RESUMO
Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.
Assuntos
Chaetomium , Histidina-tRNA Ligase , Proteínas Serina-Treonina Quinases , Chaetomium/enzimologia , Chaetomium/genética , Chaetomium/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Histidina-tRNA Ligase/metabolismo , Histidina-tRNA Ligase/química , Histidina-tRNA Ligase/genética , Estresse Fisiológico , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Cristalografia por Raios X , Modelos Moleculares , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.
Assuntos
Chaetomium , Proteínas Fúngicas , Histidina Quinase , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Chaetomium/metabolismo , Chaetomium/genética , Chaetomium/enzimologia , Histidina Quinase/metabolismo , Histidina Quinase/química , Histidina Quinase/genética , Candida albicans/metabolismo , Candida albicans/enzimologia , Fosforilação , Modelos Moleculares , Espalhamento a Baixo Ângulo , Conformação ProteicaRESUMO
The endophytic fungus Chaetomium nigricolor culture filtrate's hexane extract was used to identify a cytotoxic very long-chain fatty acid. Based on multiple spectroscopic investigations, the structure of the compound was predicted to be an unsaturated fatty acid, Nonacosenoic acid (NA). Using the MTT assay, the compound's cytotoxic potential was evaluated against MCF-7, A-431, U-251, and HEK-293 T cells. The compound was moderately cytotoxic to breast carcinoma cell line, MCF-7 cells and negligibly cytotoxic to non-cancerous cell line HEK-293 T cells. The compound exhibited mild cytotoxic activity against A-431 and U-251 cells. The compound also induced ROS generation and mitochondrial depolarization in MCF-7 cells when assessed via the NBT and JC-1 assays, respectively. This is the first report on the production of nonacosenoic acid from the endophytic fungus Chaetomium nigricolor and the assessment of its bioactivity.
Assuntos
Chaetomium , Endófitos , Ácidos Graxos Insaturados , Chaetomium/química , Humanos , Endófitos/química , Endófitos/metabolismo , Endófitos/isolamento & purificação , Ácidos Graxos Insaturados/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Caules de Planta/microbiologia , Caules de Planta/química , Sobrevivência Celular/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Linhagem CelularRESUMO
Chetocochliodin M (5) containing a rare cage-ring and chetocochliodin N (6) featuring an unusual piperazine-2,3-dione ring system together with known analogues chetomin (1), chetoseminudin C (2), chetocochliodin I (3), and oidioperazine E (4) were targeted for purification from the fungus Chaetomium cochliodes using a UPLC-Q-TOF-MS/MS approach. The structures of the new compounds were elucidated using HR-ESI-MS, NMR, and ECD spectra. Compounds 1, 3, and 6 exhibited strong cytotoxic activities against A549 and HeLa cancer cell lines.
Assuntos
Chaetomium , Espectrometria de Massas em Tandem , Chaetomium/química , Humanos , Estrutura Molecular , Espectrometria de Massas em Tandem/métodos , Células HeLa , Cromatografia Líquida de Alta Pressão/métodos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Células A549 , Piperazinas/farmacologia , Piperazinas/química , Piperazinas/isolamento & purificaçãoRESUMO
Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.
Assuntos
Chaetomium , Metilenotetra-Hidrofolato Redutase (NADPH2) , S-Adenosilmetionina , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Regulação Alostérica , Chaetomium/enzimologia , Chaetomium/metabolismo , Chaetomium/genética , Fosforilação , Humanos , Cristalografia por Raios X , Modelos Moleculares , Flavina-Adenina Dinucleotídeo/metabolismo , Flavina-Adenina Dinucleotídeo/químicaRESUMO
Rice straw breakdown is sluggish, which makes agricultural waste management difficult, however pretreatment procedures and cellulolytic fungi can address this issue. Through ITS sequencing, Chaetomium globosum C1, Aspergillus sp. F2, and Ascomycota sp. SM2 were identified from diverse sources. Ascomycota sp. SM2 exhibited the highest carboxymethyl cellulase (CMCase) activity (0.86 IU/mL) and filter-paper cellulase (FPase) activity (1.054 FPU/mL), while Aspergillus sp. F2 showed the highest CMCase activity (0.185 IU/mL) after various pretreatments of rice straw. These fungi thrived across a wide pH range, with Ascomycota sp. SM2 from pH 4 to 9, Aspergillus sp. F2, and Chaetomium globosum C1 thriving in alkaline conditions (pH 9). FTIR spectroscopy revealed significant structural changes in rice straw after enzymatic hydrolysis and solid-state fermentation, indicating lignin, cellulose, and hemicellulose degradation. Soil amendments with pretreated rice straw, cow manure, biochar, and these fungi increased root growth and soil nutrient availability, even under severe salt stress (up to 9.3 dS/m). The study emphasizes the need for a better understanding of Ascomycota sp. degradation capabilities and proposes that using cellulolytic fungus and pretreatment rice straw into soil amendments could mitigate salt-related difficulties and improve nutrient availability in salty soils.
Assuntos
Celulase , Oryza , Solo , Oryza/metabolismo , Solo/química , Celulase/metabolismo , Estresse Salino , Microbiologia do Solo , Celulose/metabolismo , Chaetomium/metabolismo , Aspergillus/metabolismo , Hidrólise , Concentração de Íons de Hidrogênio , Ascomicetos/metabolismo , Fermentação , Esterco/microbiologia , Carvão VegetalRESUMO
Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: ⢠Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. ⢠Three polyketides and one asterriquinone were isolated from HDAC deleted strain. ⢠Two different PKSs were reported in C. olivaceum SD-80A.
Assuntos
Chaetomium , Histona Desacetilases , Família Multigênica , Policetídeos , Metabolismo Secundário , Chaetomium/genética , Chaetomium/enzimologia , Chaetomium/metabolismo , Metabolismo Secundário/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Policetídeos/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Vias Biossintéticas/genética , Epigênese GenéticaRESUMO
Two new sesterterpenoids, sesterchaetins A and B (1 and 2), and two new diepoxide polyketides, chaetoketoics A and B (3 and 4), were characterized from the culture extract of Chaetomium globosum SD-347, a fungal strain derived from deep sea-sediment. Their structures and absolute configurations were unambiguously determined by detailed NMR, mass spectra, and X-ray crystallographic analysis. Compounds 1 and 2 contained a distinctive 5/8/6/5 tetracyclic carbon-ring-system, which represented a rarely occurring natural product framework. The new isolates 1-4 exhibited selective antimicrobial activities against human and aquatic pathogenic bacteria and plant-pathogenic fungi.
Assuntos
Anti-Infecciosos , Chaetomium , Policetídeos , Sesquiterpenos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Policetídeos/química , Policetídeos/isolamento & purificação , Organismos Aquáticos/química , Chaetomium/química , Bactérias/efeitos dos fármacos , Cristalografia por Raios XRESUMO
Two new cytochalasans, marcytoglobosins A (1) and B (2) were isolated from the marine sponge associated fungus Chaetomium globosum 162105, along with six known compounds (3-8). The complete structures of two new compounds were determined based on 1D/2D NMR and HR-MS spectroscopic analyses coupled with ECD calculations. All eight isolates were evaluated for their antibacterial activity. Among them, compounds 3-8 displayed antibacterial effects against Staphylococcus epidermidis, Bacillus thuringiensis, Pseudomonas syringae pv. Actinidiae, Vibrio alginolyticus, and Edwardsiella piscicida with minimum inhibitory concentration (MIC) values ranging from 10 to 25â µg/mL.
Assuntos
Antibacterianos , Chaetomium , Testes de Sensibilidade Microbiana , Poríferos , Chaetomium/química , Animais , Poríferos/microbiologia , Poríferos/química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Citocalasinas/farmacologia , Citocalasinas/química , Citocalasinas/isolamento & purificação , Conformação MolecularRESUMO
Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.
Assuntos
Aspergillus fumigatus , Gliotoxina , Piperazinas , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Piperazinas/química , Piperazinas/metabolismo , Gliotoxina/biossíntese , Gliotoxina/química , Estrutura Molecular , Chaetomium/metabolismo , Chaetomium/química , Sistemas CRISPR-CasRESUMO
By co-culturing two endophytic fungi (Chaetomium virescens and Xylaria grammica) collected from the medicinal and edible plant Smilax glabra Roxb. and analyzing them with MolNetEnhancer module on GNPS platform, seven undescribed chromone-derived polyketides (chaetoxylariones A-G), including three pairs of enantiomer ones (2a/2b, 4a/4b and 6a/6b) and four optical pure ones (1, 3, 5 and 7), as well as five known structural analogues (8-12), were obtained. The structures of these new compounds were characterized by NMR spectroscopy, single-crystal X-ray diffraction, 13C NMR calculation and DP4+ probability analyses, as well as the comparison of the experimental electronic circular dichroism (ECD) data. Structurally, compound 1 featured an unprecedented chromone-derived sulfonamide tailored by two isoleucine-derived δ-hydroxy-3-methylpentenoic acids via the acylamide and NO bonds, respectively; compound 2 represented the first example of enantiomeric chromone derivative bearing a unique spiro-[3.3]alkane ring system; compound 3 featured a decane alkyl side chain that formed an undescribed five-membered lactone ring between C-7' and C-10'; compound 4 contained an unexpected highly oxidized five-membered carbocyclic system featuring rare adjacent keto groups; compound 7 featured a rare methylsulfonyl moiety. In addition, compound 10 showed a significant inhibition towards SW620/AD300 cells with an IC50 value of PTX significantly decreased from 4.09 µM to 120 nM, and a further study uncovered that compound 10 could obviously reverse the MDR of SW620/AD300 cells.
Assuntos
Antineoplásicos , Chaetomium , Cromonas , Ensaios de Seleção de Medicamentos Antitumorais , Policetídeos , Xylariales , Cromonas/química , Cromonas/farmacologia , Cromonas/isolamento & purificação , Policetídeos/química , Policetídeos/farmacologia , Policetídeos/isolamento & purificação , Estrutura Molecular , Xylariales/química , Chaetomium/química , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral , Técnicas de Cocultura , Proliferação de Células/efeitos dos fármacosRESUMO
The disintegration of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies in vacuoles is essential to the Cvt pathway and macroautophagy in yeast. Atg15 is a vacuolar lipase required for the degradation of both Cvt and autophagic bodies. However, the molecular mechanism of their degradation by Atg15 remains poorly understood. In a recent study, we showed that recombinant Chaetomium thermophilum Atg15 (CtAtg15) possesses phospholipase activity, and that this activity is significantly elevated by proteolytic cleavage at a site away from the active center. The proteolytic cleavage of CtAtg15 causes a conformational change around the active center, resulting in the active open state. Interestingly, activated CtAtg15 can degrade not only Cvt and autophagic bodies but also organelle membranes. On the basis of these results, we propose an activation mechanism by which Atg15, as an "organellase," functions only in vacuoles.
Assuntos
Autofagia , Vacúolos , Vacúolos/metabolismo , Autofagia/fisiologia , Fosfolipases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Chaetomium/metabolismo , Membranas Intracelulares/metabolismoRESUMO
Seven new polyketides including three chromone derivatives (1-3) and four linear ones incorporating a tetrahydrofuran ring (4-7), along with three known compounds (8-10), were obtained from the fermentation of an endophytic fungus (Chaetomium sp. UJN-EF006) isolated from the leaves of Vaccinium bracteatum. The structures of these fungal metabolites have been elucidated by spectroscopic means including MS, NMR and electronic circular dichroism. A preliminary anti-inflammatory screening with the lipopolysaccharide (LPS) induced RAW264.7â cell model revealed moderate NO production inhibitory activity for compounds 1 and 4. In addition, the expression of three LPS-induced inflammatory factors IL-6, iNOS and COX-2 was also blocked by 1 and 4.
Assuntos
Chaetomium , Policetídeos , Vaccinium myrtillus , Chaetomium/química , Policetídeos/química , Lipopolissacarídeos/farmacologia , Estrutura MolecularRESUMO
Cocultivation of the high cytochalasan-producing fungi Aspergillus flavipes and Chaetomium globosum resulted in the isolation of 11 undescribed Chae-type cytochalasans. Their structures were determined by spectroscopic data and NMR data calculations. Asperchaetoglobin A (1) was the first Chae-type cytochalasan possessing an unprecedented nitrogen bridge between C-17 and C-20 to generate a surprising 5/6/12/5 multiple ring system; asperchaetoglobins B and C (2 and 3) displayed higher oxidation with an additional epoxide at the thirteen-member ring; asperchaetoglobin D (4) was the second Chae-type cytochalasin featuring a 5/6/12 tricyclic ring system. The cytotoxic activities against five human cancer cell lines and antibacterial activities against Staphylococcus aureus and Colon bacillus of selected compounds were evaluated in vitro.
Assuntos
Aspergillus , Chaetomium , Citocalasinas , Humanos , Estrutura Molecular , Técnicas de Cocultura , Citocalasinas/químicaRESUMO
Microorganisms are a unique part of our ecosystem because they affect the survival of living organisms. Although pathogenic microorganisms could be detrimental to the plants, animals, and humans, beneficial microbes have provided significant improvement in the growth and development of living organisms. In this study, the fungus Chaetomium globosium was isolated from the medicinal tree Gingko biloba, and then incorporated into a polymerization system to fabricate chitosan/acrylamide/gold (CS/Am/Au) nanocomposite hydrogels. The as-prepared hydrogel displayed increased mechanical strength due to the reinforcement of Au (gold) nanocomposites within the hydrogel matrix. Also, the equilibrium pH responsive swelling rates of the hydrogels gradually increased as the pH increases due to partial acid and basic hydrolysis occurring in the hydrogel as well as formation of hydrogen bond. In addition, the hydrogel demonstrated promising antibacterial activities against selected gram-positive (Staphylococcus epidermidis and Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) bacterial strains with an average MIC90 of 0.125 mg/mL at a dosage of 1.0 mg/L. The obtained results are quite promising towards resolving several health challenges and advancing the pharmaceutical industries.
Assuntos
Chaetomium , Quitosana , Nanocompostos , Animais , Humanos , Quitosana/química , Nanogéis , Ginkgo biloba , Ouro/farmacologia , Ecossistema , Antibacterianos/farmacologia , Antibacterianos/química , Hidrogéis/farmacologia , Hidrogéis/química , Nanocompostos/química , AcrilamidasRESUMO
Endophytic fungi live symbiotically inside plants and are hidden source of natural bioactive molecules. The present study was carried out to investigate the phytochemical analysis and antioxidant activity of endophytic fungi isolated from the ethnomedicinal plant Dillenia indica L. The ethyl acetate crude extracts of the endophytic fungal strains were preliminarily evaluated for their phytochemical analysis, and the results showed the presence of alkaloids, flavonoids, phenolics, terpene, and saponins. The crude extracts of more than 60% of the isolates showed 50-90% antioxidant activity by DPPH and H2O2 assay. The inhibition percentage of ethyl acetate extracts ranges from 34.05 to 91.5%, whereas IC50 values vary from 72.2 to 691.14%. Among all the strains, Fomitopsis meliae crude extract showed a maximum inhibition percentage, i.e., 91.5%, with an IC50 value of 88.27 µg/mL. Chaetomium globosum showed significant activity having an inhibition percentage of 89.88% and an IC50 value of 74.44 µg/mL. The total phenolic and flavonoid content in the crude extract of Chaetomium globosum was 37.4 mg gallic acid equivalent (GAE)/g DW and 31.0 mg quercetin equivalent (GAE)/g DW. GC-MS analysis of crude extract of C. globosum revealed different compounds, such as squalene; butanoic acid, 2-methyl-; hexadecanoic acid; 2-propanone, 1-phenyl-; 5-oxo-pyrrolidine-2-carboxylic acid methyl ester; 9,12-octadecadienoic acid (z)- etc. Many of these belong to phenolics, which are natural antioxidant compounds. The findings suggested that endophytic fungi associated with Dillenia indica L. can be a potential source of novel antioxidant compounds.
Assuntos
Acetatos , Chaetomium , Dilleniaceae , Antioxidantes/química , Extratos Vegetais/química , Peróxido de Hidrogênio , Compostos Fitoquímicos/farmacologia , Flavonoides/química , Fenóis/química , Fungos , Ácido GálicoRESUMO
Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar/intralysosomal autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here, we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity. The activity of CtAtg15(73-475) was markedly elevated by limited digestion. We inserted the human rhinovirus 3C protease recognition sequence and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). Our molecular dynamics simulation suggested that the cleavage facilitated conformational change around the active center of CtAtg15, resulting in an exposed state. We confirmed that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC of S. cerevisiae were disintegrated by CtAtg15. This study suggests Atg15 plays a role in disrupting any organelle membranes delivered to vacuoles by autophagy.
Assuntos
Proteínas Fúngicas , Membranas Intracelulares , Fosfolipases , Chaetomium/enzimologia , Chaetomium/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfolipases/química , Fosfolipases/genética , Fosfolipases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Domínios Proteicos , Simulação de Dinâmica Molecular , Mitocôndrias/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estrutura Terciária de Proteína , Modelos Moleculares , Ativação EnzimáticaRESUMO
The antimicrobial resistance of pathogenic microorganisms against commercial drugs has become a major problem worldwide. This study is the first of its kind to be carried out in Egypt to produce antimicrobial pharmaceuticals from isolated native taxa of the fungal Chaetomium, followed by a chemical investigation of the existing bioactive metabolites. Here, of the 155 clinical specimens in total, 100 pathogenic microbial isolates were found to be multi-drug resistant (MDR) bacteria. The Chaetomium isolates were recovered from different soil samples, and wild host plants collected from Egypt showed strong inhibitory activity against MDR isolates. Chaetomium isolates displayed broad-spectrum antimicrobial activity against C. albicans, Gram-positive, and Gram-negative bacteria, with inhibition zones of 11.3 to 25.6 mm, 10.4 to 26.0 mm, and 10.5 to 26.5 mm, respectively. As a consecutive result, the minimum inhibitory concentration (MIC) values of Chaetomium isolates ranged from 3.9 to 62.5 µg/mL. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) analysis was performed for selected Chaetomium isolates with the most promising antimicrobial potential against MDR bacteria. The LC-MS/MS analysis of Chaetomium species isolated from cultivated soil at Assuit Governate, Upper Egypt (3), and the host plant Zygophyllum album grown in Wadi El-Arbaein, Saint Katherine, South Sinai (5), revealed the presence of alkaloids as the predominant bioactive metabolites. Most detected bioactive metabolites previously displayed antimicrobial activity, confirming the antibacterial potential of selected isolates. Therefore, the Chaetomium isolates recovered from harsh habitats in Egypt are rich sources of antimicrobial metabolites, which will be a possible solution to the multi-drug resistant bacteria tragedy.
Assuntos
Anti-Infecciosos , Chaetomium , Chaetomium/química , Cromatografia Líquida , Espectrometria de Massas em Tandem , Anti-Infecciosos/metabolismo , Antibacterianos/química , Bactérias/metabolismo , Testes de Sensibilidade Microbiana , SoloRESUMO
The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.
Assuntos
Chaetomium , Açúcares , Açúcares/metabolismo , Xilose/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Saccharomyces cerevisiae/genética , Glucose/metabolismoRESUMO
Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.