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1.
Nat Commun ; 12(1): 5221, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471117

RESUMO

Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather 'open' to a 'closed' conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.


Assuntos
Antibacterianos/metabolismo , Cromossomos Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Estruturas Cromossômicas , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Família Multigênica , Transcriptoma
2.
Molecules ; 26(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34361657

RESUMO

The current review aims to summarise the biodiversity and biosynthesis of novel secondary metabolites compounds, of the phylum Actinobacteria and the diverse range of secondary metabolites produced that vary depending on its ecological environments they inhabit. Actinobacteria creates a wide range of bioactive substances that can be of great value to public health and the pharmaceutical industry. The literature analysis process for this review was conducted using the VOSviewer software tool to visualise the bibliometric networks of the most relevant databases from the Scopus database in the period between 2010 and 22 March 2021. Screening and exploring the available literature relating to the extreme environments and ecosystems that Actinobacteria inhabit aims to identify new strains of this major microorganism class, producing unique novel bioactive compounds. The knowledge gained from these studies is intended to encourage scientists in the natural product discovery field to identify and characterise novel strains containing various bioactive gene clusters with potential clinical applications. It is evident that Actinobacteria adapted to survive in extreme environments represent an important source of a wide range of bioactive compounds. Actinobacteria have a large number of secondary metabolite biosynthetic gene clusters. They can synthesise thousands of subordinate metabolites with different biological actions such as anti-bacterial, anti-parasitic, anti-fungal, anti-virus, anti-cancer and growth-promoting compounds. These are highly significant economically due to their potential applications in the food, nutrition and health industries and thus support our communities' well-being.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Produtos Biológicos , Enzimas/isolamento & purificação , Enzimas/metabolismo , Metabolismo Secundário
3.
ACS Appl Mater Interfaces ; 13(33): 39806-39818, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34387459

RESUMO

Silver nanomaterials have attracted a great deal of interest due to their broad-spectrum antimicrobial activity. However, it is still challenging to balance the high antibacterial efficiency with low damage to biological cells of silver nanostructures, especially when the diameter decreases to less than 10 nm. Here, we developed a new type of Ag nanohybrid material via a unimolecular micelle template method, which presents amazing antibacterial activities and almost noncytotoxicity. First, water-soluble multiarm star-shaped brushlike copolymer α-CD-g-[(PEO40-g-PAA50)-b-PEO5]18 was precisely synthesized and its micelle behavior in different solvents was revealed. Then, nanocrystal clusters assembled by Ag grains (Ag@Template NCs) were prepared through an in situ redox route using the unimolecular micelle of α-CD-g-[(PEO40-g-PAA50)-b-PEO5]18 as the soft template, AgNO3 as a precursor, and tetrabutylammonium borohydride (TBAB) as the reducing agent. The overall size of the achieved Ag@Template NCs is controlled by the template structure at around 40 nm (Dh in DMF), and the size of the Ag grain can be easily regulated from ∼1 to ∼5 nm by adjusting the feeding ratio of AgNO3/acrylic acid (AA) units in the template from 1:10 to 1:1. Benefitting from the structural design of the template, all Ag@Template NCs prepared here exhibit excellent dispersibility and chemical stability in different aqueous environments (neutral, pH = 5.5, and 0.9% NaCl physiological saline solution), which play a crucial role in the long-term storage and potential application in a complex physiological environment. The antibacterial and cytotoxicity tests indicate that Ag@Template NCs display much better performance than Ag nanoparticles (Ag NPs), which have a comparable overall size of ∼25 nm. The inhibitory capability of Ag@Template NCs to bacteria strongly depends on the grain size. Specifically, the Ag@Template-1 NC assembled by the smallest grains (1.6 ± 0.3 nm) presents the best antibacterial activity. For E. coli (-), the MIC value is as low as 5 µg/mL (0.36 µg/mL of Ag), while for S. aureus (+), the value is around 10 µg/mL (0.72 µg/mL of Ag). The survival rate of L02 cells and lactate dehydrogenase assay together illustrate the low cytotoxicity possessed by the prepared Ag@Template NCs. Therefore, the proposed Ag@Template NC structure successfully resolves the high reactivity, instability, and fast oxidation issues of the ultrasmall Ag nanoparticles, and integrates high antibacterial efficiency and nontoxicity to biological cells into one platform, which implies its broad potential application in biomedicine.


Assuntos
Antibacterianos/efeitos adversos , Antibacterianos/química , Nanopartículas Metálicas/química , Prata/química , Antibacterianos/metabolismo , Boroidretos/química , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Polímeros/química , Compostos de Amônio Quaternário/química , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Nanomedicina Teranóstica
4.
Nutrients ; 13(7)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34371869

RESUMO

Hibiscus sabdariffa L. (H.s.) is a polyphenolic-rich plant commonly consumed either as a beverage or spice. The aim of the present study was to evaluate the in vitro digestibility of H.s. polyphenols using an in vitro model of digestion which simulates the human stomach and small intestine. The bioaccessible polyphenols released in the digested samples were analyzed by liquid chromatography coupled to photodiode array and mass spectrometry detection. H.s. anthocyanins (cyanidin-3-O-sambubioside and delphinidin-3-O-sambubioside) content drastically dropped during the digestion process from 2.91 ± 0.03 µg g-1 and 8.53 ± 0.08 µg g-1 (w/w) CG (Cyanidin-glucoside) in the raw extract, respectively, to 0.12 ± 0.01 µg g-1 0.12 ± 0.01 µg g-1 (w/w) CG at the end of duodenal digestion. Total polyphenols also have shown a decrease from 1192.65 ± 30.37 µg g-1 (w/w) in the raw extract to 282.24 ± 7.21 µg g-1 (w/w) by the end of gastric digestion, in contrast to their increase by the end of duodenal digestion 372.91 ± 3.97 µg g-1 (w/w). On the other hand, the decrease in certain compounds (e.g., caffeoylquinicandcoumaroylquinic acids) was observed during gastric digestion resulting in an increase of quinic acid in the duodenal aliquots, thus suggesting that this compound was derived from the degradation of the more complex hydroxycinnamic acids. H.s. extract also exhibited a bacteriostatic effect against Staphylococcus aureus ATCC 6538 (MIC of 2.5 mg mL-1) and a bactericidal effect against a food isolate of Listeria monocytogenes (MBC of 2.5 mg mL-1). The undigested polyphenols of H.s. in the upper gastrointestinal tract enters the colon, where they are metabolized by the gut microbiota. The present study results showed that resistance of H.s. polyphenols during gastrointestinal digestion might affect their uptake, resulting in a decrease in their digestibility.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Digestão , Hibiscus , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Bactérias/crescimento & desenvolvimento , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Suco Gástrico/química , Hibiscus/química , Humanos , Secreções Intestinais/química , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Polifenóis/isolamento & purificação , Polifenóis/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Espectrometria de Massas em Tandem
5.
J Immunol ; 207(3): 974-984, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34282000

RESUMO

K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.


Assuntos
Antibacterianos/metabolismo , Anti-Inflamatórios/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Infecções por Escherichia coli/imunologia , Escherichia coli/fisiologia , Macrófagos/imunologia , Infecções por Pasteurella/metabolismo , Pasteurella multocida/fisiologia , Desiminases de Arginina em Proteínas/metabolismo , Animais , Antibacterianos/química , Anti-Inflamatórios/química , Peptídeos Catiônicos Antimicrobianos/química , Citrulinação , Cães , Imunidade Inata , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Ligação Proteica , Células RAW 264.7
6.
Biomolecules ; 11(7)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209734

RESUMO

Meroterpenoids are secondary metabolites formed due to mixed biosynthetic pathways which are produced in part from a terpenoid co-substrate. These mixed biosynthetically hybrid compounds are widely produced by bacteria, algae, plants, and animals. Notably amazing chemical diversity is generated among meroterpenoids via a combination of terpenoid scaffolds with polyketides, alkaloids, phenols, and amino acids. This review deals with the isolation, chemical diversity, and biological effects of 452 new meroterpenoids reported from natural sources from January 2016 to December 2020. Most of the meroterpenoids possess antimicrobial, cytotoxic, antioxidant, anti-inflammatory, antiviral, enzyme inhibitory, and immunosupressive effects.


Assuntos
Terpenos/química , Terpenos/isolamento & purificação , Terpenos/metabolismo , Alcaloides , Animais , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Antineoplásicos/metabolismo , Antioxidantes/metabolismo , Bactérias/metabolismo , Benzopiranos , Benzoquinonas , Produtos Biológicos/química , Vias Biossintéticas , Fungos/metabolismo , Humanos , Metabolismo Secundário/fisiologia , Sesquiterpenos
7.
Nat Commun ; 12(1): 4625, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330923

RESUMO

Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.


Assuntos
Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Canais Iônicos/metabolismo , Klebsiella/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Transporte Biológico , Microscopia Crioeletrônica/métodos , Canais Iônicos/química , Canais Iônicos/ultraestrutura , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica
8.
Commun Biol ; 4(1): 836, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226658

RESUMO

Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Anticorpos de Domínio Único/metabolismo , Staphylococcus aureus/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único/química , Infecções Estafilocócicas/microbiologia
9.
Nat Commun ; 12(1): 4499, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301953

RESUMO

Aurantinins (ARTs) are antibacterial polyketides featuring a unique 6/7/8/5-fused tetracyclic ring system and a triene side chain with a carboxyl terminus. Here we identify the art gene cluster and dissect ART's C-methyl incorporation patterns to study its biosynthesis. During this process, an apparently redundant methyltransferase Art28 was characterized as a malonyl-acyl carrier protein O-methyltransferase, which represents an unusual on-line methyl esterification initiation strategy for polyketide biosynthesis. The methyl ester bond introduced by Art28 is kept until the last step of ART biosynthesis, in which it is hydrolyzed by Art9 to convert inactive ART 9B to active ART B. The cryptic reactions catalyzed by Art28 and Art9 represent a protecting group biosynthetic logic to render the ART carboxyl terminus inert to unwanted side reactions and to protect producing organisms from toxic ART intermediates. Further analyses revealed a wide distribution of this initiation strategy for polyketide biosynthesis in various bacteria.


Assuntos
Proteína de Transporte de Acila/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , Policetídeos/metabolismo , Proteína de Transporte de Acila/genética , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Vias Biossintéticas/genética , Esterificação , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Metiltransferases/genética , Testes de Sensibilidade Microbiana/métodos , Modelos Químicos , Estrutura Molecular , Família Multigênica , Polienos/química , Polienos/metabolismo , Polienos/farmacologia , Policetídeos/química , Policetídeos/farmacologia
10.
Molecules ; 26(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209863

RESUMO

The fungal strain was isolated from a soil sample collected in Giza province, Egypt, and was identified as Aspergillus ochraceopetaliformis based on phenotypic and genotypic data. The ethyl acetate extract of the fungal strain exhibited promising activity levels against several pathogenic test organisms and through a series of 1H NMR guided chromatographic separations, a new α-pyrone-C-lyxofuranoside (1) along with four known compounds (2-5) were isolated. The planar structure of the new metabolite was elucidated by detailed analysis of its 1D/2D NMR and HRMS/IR/UV spectroscopic data, while the relative configuration of the sugar moiety was determined by a combined study of NOESY and coupling constants data, with the aid of theoretical calculations. The structures of the known compounds-isolated for the first time from A. ochraceopetaliformis-were established by comparison of their spectroscopic data with those in the literature. All isolated fungal metabolites were evaluated for their antibacterial and antifungal activities against six Gram-positive and Gram-negative bacteria as well as against three human pathogenic fungi.


Assuntos
Antibacterianos , Aspergillus/metabolismo , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/crescimento & desenvolvimento , Microbiologia do Solo , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Aspergillus/isolamento & purificação
11.
ACS Appl Mater Interfaces ; 13(26): 31140-31152, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34156831

RESUMO

Existing single-functional agents against dental caries are inadequate in antibacterial performance or mineralization balance. This problem can be resolved through a novel strategy, namely, the construction of an antibiofouling and mineralizing dual-bioactive tooth surface by grafting a dentotropic moiety to an antimicrobial peptide. The constructed bioactive peptide can strongly adsorb onto the tooth surface and has beneficial functions in a myriad of ways. It inhibits cariogenic bacteria Streptococcus mutans adhesion, kills planktonic S. mutans, and destroys the S. mutans biofilm on the tooth surface. It also protects teeth from demineralization in acidic environments, and induces self-healing regeneration in the remineralization environment. Molecular dynamics simulations elucidate the main adsorption mechanism that the positively charged amino acid residues in the bioactive peptide bind to phosphate groups on the tooth surface, and the main mineralization mechanism that the negative charges on the outermost layer of the bioactive peptide repel acetic acid ions and attract calcium ions as nucleation sites for remineralization. This study suggests that this in-house synthesized dual-bioactive peptide is a promising functional agent to prevent dental caries, and is effective in inducing in situ self-healing remineralization for the treatment of decayed teeth.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Incrustação Biológica/prevenção & controle , Adsorção , Antibacterianos/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Estabilidade de Medicamentos , Humanos , Hidroxiapatitas/química , Hidroxiapatitas/metabolismo , Testes de Sensibilidade Microbiana , Dente Serotino/química , Dente Serotino/microbiologia , Simulação de Dinâmica Molecular , Ligação Proteica , Saliva/metabolismo , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/fisiologia , Desmineralização do Dente/prevenção & controle , Remineralização Dentária
12.
Commun Biol ; 4(1): 729, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117352

RESUMO

The approval of plazomicin broadened the clinical library of aminoglycosides available for use against emerging bacterial pathogens. Contrarily to other aminoglycosides, resistance to plazomicin is limited; still, instances of resistance have been reported in clinical settings. Here, we present structural insights into the mechanism of plazomicin action and the mechanisms of clinical resistance. The structural data reveal that plazomicin exclusively binds to the 16S ribosomal A site, where it likely interferes with the fidelity of mRNA translation. The unique extensions to the core aminoglycoside scaffold incorporated into the structure of plazomicin do not interfere with ribosome binding, which is analogously seen in the binding of this antibiotic to the AAC(2')-Ia resistance enzyme. The data provides a structural rationale for resistance conferred by drug acetylation and ribosome methylation, i.e., the two mechanisms of resistance observed clinically. Finally, the crystal structures of plazomicin in complex with both its target and the clinically relevant resistance factor provide a roadmap for next-generation drug development that aims to ameliorate the impact of antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Sisomicina/análogos & derivados , Antibacterianos/química , Antibacterianos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Farmacorresistência Bacteriana , Metilação , Providencia/efeitos dos fármacos , Providencia/metabolismo , RNA Ribossômico 16S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Sisomicina/química , Sisomicina/metabolismo , Sisomicina/farmacologia , Relação Estrutura-Atividade
13.
J Chem Theory Comput ; 17(7): 4564-4577, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34138557

RESUMO

Antibiotics enter into bacterial cells via protein channels that serve as low-energy pathways through the outer membrane, which is otherwise impenetrable. Insights into the molecular mechanisms underlying the transport processes are vital for the development of effective antibacterials. A much-desired prerequisite is an accurate and reproducible determination of free energy surfaces for antibiotic translocation, enabling quantitative and meaningful comparisons of permeation mechanisms for different classes of antibiotics. Inefficient sampling along the orthogonal degrees of freedom, for example, in umbrella sampling and metadynamics approaches, is however a key limitation affecting the accuracy and the convergence of free energy estimates. To overcome this limitation, two sampling methods have been employed in the present study that, respectively, combine umbrella sampling and metadynamics-style biasing schemes with temperature acceleration for improved sampling along orthogonal degrees of freedom. As a model for the transport of bulky solutes, the ciprofloxacin-OmpF system has been selected. The well-tempered metadynamics approach with multiple walkers is compared with its "temperature-accelerated" variant in terms of improvements in sampling and convergence of free energy estimates. We find that the inclusion of collective variables governing solute degrees of freedom and solute-water interactions within the sampling scheme largely alleviates sampling issues. Concerning improved sampling and convergence of free energy estimates from independent simulations, the temperature-accelerated sliced sampling approach that combines umbrella sampling with temperature-accelerated molecular dynamics performs even better as shown for the ciprofloxacin-OmpF system.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Ciprofloxacina/metabolismo , Porinas/metabolismo , Simulação de Dinâmica Molecular , Permeabilidade , Termodinâmica
14.
ACS Appl Mater Interfaces ; 13(25): 29269-29280, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34143595

RESUMO

Nanoantibacterial agents based on catalytic activity were limited due to the low levels of endogenous H2O2 in the microenvironment of bacterial biofilms. However, the additional H2O2 will trigger more side effects to healthy surroundings, which is still a great challenge. Herein, we report an acid-induced self-catalyzing platform based on dextran-coated copper peroxide nanoaggregates (DCPNAs) for antibiofilm and local infection therapy applications. The dextran-functionalized DCPNAs were mediated and conveniently purified via a dextran and ethanol precipitation method, which can also cluster nanodots into nanoaggregates and show good penetrability as well as biocompatibility. Bacterial biofilms were inhibited and destroyed by the reactive oxygen species generated from the Fenton reaction between the Cu2+ and H2O2 released from DCPNAs in an acidic environment, which did not require additional H2O2. As expected, the DCPNAs exhibit low cytotoxicity and excellent acid-induced antibacterial and antibiofilm ability. Moreover, the DCPNAs realized great therapeutic outcomes in the application for in vivo wound healing. The overall excellent properties associated with the DCPNAs highlight that they could be considered as a kind of ideal antimicrobial agents for microbial biofilm infection treatment.


Assuntos
Antibacterianos , Biofilmes/efeitos dos fármacos , Cobre , Dextranos/química , Nanopartículas/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Cobre/química , Cobre/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Peróxidos/química , Salmonella/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
15.
Nat Commun ; 12(1): 3889, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188038

RESUMO

Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.


Assuntos
Antibacterianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sítio Alostérico , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Conformação Proteica , Domínios Proteicos , Especificidade por Substrato
16.
J Med Chem ; 64(12): 8644-8665, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34080858

RESUMO

Due to the poor permeability across Gram-negative bacterial membranes and the troublesome bacterial efflux mechanism, only a few GyrB/ParE inhibitors with potent activity against Gram-negative pathogens have been reported. Among them, pyrimido[4,5-b]indole derivatives represented by GP-1 demonstrated excellent broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria but were limited by hERG inhibition and poor pharmacokinetics profile. To improve their drug-like properties, we designed a series of novel pyrimido[4,5-b]indole derivatives based on the tricyclic scaffold of GP-1 and the C-7 moiety of acorafloxacin. These efforts have culminated in the discovery of a promising compound 18r with reduced hERG liability and an improved PK profile. Compound 18r exhibited superior broad-spectrum in vitro antibacterial activity compared to GP-1, including a variety of clinical multidrug G- pathogens, especially Acinetobacter baumannii, and the in vivo efficacy was also demonstrated in a neutropenic mouse thigh model of infection with multidrug-resistant A. baumannii.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Indóis/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Antibacterianos/síntese química , Antibacterianos/metabolismo , Antibacterianos/farmacocinética , DNA Girase/metabolismo , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Estabilidade de Medicamentos , Células HEK293 , Humanos , Indóis/síntese química , Indóis/metabolismo , Indóis/farmacocinética , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos , Relação Estrutura-Atividade
17.
Science ; 372(6547): 1169-1175, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112687

RESUMO

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.


Assuntos
Antibacterianos/farmacologia , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Biofilmes , Cristalografia por Raios X , Cistationina gama-Liase/química , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Descoberta de Drogas , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Tolerância a Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento
18.
Sci Rep ; 11(1): 12410, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127732

RESUMO

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.


Assuntos
Antibacterianos/química , Antivirais/química , Proteínas de Bactérias/química , Hidrolases/química , Indóis/química , Polímeros/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , COVID-19/patologia , COVID-19/virologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/metabolismo , Materiais Revestidos Biocompatíveis/farmacologia , Estabilidade de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Mycobacterium smegmatis/enzimologia , Ácido Peracético/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/efeitos dos fármacos
19.
ACS Chem Biol ; 16(5): 929-942, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33974796

RESUMO

The outer membrane of Gram-negative bacteria is a formidable permeability barrier which allows only a small subset of chemical matter to penetrate. This outer membrane barrier can hinder the study of cellular processes and compound mechanism of action, as many compounds including antibiotics are precluded from entry despite having intracellular targets. Consequently, outer membrane permeabilizing compounds are invaluable tools in such studies. Many existing compounds known to perturb the outer membrane also impact inner membrane integrity, such as polymyxins and their derivatives, making these probes nonspecific. We performed a screen of ∼140 000 diverse synthetic compounds, for those that antagonized the growth inhibitory activity of vancomycin at 15 °C in Escherichia coli, to enrich for chemicals capable of perturbing the outer membrane. This led to the discovery that liproxstatin-1, an inhibitor of ferroptosis in human cells, and MAC-0568743, a novel cationic amphiphile, could potentiate the activity of large-scaffold antibiotics with low permeation into Gram-negative bacteria at 37 °C. Liproxstatin-1 and MAC-0568743 were found to physically disrupt the integrity of the outer membrane through interactions with lipopolysaccharide in the outer leaflet of the outer membrane. We showed that these compounds selectively disrupt the outer membrane while minimally impacting inner membrane integrity, particularly at the concentrations needed to potentiate Gram-positive-targeting antibiotics. Further exploration of these molecules and their structural analogues is a promising avenue for the development of outer membrane specific probes.


Assuntos
Antibacterianos/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Parede Celular/efeitos dos fármacos , Vancomicina/química , Acinetobacter baumannii/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Permeabilidade da Membrana Celular , Parede Celular/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Ensaios de Triagem em Larga Escala , Klebsiella pneumoniae/metabolismo , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Polimixinas/química , Polimixinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Quinoxalinas/química , Quinoxalinas/metabolismo , Compostos de Espiro/química , Compostos de Espiro/metabolismo , Vancomicina/metabolismo , Vancomicina/farmacologia
20.
J Biol Chem ; 296: 100799, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34022225

RESUMO

The Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a common source of antibiotic resistance in Gram-negative bacterial infections. KPC-2 is a class A ß-lactamase that exhibits a broad substrate profile and hydrolyzes most ß-lactam antibiotics including carbapenems owing to rapid deacylation of the covalent acyl-enzyme intermediate. However, the features that allow KPC-2 to deacylate substrates more rapidly than non-carbapenemase enzymes are not clear. The active-site residues in KPC-2 are largely conserved in sequence and structure compared with non-carbapenemases, suggesting that subtle alterations may collectively facilitate hydrolysis of carbapenems. We utilized a nonbiased genetic approach to identify mutants deficient in carbapenem hydrolysis but competent for ampicillin hydrolysis. Subsequent pre-steady-state enzyme kinetics analyses showed that the substitutions slow the rate of deacylation of carbapenems. Structure determination via X-ray diffraction indicated that a F72Y mutant forms a hydrogen bond between the tyrosine hydroxyl group and Glu166, which may lower basicity and impair the activation of the catalytic water for deacylation, whereas several mutants impact the structure of the Q214-R220 active site loop. A T215P substitution lowers the deacylation rate and drastically alters the conformation of the loop, thereby disrupting interactions between the enzyme and the carbapenem acyl-enzyme intermediate. Thus, the environment of the Glu166 general base and the precise placement and conformational stability of the Q214-R220 loop are critical for efficient deacylation of carbapenems by the KPC-2 enzyme. Therefore, the design of carbapenem antibiotics that interact with Glu166 or alter the Q214-R220 loop conformation may disrupt enzyme function and overcome resistance.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Carbapenêmicos/metabolismo , Klebsiella pneumoniae/metabolismo , beta-Lactamases/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Hidrólise , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/química , Modelos Moleculares , Conformação Proteica , beta-Lactamases/química
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