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1.
PLoS One ; 15(5): e0232927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396566

RESUMO

Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.


Assuntos
Proteínas de Bactérias/genética , Reatores Biológicos/microbiologia , Macrolídeos/metabolismo , Mutação , Streptomyces/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Fermentação , Engenharia Metabólica , Metiltransferases/genética , Proteínas Ribossômicas/genética , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/metabolismo
2.
Nat Commun ; 11(1): 140, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919415

RESUMO

Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Macrolídeos/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Peptídeos/metabolismo , Sítios de Ligação/fisiologia , Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/fisiologia , Macrolídeos/farmacologia , Peptídeos/farmacologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína
3.
Nat Chem Biol ; 16(2): 206-213, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932720

RESUMO

Genetic screens in cultured human cells represent a powerful unbiased strategy to identify cellular pathways that determine drug efficacy, providing critical information for clinical development. We used insertional mutagenesis-based screens in haploid cells to identify genes required for the sensitivity to lasonolide A (LasA), a macrolide derived from a marine sponge that kills certain types of cancer cells at low nanomolar concentrations. Our screens converged on a single gene, LDAH, encoding a member of the metabolite serine hydrolase family that is localized on the surface of lipid droplets. Mechanistic studies revealed that LasA accumulates in lipid droplets, where it is cleaved into a toxic metabolite by LDAH. We suggest that selective partitioning of hydrophobic drugs into the oil phase of lipid droplets can influence their activation and eventual toxicity to cells.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Gotículas Lipídicas/metabolismo , Macrolídeos/farmacocinética , Macrolídeos/toxicidade , Proteínas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Haploidia , Humanos , Inativação Metabólica , Gotículas Lipídicas/efeitos dos fármacos , Macrolídeos/metabolismo , Proteínas/genética
4.
J Biosci Bioeng ; 129(5): 558-564, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31924510

RESUMO

ε-Poly-l-lysine (ε-PL) produced as a secondary metabolite of Streptomyces albulus has long been used as a natural food preservative in a number of countries, including Japan, the United States, South Korea, and China. To date, numerous studies employing classical biotechnological approaches have been carried out to improve its productivity. Here we report a modern and rational genetic approach to enhancing metabolic flux toward ε-PL biosynthesis. Based on in silico genome analyses, we revealed that S. albulus NBRC14147 produces five antifungal polyene antibiotics-tetramycin A and B, tetrin A and B, and a trace amount of nystatin A1-concomitantly with antimicrobial ε-PL. Targeted inactivation of the biosynthetic gene cluster for tetramycins and tetrins in a nystatin A1 production-deficient mutant completely abolished the production of polyene macrolides, which in turn led to an approximately 20% improvement in ε-PL production that closely correlated with the polyene defects. The biosynthetic flux for ε-PL was thus successfully enhanced by inactivation of the concomitant secondary metabolite biosynthetic pathways. Since this elimination of concomitantly produced metabolites also allows for simpler purification after fermentation production of ε-PL, the rational strain engineering strategy we show here will improve its industrial production.


Assuntos
Macrolídeos/metabolismo , Polienos/metabolismo , Polilisina/biossíntese , Streptomyces/metabolismo , Fermentação , Conservantes de Alimentos/metabolismo , Macrolídeos/química , Polienos/química , Streptomyces/química , Streptomyces/genética
5.
Nat Prod Res ; 34(5): 710-713, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30445822

RESUMO

The correlation between the allocation of trisoxazole macrolides in the capitums, appendages, and bases of the sponge Penares cf. nux and the surface-attached bacteria on the corresponding parts was examined. The kabiramide contents were highest in the capitums, followed by the appendages and bases. Conversely, direct counts of cultivable surface-attached bacteria showed that the bacteria aggregate more densely on the surfaces of the bases. This suggested the repelling effects of the kabiramides against the fouling bacteria, particularly on the capitums and appendages. Twenty-two bacterial strains were isolated and identified to 15 species; however, none has shown the potentials as a producer of any secondary metabolites in the sponge P. nux.


Assuntos
Antibacterianos/metabolismo , Macrolídeos/farmacologia , Poríferos/metabolismo , Animais , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Aderência Bacteriana/fisiologia , Macrolídeos/metabolismo , Oxazóis/metabolismo , Oxazóis/farmacologia , Poríferos/química
6.
J Microbiol Biotechnol ; 29(12): 1931-1937, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31693835

RESUMO

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.


Assuntos
Cromossomos Artificiais , Daptomicina/biossíntese , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , Daptomicina/farmacologia , Furanos/metabolismo , Genes Bacterianos , Vetores Genéticos , Lipídeos , Macrolídeos/metabolismo , Peptídeo Sintases , Policetídeos/metabolismo , Staphylococcus aureus/efeitos dos fármacos
7.
Environ Microbiol ; 21(12): 4755-4772, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31600864

RESUMO

Myxococcus xanthus kills susceptible bacteria using myxovirescin A (TA) during predation. However, whether prey cells in nature can escape M. xanthus by developing resistance to TA is unknown. We observed that many field-isolated Bacillus licheniformis strains could survive encounters with M. xanthus, which was correlated to their TA resistance. A TA glycoside was identified in the broth of predation-resistant B. licheniformis J32 co-cultured with M. xanthus, and a glycosyltransferase gene (yjiC) was up-regulated in J32 after the addition of TA. Hetero-expressed YjiC-modified TA to a TA glucoside (TA-Gluc) by conjugating a glucose moiety to the C-21 hydroxyl group, and the resulting compound was identical to the TA glycoside present in the co-culture broth. TA-Gluc exhibited diminished bactericidal activity due to its weaker binding with LspA, as suggested by in silico docking data. Heterologous expression of the yjiC gene conferred both TA and M. xanthus-predation resistance to the host Escherichia coli cells. Furthermore, under predatory pressure, B. licheniformis Y071 rapidly developed predation resistance by acquiring TA resistance through the overexpression of yjiC and lspA genes. These results suggest that M. xanthus predation resistance in B. licheniformis is due to the TA deactivation by glucosylation, which is induced in a predator-mediated manner.


Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Macrolídeos/metabolismo , Myxococcus xanthus/metabolismo , Bacillus licheniformis/metabolismo , Proteínas de Bactérias/genética , Glicosilação , Glicosiltransferases/genética , Macrolídeos/química , Myxococcus xanthus/química , Myxococcus xanthus/genética
8.
J Agric Food Chem ; 67(34): 9667-9682, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31415166

RESUMO

This study assessed the feasibility of an NMR metabolomics approach coupled to multivariate data analysis to monitor the naturally present or stresses-elicited metabolites from a long-term (>170 days) culture of the dinoflagellate marine microalgae Amphidinium carterae grown in a fiberglass paddlewheel-driven raceway photobioreactor. Metabolic contents, in particular, in two members of the amphidinol family, amphidinol A and its 7-sulfate derivative amphidinol B (referred as APDs), and other compounds of interest (fatty acids, carotenoids, oxylipins, etc.) were evaluated by altering concentration levels of the f/2 medium nutrients and daily mean irradiance. Operating with a 24 h sinusoidal light cycle allowed a 3-fold increase in APD production, which was also detected by an increase in hemolytic activity of the methanolic extract of A. carterae biomass. The presence of APDs was consistent with the antitumoral activity measured in the methanolic extracts of the biomass. Increased daily irradiance was accompanied by a general decrease in pigments and an increase in SFAs (saturated fatty acids), MUFAs (monounsaturated fatty acids), and DHA (docosahexaenoic acid), while increased nutrient availability lead to an increase in sugar, amino acid, and PUFA ω-3 contents and pigments and a decrease in SFAs and MUFAs. NMR-based metabolomics is shown to be a fast and suitable method to accompany the production of APD and bioactive compounds without the need of tedious isolation methods and bioassays. The two APD compounds were chemically identified by spectroscopic NMR and spectrometric ESI-IT MS (electrospray ionization ion trap mass spectrometry) and ESI-TOF MS (ESI time-of-flight mass spectrometry) methods.


Assuntos
Dinoflagelados/metabolismo , Macrolídeos/química , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodos , Microalgas/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Dinoflagelados/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Macrolídeos/metabolismo , Microalgas/química , Análise Multivariada
9.
Microb Cell Fact ; 18(1): 137, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409353

RESUMO

Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria is associated with particular biosynthetic gene clusters (BGCs) in these microorganisms. The majority of these BGCs include polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) or a combination of both PKS and NRPS. Macrolides compounds contain a core macro-lactone ring (aglycone) decorated with diverse functional groups in their chemical structures. The aglycon is generated by megaenzyme polyketide synthases (PKSs) from diverse acyl-CoA as precursor substrates. Further, post-PKS enzymes are responsible for allocating the structural diversity and functional characteristics for their biological activities. Macrolides are biologically important for their uses in therapeutics as antibiotics, anti-tumor agents, immunosuppressants, anti-parasites and many more. Thus, precise genetic/metabolic engineering of actinobacteria along with the application of various chemical/biological approaches have made it plausible for production of macrolides in industrial scale or generation of their novel derivatives with more effective biological properties. In this review, we have discussed versatile approaches for generating a wide range of macrolide structures by engineering the PKS and post-PKS cascades at either enzyme or cellular level in actinobacteria species, either the native or heterologous producer strains.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/genética , Macrolídeos/metabolismo , Policetídeos/metabolismo , Produtos Biológicos/metabolismo , Engenharia Genética , Família Multigênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo
10.
Ecotoxicol Environ Saf ; 183: 109489, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31394379

RESUMO

Avermectins and moxidectin are antiparasitics widely used as active pharmaceutical ingredients in veterinary medicine, as well as in pesticide formulations for pest control in agriculture. Although the use of these compounds provides benefits to agribusiness, they can impact the environment, since a large part of these substances may reach the soil and water from the excreta of treated animals and following direct applications to crops. The present work had the objective of evaluating the dissipation behaviors of abamectin, doramectin, eprinomectin, ivermectin, and moxidectin in four native Brazilian soils of different textural classes (clay, sandy-clay, sandy, and sandy-clay-loam), following OECD Guideline 307. The studies were conducted in a climate chamber at 22 °C, 71% relative humidity, and protected from light. The dissipation studies were carried out with all drugs together, since no difference was verified when studies were done with each drug separately. The concentrations of the drugs in the soils were determined using an ultra-high performance liquid chromatograph coupled to a fluorescence detector or a tandem mass spectrometer. The dissipation half-life (DT50) values ranged from 9 to 16 days and the calculated GUS index values were in the range from -1.10 to 0.08, indicating low mobility of the drugs in the soils evaluated and low tendency for leaching. In addition, a field study was carried out to evaluate the dissipation of abamectin after application of a foliar pesticide in an orange crop. A DT50 of 9 days was determined, which was similar to that obtained under controlled conditions in the climate chamber (12 days), indicating that biotransformation was the primary process influencing the overall dissipation.


Assuntos
Antiparasitários/química , Ivermectina/análogos & derivados , Macrolídeos/metabolismo , Praguicidas/química , Poluentes do Solo/química , Solo/química , Antiparasitários/análise , Brasil , Monitoramento Ambiental , Meia-Vida , Ivermectina/análise , Ivermectina/química , Ivermectina/metabolismo , Macrolídeos/análise , Macrolídeos/química , Praguicidas/análise , Poluentes do Solo/análise
12.
J Agric Food Chem ; 67(26): 7266-7273, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244199

RESUMO

Chemical investigation of fungus Pochonia chlamydosporia strain 170, derived from rice fermentation sediment samples, afforded seven radicicol analogues, including two new compounds, monocillin VI (1) and monocillin VII (2), and five known compounds, monocillin II (3), monorden D (4), monocillin IV (5), monocillin V (6), and pochonin M (7). The structures of compounds 1-7 were established primarily by analysis of nuclear magnetic resonance data, and the absolute configurations of the secondary alcohol in compounds 1 and 2 were assigned by the modified Mosher method. All seven compounds have modest antibacterial activities, with a minimal inhibitory concentration (MIC) of 25.6 µg/mL for compounds 1 and 3-7 and 51.2 µg/mL for compound 2, on inhibition of the growth of the plant pathogen Xanthomonas campestris (the positive control ampicillin showed a MIC value of 12.8 µg/mL), indicating that the fungus has the potential to control bacterial disease. The biosynthetic gene cluster and putative biosynthetic pathways of these radicicol analogues in the P. chlamydosporia genome were proposed. These findings increase our knowledge of the chemical potential of P. chlamydosporia and may allow us to better utilize the fungus as a biological control agent.


Assuntos
Antibacterianos/química , Hypocreales/metabolismo , Macrolídeos/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Vias Biossintéticas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/química , Hypocreales/genética , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica , Xanthomonas campestris/efeitos dos fármacos , Xanthomonas campestris/crescimento & desenvolvimento
13.
J Microbiol ; 57(9): 795-802, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31187417

RESUMO

Myxococcus xanthus, a myxobacterium, displays phase variation between yellow phase and tan phase. We found that deletion of the encA gene encoding encapsulin and the encF gene encoding a metalloprotease causes formation of tan colonies that never transform into yellow colonies. The encA and encF mutants were defective in the production of DK-xanthene and myxovirescin. They did not produce extracellular polysaccharides; hence, the cells did not aggregate in liquid and showed reduced swarming on agar plates. The mutants had defective sporulation, but were rescued extracellularly by wild type cells. All these traits indicate that the encA and encF mutants are likely to be tan-phase-locked, and encapsulin has a close relationship with phase variation in M. xanthus. The encA and encF genes are localized in the same gene cluster, encBAEFG (MXAN_3557~MXAN_3553). Unlike the encA and encF genes, deletion of other genes in the cluster did not show tan-phase-locked phenotype.


Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/crescimento & desenvolvimento , Myxococcus xanthus/genética , Proteínas de Bactérias/genética , Cor , Deleção de Genes , Macrolídeos/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Mutação , Myxococcus xanthus/metabolismo , Fenótipo , Polissacarídeos Bacterianos/biossíntese , Xantenos/metabolismo
14.
PLoS One ; 14(4): e0215958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31039188

RESUMO

Ossamycin from Streptomyces hygroscopicus var. ossamyceticus is an antifungal and cytotoxic polyketide and a potent inhibitor of the mitochondrial ATPase. Analysis of a near-complete genome sequence of the ossamycin producer has allowed the identification of the 127-kbp ossamycin biosynthetic gene cluster. The presence in the cluster of a specific crotonyl-CoA carboxylase/reductase homologue suggests that the 5-methylhexanoate extension unit used in construction of the macrocyclic core is incorporated intact from the unusual precursor isobutyrylmalonyl-CoA. Surprisingly, the modular polyketide synthase uses only 14 extension modules to accomplish 15 cycles of polyketide chain extension, a rare example of programmed iteration on a modular polyketide synthase. Specific deletion of genes encoding cytochrome P450 enzymes has given insight into the late-stage tailoring of the ossamycin macrocycle required for the attachment of the unusual 2,3,4,6-deoxyaminohexose sugar l-ossamine to C-8 of the ossamycin macrocycle. The ossamycin cluster also encodes a putative spirocyclase enzyme, OssO, which may play a role in establishing the characteristic spiroketal moiety of the natural product.


Assuntos
Vias Biossintéticas , Macrolídeos/química , Macrolídeos/metabolismo , Compostos de Espiro/química , Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Bacterianos , Família Multigênica , Policetídeo Sintases/genética , Streptomyces/genética
15.
ACS Chem Biol ; 14(6): 1305-1309, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31095370

RESUMO

Pentamycin is a polyene antibiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomoniasis, and mixed infections. Chemical instability has hindered its widespread application and development as a drug. Here, we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Genome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsible for filipin III biosynthesis. The ptnJ gene, which is absent from the filipin III biosynthetic gene cluster, was shown to encode a cytochrome P450 capable of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin biosynthesis, paving the way for biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsible for the assembly of filipin III and pentamycin, suggesting pentamycin production may be common in Streptomyces species.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Macrolídeos/metabolismo , Streptomyces/metabolismo , Vias Biossintéticas , Catálise , Genes Bacterianos , Família Multigênica , Polienos/metabolismo , Streptomyces/genética
16.
Toxins (Basel) ; 11(3)2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934618

RESUMO

Ulcers due to infections with Mycobacterium ulcerans are characterized by complete lack of wound healing processes, painless, an underlying bed of host dead cells and undermined edges due to necrosis. Mycolactone, a macrolide produced by the mycobacterium, is believed to be the toxin responsible. Of interest and relevance is the knowledge that Buruli ulcer (BU) patients remember experiencing trauma previously at the site of the ulcers, suggesting an impairment of wound healing processes, the plausible effect due to the toxin. Wound healing processes involve activation of the blood platelets to release the contents of the dense granules mainly serotonin, calcium ions, and ADP/ATP by exocytosis into the bloodstream. The serotonin release results in attracting more platelets and mast cells to the wound site, with the mast cells also undergoing degranulation, releasing compounds into the bloodstream by exocytosis. Recent work has identified interference in the co-translational translocation of many secreted proteins via the endoplasmic reticulum and cell death involving Wiskott-Aldrich syndrome protein (WASP), Sec61, and angiotensin II receptors (AT2R). We hypothesized that mycolactone by being lipophilic, passively crosses cell membranes and binds to key proteins that are involved in exocytosis by platelets and mast cells, thus inhibiting the initiation of wound healing processes. Based on this, molecular docking studies were performed with mycolactone against key soluble n-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and regulators, namely Vesicle-associated membrane protein (VAMP8), Synaptosomal-associated protein (SNAP23, syntaxin 11, Munc13-4 (its isoform Munc13-1 was used), and Munc18b; and also against known mycolactone targets (Sec61, AT2R, and WASP). Munc18b was shown to be a plausible mycolactone target after the molecular docking studies with binding affinity of -8.5 kcal/mol. Structural studies and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding energy calculations of the mycolactone and Munc18b complex was done with 100 ns molecular dynamics simulations using GROMACS. Mycolactone binds strongly to Munc18b with an average binding energy of -247.571 ± 37.471 kJ/mol, and its presence elicits changes in the structural conformation of the protein. Analysis of the binding interactions also shows that mycolactone interacts with Arg405, which is an important residue of Munc18b, whose mutation could result in impaired granule exocytosis. These findings consolidate the possibility that Munc18b could be a target of mycolactone. The implication of the interaction can be experimentally evaluated to further understand its role in granule exocytosis impairment in Buruli ulcer.


Assuntos
Macrolídeos/metabolismo , Proteínas Munc18/metabolismo , Plaquetas/metabolismo , Úlcera de Buruli , Exocitose , Humanos , Macrolídeos/química , Mastócitos/metabolismo , Simulação de Acoplamento Molecular , Proteínas Munc18/química , Ligação Proteica
17.
J Agric Food Chem ; 67(17): 4782-4792, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30973721

RESUMO

Polyketides represent an important class of biologically active and structurally diverse compounds found in nature. They are biosynthesized from acyl CoA precursors by polyketide synthases (PKSs). The use of combinatorial biosynthesis to form hybrid PKSs is considered to be an excellent approach for the development of novel polyketides. Here, 10 new 16-membered macrolide compounds were isolated from the broth of the genetically engineered strain Streptomyces avermitilis TM24, in which the PKS gene aveA3 was seamlessly replaced by the milbemycin PKS gene milA3. Their structures were elucidated on the basis of NMR and MS spectroscopic analyses. The acaricidal and nematicidal activities of them against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. The results indicated that compound 1 had potent acaricidal activity against adult mites with an LC50 value of 0.0022 mg L-1, while compounds 5 and 7 possessed potent nematicidal activity with LC50 values of 4.56 and 4.30 mg L-1, respectively.


Assuntos
Acaricidas/farmacologia , Antinematódeos/farmacologia , Proteínas de Bactérias/genética , Macrolídeos/farmacologia , Streptomyces/química , Streptomyces/genética , Acaricidas/química , Acaricidas/isolamento & purificação , Acaricidas/metabolismo , Animais , Antinematódeos/química , Antinematódeos/isolamento & purificação , Antinematódeos/metabolismo , Proteínas de Bactérias/metabolismo , Feminino , Engenharia Genética , Macrolídeos/química , Macrolídeos/isolamento & purificação , Macrolídeos/metabolismo , Masculino , Estrutura Molecular , Streptomyces/metabolismo , Tetranychidae/efeitos dos fármacos , Tylenchida/efeitos dos fármacos
18.
Toxins (Basel) ; 11(3)2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836720

RESUMO

Buruli ulcer is a neglected tropical infectious disease, produced by the environmentally persistent pathogen Mycobacterium ulcerans (MU). Neither the ecological niche nor the exact mode of transmission of MU are completely elucidated. However, some environmental factors, such as the concentration in chitin and pH values, were reported to promote MU growth in vitro. We pursued this research using next generation sequencing (NGS) and mRNA sequencing to investigate potential changes in MU genomic expression profiles across in vitro environmental conditions known to be suitable for MU growth. Supplementing the growth culture medium in either chitin alone, calcium alone, or in both chitin and calcium significantly impacted the MU transcriptome and thus several metabolic pathways, such as, for instance, those involved in DNA synthesis or cell wall production. By contrast, some genes carried by the virulence plasmid and necessary for the production of the mycolactone toxin were expressed neither in control nor in any modified environments. We hypothesized that these genes are only expressed in stressful conditions. Our results describe important environmental determinants playing a role in the pathogenicity of MU, helping the understanding of its complex natural life cycle and encouraging further research using genomic approaches.


Assuntos
Macrolídeos/metabolismo , Mycobacterium ulcerans/genética , Mycobacterium ulcerans/metabolismo , Transcriptoma , Técnicas Bacteriológicas , Meio Ambiente , Sequenciamento de Nucleotídeos em Larga Escala
19.
Bioorg Med Chem ; 27(8): 1677-1682, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30878192

RESUMO

Molecular behavior under bilayer membrane environments is one of the important research topics concerning how organic molecules exert their biological activities when interacting with cellular membranes. However, chemistry-based approaches to this property have not been successful when compared with the structural biological strategy on ligand-receptor interactions. Here, we investigated the molecular behavior of the lipophilic ATPase inhibitor bafilomycin A1 and its derivatives under a lipid environment from a chemical point of view. Our results revealed significant differences in membrane affinity and dynamics among ligands having different inhibitory potencies, suggesting the specific contribution of ligand-membrane interactions to their biological activity.


Assuntos
Membrana Celular/química , Ligantes , Macrolídeos/química , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Flúor/química , Ligação de Hidrogênio , Cinética , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Espectroscopia de Ressonância Magnética
20.
Mol Microbiol ; 111(6): 1493-1509, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30825339

RESUMO

Bacterial pathogen Dickeya zeae strain EC1 produces antibiotics-like phytotoxins called zeamines, which are major virulence determinants encoded by the zms gene cluster. In this study, we identified a zeamine-deficient mutant with a Tn5 insertion in a gene designated as vfmI encoding a two-component system (TCS) sensor histidine kinase (HK), which is accompanied by vfmH encoding a response regulator (RR) at the same genetic locus. Domain analysis shows this TCS is analogous to the VfmIH of D. dadantii, with typical characteristics of sensor HK and RR, respectively, and sharing the same operon. Deletion of either vfmI or vfmH resulted in decreased production of zeamines and cell wall degrading enzymes (CWDEs), and alleviated virulence on rice seeds and potato tubers. In D. dadantii 3937, VfmH was shown to bind to the promoters of vfmA and vfmE, while in D. zeae EC1, VfmH could bind to the promoters of vfmA, vfmE and vfmF. RNA-seq analysis of strain EC1 and its vfmH mutant also showed that the TCS positively regulated a range of virulence genes, including zms, T1SS, T2SS, T3SS, T6SS, flagellar and CWDE genes.


Assuntos
Proteínas de Bactérias/genética , Gammaproteobacteria/genética , Gammaproteobacteria/patogenicidade , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Histidina Quinase/genética , Macrolídeos/metabolismo , Família Multigênica , Óperon , Fenótipo , Doenças das Plantas/microbiologia , Poliaminas/metabolismo , Regiões Promotoras Genéticas , Percepção de Quorum , Análise de Sequência de RNA , Virulência/genética
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