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1.
Molecules ; 26(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926006

RESUMO

This study was performed to clarify the inhibitory effects of cycloheterophyllin on melanin synthesis. In order to elucidate the inhibitory effects of cycloheterophyllin on the B16F10 cell line, cell viability, messenger ribonucleic acid (mRNA) expressions, tyrosinase activity assay, and melanin production assay were measured. The effects of cycloheterophyllin on tyrosinase-related protein 1 (TYRP1)/TYRP2/tyrosinase (TYR)/microphthalmia-associated transcription factor (MITF) mRNA expressions and melanin content were determined. Quantitative real-time RT-PCR showed that cycloheterophyllin decreased the mRNA expression level of TYRP1/TYRP2/TYR/MITF genes and melanin production contents than α-MSH-treated B16F10 cells. The tyrosinase activity assay revealed that cycloheterophyllin decreased the melanin production in the B16F10 cells. These data show that cycloheterophyllin increases the whitening effects in the B16F10 cells; thus, cycloheterophyllin is a potent ingredient for skin whitening. Thus, further research on the mechanism of action of cycloheterophyllin for the development of functional materials should be investigated.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Flavonoides/farmacologia , Melaninas/biossíntese , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Relação Dose-Resposta a Droga , Flavonoides/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melaninas/genética , Melanoma Experimental , Camundongos
2.
Molecules ; 26(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925414

RESUMO

Natural products (NPs) are evolutionarily optimized as drug-like molecules and remain the most consistently successful source of drugs and drug leads. They offer major opportunities for finding novel lead structures that are active against a broad spectrum of assay targets, particularly those from secondary metabolites of microbial origin. Due to traditional discovery approaches' limitations relying on untargeted screening methods, there is a growing trend to employ unconventional secondary metabolomics techniques. Aided by the more in-depth understanding of different biosynthetic pathways and the technological advancement in analytical instrumentation, the development of new methodologies provides an alternative that can accelerate discoveries of new lead-structures of natural origin. This present mini-review briefly discusses selected examples regarding advancements in bioinformatics and genomics (focusing on genome mining and metagenomics approaches), as well as bioanalytics (mass-spectrometry) towards the microbial NPs-based drug discovery and development. The selected recent discoveries from 2015 to 2020 are featured herein.


Assuntos
Bactérias/genética , Produtos Biológicos/uso terapêutico , Metabolômica , Metagenômica , Vias Biossintéticas/efeitos dos fármacos , Biologia Computacional , Descoberta de Drogas , Genoma Bacteriano/genética , Humanos
3.
Int J Food Microbiol ; 348: 109207, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-33930837

RESUMO

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 23-40% and the production of aflatoxins by 93.9-99.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Glicolipídeos/farmacologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Vias Biossintéticas/efeitos dos fármacos , Produtos Agrícolas , Genes Fúngicos/genética , Humanos , Hifas/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Pseudomonas aeruginosa/metabolismo
4.
Molecules ; 26(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670642

RESUMO

We investigated the relationship between the blue-light photoreceptor cryptochrome (CRY) and melatonin biosynthesis by generating RNA interference (RNAi) transgenic rice plants that suppress the cryptochrome 1b gene (CRY1b). The resulting CRY1b RNAi rice lines expressed less CRY1b mRNA, but not CRY1a or CRY2 mRNA, suggesting that the suppression is specific to CRY1b. The growth of CRY1b RNAi rice seedlings was enhanced under blue light compared to wild-type growth, providing phenotypic evidence for impaired CRY function. When these CRY1b RNAi rice plants were challenged with cadmium to induce melatonin, wild-type plants produced 100 ng/g fresh weight (FW) melatonin, whereas CRY1b RNAi lines produced 60 ng/g FW melatonin on average, indicating that melatonin biosynthesis requires the CRY photoreceptor. Due to possible feedback regulation, the expression of melatonin biosynthesis genes such as T5H, SNAT1, SNAT2, and COMT was elevated in the CRY1b RNAi lines compared to the wild-type plants. In addition, laminar angles decreased in the CRY1b RNAi lines via the suppression of brassinosteroid (BR) biosynthesis genes such as DWARF. The main cause of the BR decrease in the CRY1b RNAi lines seems to be the suppression of CRY rather than decreased melatonin because the melatonin decrease suppressed DWARF4 rather than DWARF.


Assuntos
Vias Biossintéticas/genética , Brassinosteroides/biossíntese , Criptocromos/genética , Genes de Plantas , Melatonina/biossíntese , Oryza/genética , Tolerância ao Sal/genética , Vias Biossintéticas/efeitos dos fármacos , Criptocromos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/efeitos dos fármacos , Fenótipo , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tolerância ao Sal/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/genética , Serotonina/metabolismo , Cloreto de Sódio/farmacologia
5.
Mol Biol Rep ; 48(2): 1707-1715, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33611780

RESUMO

Saffron stigmas are widely used as food additives and as traditional medicine in Iran and many other countries. The unique taste, flavor and pharmaceutical properties of saffron stigmas are due to the presence of three apocarotenoids secondary metabolites crocin, picrocrocin and safranal. There is limited knowledge about the effect of environmental stresses on the metabolism of apocarotenoids in saffron. We analyzed the content of crocin and picrocrocin and the expression of key genes of apocarotenoid biosynthesis pathways (CsCCD2, CsCCD4, CsUGT2, CsCHY-ß and CsLCYB) in saffron plants exposed to moderate (90 mM) and high (150 mM) salt (NaCl) concentrations. Measuring ion concentrations in leaves showed an increased accumulation of Na+ and decreased uptake of K+ in salt treated compared to control plants indicating an effective salt stress. HPLC analysis of apocarotenoids revealed that crocin production was significantly halted (P < 0.05) with increasing salt concentration while picrocrocin level did not change with moderate salt but significantly dropped by high salt concentration. Real-time PCR analysis revealed a progressive decrease in transcript levels of CsUGT2 and CsLCYB genes with increasing salt concentration (P < 0.05). The expression of CsCCD2 and CsCHY-ß tolerated moderate salt concentration but significantly downregulated with high salt concentration. CsCCD4 however responded differently to salt concentration being decreased with moderate salt but increased at higher salt concentration. Our result suggested that salt stress had an adverse effect on the production of saffron apocarotenoids and it is likely influencing the quality of saffron stigma produced.


Assuntos
Carotenoides/metabolismo , Crocus/química , Crocus/metabolismo , Cicloexenos/metabolismo , Estresse Salino/genética , Terpenos/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Cromatografia Líquida de Alta Pressão , Crocus/efeitos dos fármacos , Crocus/genética , Regulação da Expressão Gênica de Plantas/genética , Glucosídeos/metabolismo , Folhas de Planta/química , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Potássio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sódio/metabolismo , Cloreto de Sódio/toxicidade
6.
Cell ; 184(1): 106-119.e14, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33333024

RESUMO

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.


Assuntos
COVID-19/genética , Infecções por Coronavirus/genética , Coronavirus/fisiologia , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Células A549 , Animais , Vias Biossintéticas/efeitos dos fármacos , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Colesterol/biossíntese , Colesterol/metabolismo , Análise por Conglomerados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resfriado Comum/genética , Resfriado Comum/virologia , Coronavirus/classificação , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos , Fosfatidilinositóis/biossíntese , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral
7.
J Med Chem ; 64(1): 797-811, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33369426

RESUMO

In the kynurenine pathway for tryptophan degradation, an unstable metabolic intermediate, α-amino-ß-carboxymuconate-ε-semialdehyde (ACMS), can nonenzymatically cyclize to form quinolinic acid, the precursor for de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+). In a competing reaction, ACMS is decarboxylated by ACMS decarboxylase (ACMSD) for further metabolism and energy production. Therefore, the inhibition of ACMSD increases NAD+ levels. In this study, an Food and Drug Administration (FDA)-approved drug, diflunisal, was found to competitively inhibit ACMSD. The complex structure of ACMSD with diflunisal revealed a previously unknown ligand-binding mode and was consistent with the results of inhibition assays, as well as a structure-activity relationship (SAR) study. Moreover, two synthesized diflunisal derivatives showed half-maximal inhibitory concentration (IC50) values 1 order of magnitude better than diflunisal at 1.32 ± 0.07 µM (22) and 3.10 ± 0.11 µM (20), respectively. The results suggest that diflunisal derivatives have the potential to modulate NAD+ levels. The ligand-binding mode revealed here provides a new direction for developing inhibitors of ACMSD.


Assuntos
Carboxiliases/metabolismo , Diflunisal/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Vias Biossintéticas/efeitos dos fármacos , Carboxiliases/antagonistas & inibidores , Domínio Catalítico , Cristalografia por Raios X , Diflunisal/análogos & derivados , Diflunisal/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Cinurenina/metabolismo , Simulação de Acoplamento Molecular , NAD/metabolismo , Pseudomonas fluorescens/enzimologia , Relação Estrutura-Atividade , Triptofano/metabolismo
8.
Nat Chem Biol ; 16(8): 912-919, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32541965

RESUMO

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.


Assuntos
Vias Biossintéticas/fisiologia , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Vias Biossintéticas/efeitos dos fármacos , Biotecnologia/métodos , Sistema Livre de Células/metabolismo , Redes e Vias Metabólicas/fisiologia , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia
9.
Anticancer Res ; 40(5): 2675-2685, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366412

RESUMO

BACKGROUND/AIM: To evaluate the anti-cancer mechanism of N-Farnesyl-norcantharimide (NC15). MATERIALS AND METHODS: The viability of NC15-treated human leukemic Jurkat T (JKT) cells was assessed using the Kit-8 cell counting method. Flow cytometry analysis, human apoptosis antibody array assay, and whole genome sequencing were adopted to investigate the mechanism underlying the anti-cancer activity of NC15 in JKT cells. RESULTS: The growth inhibition rates of NC15 in JKT cells were about 80% and 95% after treatment with 8 µmol/l NC15 for 24 and 48 h, respectively. The percentages of NC15-treated JKT cells in the sub-G1 phase at 24 and 48 h were 22.0% and 34.3%, respectively, in contrast to the 1.5% in the control. Next-generation sequencing showed that many tumor suppressor genes (TSG) were up-regulated, while many genes associated with steroid biosynthesis, metabolic pathways, and fatty acid metabolism were down-regulated. CONCLUSION: NC15 can reduce the cell viability and increase the percentage of JKT cells in the sub-G1 phase by up-regulating TSG and related genes, and down-regulating the genes for steroid biosynthesis, metabolic pathways and fatty acid metabolism, instead of through apoptosis.


Assuntos
Cantaridina/análogos & derivados , Regulação para Baixo/efeitos dos fármacos , Ácidos Graxos/metabolismo , Genes Supressores de Tumor , Redes e Vias Metabólicas/genética , Esteroides/biossíntese , Linfócitos T/citologia , Regulação para Cima/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Cantaridina/química , Cantaridina/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Regulação para Baixo/genética , Humanos , Células Jurkat , Redes e Vias Metabólicas/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Regulação para Cima/genética
10.
Proc Natl Acad Sci U S A ; 117(18): 9964-9972, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312817

RESUMO

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, served as a potent Nrf2 inhibitor, which exhibited selective cytotoxicity to patient-derived IDH1-mutated glioma cells in vitro and in vivo. Mechanistically, triptolide compromised the expression of GCLC, GCLM, and SLC7A11, which disrupted glutathione metabolism and established synthetic lethality with reactive oxygen species derived from IDH1 mutant neomorphic activity. Our findings highlight triptolide as a valuable therapeutic approach for IDH1-mutated malignancies by targeting the Nrf2-driven glutathione synthesis pathway.


Assuntos
Diterpenos/farmacologia , Glioma/tratamento farmacológico , Isocitrato Desidrogenase/genética , Fator 2 Relacionado a NF-E2/genética , Fenantrenos/farmacologia , Sistema y+ de Transporte de Aminoácidos/genética , Animais , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular Tumoral , Compostos de Epóxi/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/patologia , Glutamato-Cisteína Ligase/genética , Glutationa/metabolismo , Humanos , Camundongos , Mutação/genética , Espécies Reativas de Oxigênio/metabolismo , Mutações Sintéticas Letais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Commun ; 11(1): 1830, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286350

RESUMO

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.


Assuntos
Diterpenos/farmacologia , Fungos/química , Naftalenos/farmacologia , Pironas/farmacologia , Biologia Sintética , Peptídeos beta-Amiloides/metabolismo , Animais , Fármacos Anti-HIV/farmacologia , Aspergillus/química , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Proliferação de Células/efeitos dos fármacos , Diterpenos/química , Drosophila/efeitos dos fármacos , Fungos/genética , Genoma Fúngico , HIV-1/efeitos dos fármacos , Humanos , Células MCF-7 , Naftalenos/química , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Agregados Proteicos , Pironas/química , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Estereoisomerismo
12.
Nat Commun ; 11(1): 1455, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193379

RESUMO

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Daptomicina/farmacologia , Lipídeos de Membrana/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Humanos , Membranas Artificiais , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Fosfatidilgliceróis/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
13.
Sci Rep ; 10(1): 5563, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221330

RESUMO

The world is in the midst of an antimicrobial resistance crisis, driving a need to discover novel antibiotic substances. Using chemical cues as inducers to unveil a microorganism's full metabolic potential is considered a successful strategy. To this end, we investigated an inducible antagonistic behavior in multiple isolates of the order Bacillales, where large inhibition zones were produced against Ralstonia solanacearum only when grown in the presence of the indicator triphenyl tetrazolium chloride (TTC). This bioactivity was produced in a TTC-dose dependent manner. Escherichia coli and Staphylococcus sp. isolates were also inhibited by Bacillus sp. strains in TTC presence, to a lesser extent. Knockout mutants and transcriptomic analysis of B. subtilis NCIB 3610 cells revealed that genes from the L-histidine biosynthetic pathway, the purine, pyrimidine de novo synthesis and salvage and interconversion routes, were significantly upregulated. Chemical space studied through metabolomic analysis, showed increased presence of nitrogenous compounds in extracts from induced bacteria. The metabolites orotic acid and L-phenylalaninamide were tested against R. solanacearum, E. coli, Staphylococcus sp. and B. subtilis, and exhibited activity against pathogens only in the presence of TTC, suggesting a biotransformation of nitrogenous compounds in Bacillus sp. cells as the plausible cause of the inducible antagonistic behavior.


Assuntos
Antibacterianos/farmacologia , Bacillales/metabolismo , Bactérias/efeitos dos fármacos , Sais de Tetrazólio/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Testes de Sensibilidade Microbiana
14.
mBio ; 11(1)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098820

RESUMO

Low doses of antibiotics can trigger secondary metabolite biosynthesis in bacteria, but the underlying mechanisms are generally unknown. We sought to better understand this phenomenon by studying how the antibiotic trimethoprim activates the synthesis of the virulence factor malleilactone in Burkholderia thailandensis Using transcriptomics, quantitative multiplexed proteomics, and primary metabolomics, we systematically mapped the changes induced by trimethoprim. Surprisingly, even subinhibitory doses of the antibiotic resulted in broad transcriptional and translational alterations, with ∼8.5% of the transcriptome and ∼5% of the proteome up- or downregulated >4-fold. Follow-up studies with genetic-biochemical experiments showed that the induction of malleilactone synthesis can be sufficiently explained by the accumulation of methionine biosynthetic precursors, notably homoserine, as a result of inhibition of the folate pathway. Homoserine activated the malleilactone gene cluster via the transcriptional regulator MalR and gave rise to a secondary metabolome which was very similar to that generated by trimethoprim. Our work highlights the expansive changes that low-dose trimethoprim induces on bacterial physiology and provides insights into its stimulatory effect on secondary metabolism.IMPORTANCE The discovery of antibiotics ranks among the most significant accomplishments of the last century. Although the targets of nearly all clinical antibiotics are known, our understanding regarding their natural functions and the effects of subinhibitory concentrations is in its infancy. Stimulatory rather than inhibitory functions have been attributed to low-dose antibiotics. Among these, we previously found that antibiotics activate silent biosynthetic genes and thereby enhance the metabolic output of bacteria. The regulatory circuits underlying this phenomenon are unknown. We take a first step toward elucidating these circuits and show that low doses of trimethoprim (Tmp) have cell-wide effects on the saprophyte Burkholderia thailandensis Most importantly, inhibition of one-carbon metabolic processes by Tmp leads to an accumulation of homoserine, which induces the production of an otherwise silent cytotoxin via a LuxR-type transcriptional regulator. These results provide a starting point for uncovering the molecular basis of the hormetic effects of antibiotics.


Assuntos
Antibacterianos/farmacologia , Burkholderia/efeitos dos fármacos , Burkholderia/metabolismo , Metabolismo Secundário/efeitos dos fármacos , Proteínas de Bactérias , Produtos Biológicos/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Burkholderia/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homosserina/metabolismo , Lactonas/química , Lactonas/metabolismo , Família Multigênica , Metabolismo Secundário/genética , Trimetoprima/farmacologia , Fatores de Virulência/metabolismo
15.
Sci Rep ; 10(1): 2918, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075995

RESUMO

Microbial flocculant (MBF), an environmentally friendly water treatment agent, can be widely used in various water treatments. However, its use is limited by low yield and high cost. This problem can be solved by clarifying its biosynthesis mechanism and regulating it. Paenibacillus shenyangensis A9, a flocculant-producing bacterium, was used to produce polysaccharide-type MBFA9 by regulating the nitrogen source (nitrogen adequacy/nitrogen deficiency). In this study, RNA-Seq high-throughput sequencing technology and bioinformatic approaches were used to investigate the fermentation and biosynthesis of polysaccharide-type MBFA9 by regulating the nitrogen source (high nitrogen/low nitrogen) in the flocculant-producing bacteria Paenibacillus shenyangensis A9. Differentially expressed genes, functional clustering, and functional annotation of key genes were assessed. Then the MBFA9 biosynthesis and metabolic pathway were reconstructed. Our results showed that when cultured under different nitrogen conditions, bacterial strain A9 had a greater ability to synthesize polysaccharide-type MBFA9 under low nitrogen compared to high nitrogen conditions, with the yield of MBFA9 reaching 4.2 g/L at 36 h of cultivation. The quality of transcriptome sequencing data was reliable, with a matching rate of 85.38% and 85.48% when L36/H36 was mapped to the reference genome. The total expressed genes detected were 4719 and 4730, with 265 differentially expressed genes. The differentially expressed genes were classified into 3 categories: molecular function (MF), cell component (CC), and biological process (BP), and can be further divided into 22 subcategories. There were 192 upregulated genes and 73 downregulated genes, with upregulation being predominant under low nitrogen. UDP-Gal, UDP-Glc, UDP-GlcA, and UDP-GlcNAc, which are in the polysaccharide metabolic pathway, could all be used as precursors for MBFA9 biosynthesis, and murA, wecB, pgm, galU/galF, fcl, gmd, and glgC were the main functional genes capable of affecting the growth of bacteria and the biosynthesis of MBF. Results from this study provide evidence that high-level expression of key genes in MBFA9 biosynthesis, regulation, and control can achieve MBFA9 directional synthesis for large-scale applications.


Assuntos
Perfilação da Expressão Gênica , Nitrogênio/farmacologia , Paenibacillus/química , Paenibacillus/genética , Polissacarídeos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Carbono/farmacologia , Floculação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Genes Bacterianos , Paenibacillus/efeitos dos fármacos , Paenibacillus/crescimento & desenvolvimento
16.
Int J Mol Sci ; 21(4)2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085660

RESUMO

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.


Assuntos
Vias Biossintéticas , Reparo do DNA , Proteínas Fúngicas/metabolismo , Pirimidinas/biossíntese , Verticillium/metabolismo , Verticillium/patogenicidade , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Reparo do DNA/efeitos dos fármacos , Fluorescência , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Tiamina/farmacologia , Tabaco/microbiologia , Raios Ultravioleta , Verticillium/efeitos dos fármacos , Verticillium/crescimento & desenvolvimento , Virulência/efeitos dos fármacos , Virulência/genética , Virulência/efeitos da radiação
17.
PLoS One ; 15(2): e0229490, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32107496

RESUMO

Application of plant growth regulators has become one of the most important means of improving yield and quality of medicinal plants. To understand the molecular basis of phytohormone-regulated oleanolic acid metabolism, RNA-seq was used to analyze global gene expression in Achyranthes bidentata treated with 2.0 mg/L 1-naphthaleneacetic acid (NAA) and 1.0 mg/L 6-benzyladenine (6-BA). Compared with untreated controls, the expression levels of 20,896 genes were significantly altered with phytohormone treatment. We found that 13071 (62.5%) unigenes were up-regulated, and a lot of differentially expressed genes involved in hormone or terpenoid biosynthesis, or transcription factors were significantly up-regulated. These results suggest that oleanolic acid biosynthesis induced by NAA and 6-BA occurs due to the expression of key genes involved in jasmonic acid signal transduction. This study is the first to analyze the production and hormonal regulation of medicinal A. bidentata metabolites at the molecular level. The results herein contribute to a better understanding of the regulation of oleanane-type triterpenoid saponins accumulation and define strategies to improve the yield of these useful metabolites.


Assuntos
Achyranthes/efeitos dos fármacos , Achyranthes/metabolismo , Compostos de Benzil/farmacologia , Ciclopentanos/metabolismo , Ácidos Naftalenoacéticos/farmacologia , Ácido Oleanólico/biossíntese , Oxilipinas/metabolismo , Purinas/farmacologia , Achyranthes/crescimento & desenvolvimento , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Medicina Tradicional Chinesa , Filogenia , Reguladores de Crescimento de Plantas/metabolismo , Plantas Medicinais/efeitos dos fármacos , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , RNA-Seq , Saponinas/metabolismo
18.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952262

RESUMO

Hyperlipidemia is a chronic disorder that plays an important role in the development of cardiovascular diseases, type II diabetes, atherosclerosis, hypertension, and non-alcoholic fatty liver disease. Hyperlipidemias have created a worldwide health crisis and impose a substantial burden not only on personal health but also on societies and economies. Transcription factors in the sterol regulatory element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. SREBPs regulate lipid homeostasis by controlling the expression of a range of enzymes required for the synthesis of endogenous cholesterol, fatty acids, triacylglycerol, and phospholipids. Thereby, SREBPs have been considered as targets for the treatment of metabolic diseases. The aim of this study was to investigate the beneficial functions and the possible underlying molecular mechanisms of SREBP decoy ODN, which is a novel inhibitor of SREBPs, in high-fat diet (HFD)-fed hyperlipidemic mice. Our studies using HFD-induced hyperlipidemia animal model revealed that SREBB decoy ODN inhibited the increased expression of fatty acid synthetic pathway, such as SREBP-1c, FAS, SCD-1, ACC1, and HMGCR. In addition, SREBP decoy ODN decreased pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-8, and IL-6 expression. These results suggest that SREBP decoy ODN exerts its anti-hyperlipidemia effects in HFD-induced hyperlipidemia mice by regulating their lipid metabolism and inhibiting lipogenesis through inactivation of the SREPB pathway.


Assuntos
Modelos Animais de Doenças , Hiperlipidemias/prevenção & controle , Oligodesoxirribonucleotídeos/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperlipidemias/etiologia , Hiperlipidemias/genética , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Masculino , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
19.
Molecules ; 25(2)2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936318

RESUMO

Whole-genome sequence data of the genus Streptomyces have shown a far greater chemical diversity of metabolites than what have been discovered under typical laboratory fermentation conditions. In our previous natural product discovery efforts on Streptomyces sp. MA37, a bacterium isolated from the rhizosphere soil sample in Legon, Ghana, we discovered a handful of specialised metabolites from this talented strain. However, analysis of the draft genome of MA37 suggested that most of the encoded biosynthetic gene clusters (BGCs) remained cryptic or silent, and only a small fraction of BGCs for the production of specialised metabolites were expressed when cultured in our laboratory conditions. In order to induce the expression of the seemingly silent BGCs, we have carried out a co-culture experiment by growing the MA37 strain with the Gram-negative bacterium Pseudomonas sp. in a co-culture chamber that allows co-fermentation of two microorganisms with no direct contact but allows exchange of nutrients, metabolites, and other chemical cues. This co-culture approach led to the upregulation of several metabolites that were not previously observed in the monocultures of each strain. Moreover, the co-culture induced the expression of the cryptic indole alkaloid BGC in MA37 and led to the characterization of the known indolocarbazole alkaloid, BE-13793C 1. Neither bacterium produced compound 1 when cultured alone. The structure of 1 was elucidated by Nuclear Magnetic Resonance (NMR), mass spectrometry analyses and comparison of experimental with literature data. A putative biosynthetic pathway of 1 was proposed. Furthermore, BE-13793C 1 showed strong anti-proliferative activity against HT-29 (ATCC HTB-38) cells but no toxic effect to normal lung (ATCC CCL-171) cells. To the best of our knowledge, this is the first report for the activity of 1 against HT-29. No significant antimicrobial and anti-trypanosomal activities for 1 were observed. This research provides a solid foundation for the fact that a co-culture approach paves the way for increasing the chemical diversity of strain MA37. Further characterization of other upregulated metabolites in this strain is currently ongoing in our laboratory.


Assuntos
Vias Biossintéticas , Técnicas de Cocultura/métodos , Alcaloides Indólicos/metabolismo , Metaboloma , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Bioensaio , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Células HT29 , Humanos , Alcaloides Indólicos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica , Espectroscopia de Prótons por Ressonância Magnética , Pseudomonas/efeitos dos fármacos , Pseudomonas/metabolismo , Trypanosoma/efeitos dos fármacos
20.
Toxicol Appl Pharmacol ; 388: 114875, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31884101

RESUMO

Phthalates are used as solvents and plasticizers in a wide variety of consumer products. Most people are exposed to phthalates as parent compounds through ingestion, inhalation, and dermal contact. However, these parent compounds are quickly metabolized to more active compounds in several tissues. Although studies indicate that phthalate metabolites reach the ovary, little is known about whether they are ovarian toxicants. Thus, this study tested the hypothesis that phthalate metabolites influence the expression of genes involved in sex steroid synthesis, cell cycle regulation, cell death, oxidative stress, and key receptors, as well as production of sex steroid hormones by mouse antral follicles. The selected metabolite mixture consisted of 36.7% monoethyl phthalate (MEP), 19.4% mono(2-ethylhexyl) phthalate (MEHP), 15.3% monobutyl phthalate (MBP), 10.2% monoisobutyl phthalate (MiBP), 10.2% monoisononyl phthalate (MiNP), and 8.2% monobenzyl phthalate (MBzP). Antral follicles from adult CD-1 mice were cultured for 96 h with vehicle control (DMSO) or metabolite mixture (0.065-325 µg/mL). Growth of follicles in culture was monitored every 24 h. Total RNA was isolated after 24 and 96 h and used for gene expression analysis. Media were collected and subjected to hormone analysis. Exposure to the phthalate mixture inhibited follicle growth, decreased expression of steroidogenic enzymes, and altered the levels of sex steroids relative to control. The mixture, primarily at the two highest doses, also altered expression of cell cycle regulators, apoptotic factors, oxidative stress genes, and some receptors. Collectively, these data suggest that mixtures of phthalate metabolites can directly impact follicle health.


Assuntos
Exposição Ambiental/efeitos adversos , Hormônios Esteroides Gonadais/biossíntese , Folículo Ovariano/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Feminino , Perfilação da Expressão Gênica , Camundongos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ácidos Ftálicos/metabolismo , Técnicas de Cultura de Tecidos , Testes de Toxicidade Aguda/métodos
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