the FgMFS1 protein was consistent with the schematic for the antiporter. The subcellular localization experiment indicated that FgMFS1 was usually expressed in the vacuole of hyphae, but was alternatively distributed in the cell membrane under SA treatment, indicating an element of F. graminearum in response to SA. ΔFgMFS1 (loss of function mutant of FgMFS1) showed enhanced sensitivity to SA, less pathogenicity towards wheat, and reduced DON production under SA stress. Re-introduction of a functional FgMFS1 gene into ∆FgMFS1 recovered the mutant phenotypes. Wheat spikes inoculated with ΔFgMFS1 accumulated more SA when compared to those inoculated with the wild-type strain. Ecotopic expression of FgMFS1 in yeast enhanced its tolerance to SA as expected, further demonstrating that FgMFS1 functions as an SA exporter. In conclusion, FgMFS1 encodes an SA exporter in F. graminearum, which is critical for its response to wheat endogenous SA and pathogenicity towards wheat.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Genes Fúngicos , Doenças das Plantas/microbiologia , Ácido Salicílico/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Triticum/microbiologia , Proteínas de Transporte/genética , Proteínas Fúngicas/genética , Fusarium/genética
4.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361767

RESUMO

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Xilanos/metabolismo , Sequência de Aminoácidos , Arabinose/química , Arabinose/metabolismo , Sequência de Carboidratos , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Glucuronatos/química , Glucuronatos/metabolismo , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Talaromyces/química , Talaromyces/genética , Xilanos/química
5.
J Microbiol ; 59(9): 819-826, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34382148

RESUMO

Phosphate solubilizing fungi (PSF) have been widely applied to dissolve insoluble phosphates (IPs). However, the PSF usually demonstrates a different phosphate solubilizing capacity for various IPs. This study explored the mechanisms of Aspergillus niger for the dissolution of ferric phosphate (FePO4, Fe-P), and tricalcium phosphate (Ca3[PO4]2, Ca-P) regarding the tricarboxylic acid (TCA) cycle. Aspergillus niger has higher phosphorus (P) content released from Ca-P, reached the maximum value of 861 mg/L after seven days of incubation, compared with the 169 mg/L from Fe-P. Oxalic acid promoted the release of P from Ca-P through the formation of calcium oxalate. The presence of Fe-P can stimulate A. niger to secrete large amounts of citric acid, confirmed by the enhancement of citrate synthase (CS) activity. However, citric acid only promotes 0.5% of P released from Fe-P. Meanwhile, although oxalic acid still dominates the release of P from Fe-P, its abundance was significantly declined. In contrast, oxalic acid also shows a higher P release ratio in Ca-P than citric acid, i.e., 36% vs. 22%. This study points to the future usage of A. niger to dissolve IPs in soil required to enhance oxalic acid secretion.


Assuntos
Aspergillus niger/metabolismo , Fosfatos de Cálcio/metabolismo , Compostos Férricos/metabolismo , Aspergillus niger/genética , Fosfatos de Cálcio/química , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Ácido Cítrico/metabolismo , Compostos Férricos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Oxálico/metabolismo , Solo/química
6.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445420

RESUMO

Fungal secondary metabolites are renowned toxins as well as valuable sources of antibiotics, cholesterol-lowering drugs, and immunosuppressants; hence, great efforts were levied to understand how these compounds are genetically regulated. The genes encoding for the enzymes required for synthesizing secondary metabolites are arranged in biosynthetic gene clusters (BGCs). Often, BGCs contain a pathway specific transcription factor (PSTF), a valuable tool in shutting down or turning up production of the BGC product. In this review, we present an in-depth view of PSTFs by examining over 40 characterized BGCs in the well-studied fungal species Aspergillus nidulans and Aspergillus fumigatus. Herein, we find BGC size is a predictor for presence of PSTFs, consider the number and the relative location of PSTF in regard to the cluster(s) regulated, discuss the function and the evolution of PSTFs, and present application strategies for pathway specific activation of cryptic BGCs.


Assuntos
Aspergillus nidulans/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aspergillus nidulans/genética , Vias Biossintéticas , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Família Multigênica , Metabolismo Secundário
7.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361714

RESUMO

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.


Assuntos
Antracenos/química , Proteínas Fúngicas/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/química , Hipoglicemiantes/química , Perileno/análogos & derivados , alfa-Glucosidases/química , Antracenos/metabolismo , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Inibidores de Glicosídeo Hidrolases/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Hipoglicemiantes/metabolismo , Cinética , Simulação de Acoplamento Molecular , Nitrofenilgalactosídeos/química , Nitrofenilgalactosídeos/metabolismo , Perileno/química , Perileno/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/enzimologia , Ressonância de Plasmônio de Superfície , alfa-Glucosidases/metabolismo
8.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361722

RESUMO

Off-flavors produced by undesirable microbial spoilage are a major concern in wineries, as they affect wine quality. This situation is worse in warm areas affected by global warming because of the resulting higher pHs in wines. Natural biotechnologies can aid in effectively controlling these processes, while reducing the use of chemical preservatives such as SO2. Bioacidification reduces the development of spoilage yeasts and bacteria, but also increases the amount of molecular SO2, which allows for lower total levels. The use of non-Saccharomyces yeasts, such as Lachancea thermotolerans, results in effective acidification through the production of lactic acid from sugars. Furthermore, high lactic acid contents (>4 g/L) inhibit lactic acid bacteria and have some effect on Brettanomyces. Additionally, the use of yeasts with hydroxycinnamate decarboxylase (HCDC) activity can be useful to promote the fermentative formation of stable vinylphenolic pyranoanthocyanins, reducing the amount of ethylphenol precursors. This biotechnology increases the amount of stable pigments and simultaneously prevents the formation of high contents of ethylphenols, even when the wine is contaminated by Brettanomyces.


Assuntos
Brettanomyces/metabolismo , Aromatizantes/metabolismo , Tecnologia de Alimentos/métodos , Odorantes/análise , Saccharomycetales/metabolismo , Vinho/análise , Antocianinas/metabolismo , Carboxiliases/metabolismo , Fermentação , Proteínas Fúngicas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Dióxido de Enxofre/farmacologia , Vitis/metabolismo , Vitis/microbiologia , Vinho/microbiologia
9.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360577

RESUMO

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s' interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.


Assuntos
Aminoácidos/metabolismo , Azóis/metabolismo , Basidiomycota/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fluconazol/metabolismo , Proteínas Fúngicas/metabolismo , Itraconazol/metabolismo , Aminoácidos/química , Antifúngicos/química , Antifúngicos/metabolismo , Azóis/química , Simulação por Computador , Sistema Enzimático do Citocromo P-450/química , Fluconazol/química , Proteínas Fúngicas/química , Itraconazol/química , Ligantes , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
10.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360609

RESUMO

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic-hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.


Assuntos
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/genética , Propriedades de Superfície
11.
Sci Rep ; 11(1): 14748, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285303

RESUMO

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.


Assuntos
Candida albicans/enzimologia , Farmacorresistência Fúngica , Proteínas Fúngicas/metabolismo , Lipossomos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Cinética , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/genética , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
J Chem Inf Model ; 61(7): 3604-3614, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34251205

RESUMO

Environmentally friendly processes are nowadays a trending topic to get highly desired chemical compounds and, in this sense, the use of enzyme-catalyzed routes is becoming a promising alternative to traditional synthetic methods. In the present paper, a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study on the epoxidation of alkenes catalyzed by the Ser105Ala variant of the promiscuous Candida antarctica lipase B (CALB) is presented in an attempt to search for alternative paths to get useful intermediates in industries. The catalyzed reaction, described at the atomistic level with a model of the full solvated in a box of water molecules, is compared with the alternative epoxidation of alkenes by peroxy acids in chloroform. Free-energy profiles obtained at the density functional theory (DFT)/MM level show how Ser105Ala CALB is capable of epoxide short alkenes in a two-step process with free-energy barriers, in agreement with available experimental data, that are significantly lower than those of the single-step reaction in solution. The possible (R)-enantioselectivity dictated by the binding step, explored by means of alchemical QM/MM free-energy perturbation (FEP) methods, and the preference for the (S)-enantiomer derived from the free-energy landscape of the chemical steps would cancel out, thus predicting the lack of enantioselectivity experimentally observed. In general, our results provide general information on the molecular mechanism employed by a highly promiscuous enzyme, with potential applications in biotechnology.


Assuntos
Compostos de Epóxi , Lipase , Basidiomycota , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Simulação de Dinâmica Molecular , Estereoisomerismo
13.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299357

RESUMO

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine161, sumoylation of lysine242 and phosphorylation of threonine319. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A. fumigatus.


Assuntos
Aspergillus fumigatus/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Homeostase/genética , Ferro/metabolismo , Processamento de Proteína Pós-Traducional/genética , Adaptação Fisiológica/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Mutação Puntual/genética , Sideróforos/genética , Treonina/genética , Virulência/genética
14.
Nat Commun ; 12(1): 4290, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257288

RESUMO

Intestinal intraepithelial lymphocytes (IEL) are an abundant population of tissue-resident T cells that protect and maintain the intestinal barrier. IEL respond to epithelial cell-derived IL-15, which is complexed to the IL-15 receptor α chain (IL-15/Rα). IL-15 is essential both for maintaining IEL homeostasis and inducing IEL responses to epithelial stress, which has been associated with Coeliac disease. Here, we apply quantitative mass spectrometry to IL-15/Rα-stimulated IEL to investigate how IL-15 directly regulates inflammatory functions of IEL. IL-15/Rα drives IEL activation through cell cycle regulation, upregulation of metabolic machinery and expression of a select repertoire of cell surface receptors. IL-15/Rα selectively upregulates the Ser/Thr kinases PIM1 and PIM2, which are essential for IEL to proliferate, grow and upregulate granzyme B in response to inflammatory IL-15. Notably, IEL from patients with Coeliac disease have high PIM expression. Together, these data indicate PIM kinases as important effectors of IEL responses to inflammatory IL-15.


Assuntos
Interleucina-15/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Granzimas/genética , Granzimas/metabolismo , Humanos , Interleucina-15/genética , Linfócitos Intraepiteliais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
15.
Appl Microbiol Biotechnol ; 105(14-15): 5915-5929, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34292355

RESUMO

Arginine is an important amino acid involved in processes such as cell signal transduction, protein synthesis, and sexual reproduction. To understand the biological roles of arginine biosynthesis in pathogenic fungi, we used Cpa1, the carbamoyl phosphate synthase arginine-specific small chain subunit in Saccharomyces cerevisiae as a query to identify its ortholog in the Magnaporthe oryzae genome and named it MoCpa1. MoCpa1 is a 471-amino acid protein containing a CPSase_sm_chain domain and a GATase domain. MoCpa1 transcripts were highly expressed at the conidiation, early-infection, and late-infection stages of the fungus. Targeted deletion of the MoCPA1 gene resulted in a ΔMocpa1 mutant exhibiting arginine auxotrophy on minimum culture medium (MM), confirming its role in de novo arginine biosynthesis. The ΔMocpa1 mutant presented significantly decreased sporulation with some of its conidia being defective in morphology. Furthermore, the ΔMocpa1 mutant was nonpathogenic on rice and barley leaves, which was a result of defects in appressorium-mediated penetration and restricted invasive hyphal growth within host cells. Addition of exogenous arginine partially rescued conidiation and pathogenicity defects on the barley and rice leaves, while introduction of the MoCPA1 gene into the ΔMocpa1 mutant fully complemented the lost phenotype. Further confocal microscopy examination revealed that MoCpa1 is localized in the mitochondria. In summary, our results demonstrate that MoCpa1-mediated arginine biosynthesis is crucial for fungal development, conidiation, appressorium formation, and infection-related morphogenesis in M. oryzae, thus serving as an attractive target for mitigating obstinate fungal plant pathogens. KEY POINTS: • MoCpa1 is important for aerial hyphal growth and arginine biosynthesis. • MoCpa1 is pivotal for conidial morphogenesis and appressorium formation. • MoCpa1 is crucial for full virulence in M. oryzae.


Assuntos
Magnaporthe , Oryza , Arginina , Ascomicetos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Magnaporthe/metabolismo , Oryza/metabolismo , Doenças das Plantas , Esporos Fúngicos/metabolismo
16.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200464

RESUMO

Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca2+ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.


Assuntos
Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Cryptococcus neoformans/efeitos dos fármacos , Timol/farmacologia , Calcineurina/metabolismo , Criptococose/metabolismo , Cryptococcus neoformans/metabolismo , Ergosterol/farmacologia , Proteínas Fúngicas/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monoterpenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Thymus (Planta)/química
17.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207287

RESUMO

Gastrodia elata is a well-known medicinal and heterotrophic orchid. Its germination, limited by the impermeability of seed coat lignin and inhibition by abscisic acid (ABA), is triggered by symbiosis with fungi such as Mycena spp. However, the molecular mechanisms of lignin degradation by Mycena and ABA biosynthesis and signaling in G. elata remain unclear. In order to gain insights into these two processes, this study analyzed the transcriptomes of these organisms during their dynamic symbiosis. Among the 25 lignin-modifying enzyme genes in Mycena, two ligninolytic class II peroxidases and two laccases were significantly upregulated, most likely enabling Mycena hyphae to break through the lignin seed coats of G. elata. Genes related to reduced virulence and loss of pathogenicity in Mycena accounted for more than half of annotated genes, presumably contributing to symbiosis. After coculture, upregulated genes outnumbered downregulated genes in G. elata seeds, suggesting slightly increased biological activity, while Mycena hyphae had fewer upregulated than downregulated genes, indicating decreased biological activity. ABA biosynthesis in G. elata was reduced by the downregulated expression of 9-cis-epoxycarotenoid dioxygenase (NCED-2), and ABA signaling was blocked by the downregulated expression of a receptor protein (PYL12-like). This is the first report to describe the role of NCED-2 and PYL12-like in breaking G. elata seed dormancy by reducing the synthesis and blocking the signaling of the germination inhibitor ABA. This study provides a theoretical basis for screening germination fungi to identify effective symbionts and for reducing ABA inhibition of G. elata seed germination.


Assuntos
Ácido Abscísico/metabolismo , Agaricales/patogenicidade , Proteínas Fúngicas/genética , Gastrodia/microbiologia , Lignina/metabolismo , Proteínas de Plantas/genética , Agaricales/genética , Agaricales/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Proteínas Fúngicas/metabolismo , Gastrodia/genética , Gastrodia/crescimento & desenvolvimento , Gastrodia/metabolismo , Germinação , Lacase/genética , Lacase/metabolismo , Lignina/genética , Peroxidases/genética , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Simbiose , Transcriptoma
18.
Mol Genet Genomics ; 296(5): 1135-1145, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34196769

RESUMO

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.


Assuntos
Proteínas Fúngicas/metabolismo , Histidina Quinase/química , Histidina Quinase/metabolismo , Magnaporthe/enzimologia , Dioxóis/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Teste de Complementação Genética , Histidina Quinase/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microrganismos Geneticamente Modificados , Mutação , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pirróis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Nat Commun ; 12(1): 4417, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285212

RESUMO

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.


Assuntos
Aspergillus/enzimologia , Biocatálise , Proteínas Fúngicas/metabolismo , Oxigenases/metabolismo , Peróxidos/metabolismo , Aspergillus/genética , Domínio Catalítico , Cristalografia por Raios X , Compostos Ferrosos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Ácidos Cetoglutáricos/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Oxigenases/genética , Oxigenases/isolamento & purificação , Oxigenases/ultraestrutura , Conformação Proteica em Folha beta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Terpenos/metabolismo
20.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281276

RESUMO

Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and in remediation is considerable. The analysis of fungal EVs opens new horizons for diagnosing fungal infections and developing vaccines against mycoses and novel applications of nanotherapy and sensors in industrial processes.


Assuntos
Vesículas Extracelulares/fisiologia , Fungos/fisiologia , Transporte Biológico Ativo , Vesículas Extracelulares/genética , Vesículas Extracelulares/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/patogenicidade , Genes Fúngicos , Interações entre Hospedeiro e Microrganismos/imunologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Micoses/diagnóstico , Micoses/microbiologia , RNA Fúngico/genética , RNA Fúngico/metabolismo
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