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Historically, small molecules biosynthesised by bacteria have been an excellent source for antibacterial drugs. Today, however, the rediscovery of known compounds is a significant hurdle to developing new antimicrobials. Here we use a genome mining and synthetic biology approach to discover the ambocidins: calcium-dependent lipodepsipeptides that are active against drug-resistant Gram-positive pathogens. By cloning a silent biosynthetic gene cluster (the amb cluster) from Streptomyces ambofaciens ATCC 2387 and integrating this into the chromosome of Streptomyces avermitilis we induce expression of ambocidin A and B: two new Nε-hydroxyarginine-containing cyclic lipodepsipeptides active against drug-resistant Gram-positive pathogens. Using a panel of Streptomyces host strains, we show that the choice of heterologous host is critical for producing the biologically active compounds, and that inappropriate host choice leads to aberrant production inactive derivatives. We show that Nε-hydroxyarginine is the product of a haem-dependent oxygenase and that it enhances biological activity. Ambocidin A inhibits cell wall biosynthesis by binding to Lipid II at a different site than vancomycin. Unlike daptomycin, ambocidin A retains antimicrobial activity in the presence of lung-surfactant, giving it the potential to treat bacterial pneumonia. Our work expands the family of calcium-dependent lipopeptide antibiotics with new members exhibiting a distinct mechanism of action.
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Correction for 'Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis' by Rory F. Little et al., Nat. Prod. Rep., 2021, DOI: 10.1039/d1np00035g.
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Review covering up to mid-2021The structure of polyketide and non-ribosomal peptide natural products is strongly influenced by how they are released from their biosynthetic enzymes. As such, Nature has evolved a diverse range of release mechanisms, leading to the formation of bioactive chemical scaffolds such as lactones, lactams, diketopiperazines, and tetronates. Here, we review the enzymes and mechanisms used for chain release in polyketide and non-ribosomal peptide biosynthesis, how these mechanisms affect natural product structure, and how they could be utilised to introduce structural diversity into the products of engineered biosynthetic pathways.
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Biossíntese de Peptídeos Independentes de Ácido Nucleico , Policetídeos/metabolismo , Vias Biossintéticas , Engenharia Metabólica , Estrutura MolecularRESUMO
Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously-overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol-derived C3 building block. The key role of an A-factor synthase (AfsA)-like offloading domain was corroborated by CRISPR-Cas-mediated gene editing, which facilitated precise excision within a PKS domain.
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4-Butirolactona/análogos & derivados , Antifúngicos/farmacologia , Burkholderia/química , Hypocreales/efeitos dos fármacos , Policetídeos/farmacologia , 4-Butirolactona/biossíntese , 4-Butirolactona/química , 4-Butirolactona/farmacologia , Animais , Antifúngicos/química , Antifúngicos/metabolismo , Burkholderia/genética , Burkholderia/metabolismo , Besouros , Testes de Sensibilidade Microbiana , Policetídeos/química , Policetídeos/metabolismoRESUMO
This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.
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Aziridinas/uso terapêutico , Proteínas de Escherichia coli , Terapia Genética/métodos , Neoplasias Experimentais/terapia , Nitrorredutases , Pró-Fármacos/uso terapêutico , Animais , Evolução Molecular Direcionada , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/uso terapêutico , Humanos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Nitrorredutases/biossíntese , Nitrorredutases/genética , Nitrorredutases/uso terapêuticoRESUMO
Cyclopropanol rings are highly reactive and may function as molecular "warheads" that affect natural product bioactivity. Yet, knowledge on their biosynthesis is limited. Using gene cluster analyses, isotope labeling, and in vitro enzyme assays, we shed first light on the biosynthesis of the cyclopropanol-substituted amino acid cleonine, a residue in the antimicrobial depsipeptide valgamicin C and the cytotoxic glycopeptide cleomycin A2. We decipher the biosynthetic origin of valgamicin C and show that the cleonine cyclopropanol ring is derived from dimethylsulfoniopropionate (DMSP). Furthermore, we demonstrate that part of the biosynthesis is analogous to the formation of malleicyprol polyketides in pathogenic bacteria. By genome mining and metabolic profiling, we identify the potential to produce cyclopropanol rings in other bacterial species. Our results reveal a general mechanism for cyclopropyl alcohol biosynthesis across diverse natural products that may be harnessed for bioengineering and drug discovery.
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Aminoácidos , Produtos Biológicos , Vias Biossintéticas , Ciclopropanos , Depsipeptídeos , Éteres Cíclicos , Furanos , Policetídeos , Família MultigênicaRESUMO
Bacterial pathogens of the Burkholderia pseudomallei (BP) group cause life-threatening infections in both humans and animals. Critical for the virulence of these often antibiotic-resistant pathogens is the polyketide hybrid metabolite malleicyprol, which features two chains, a short cyclopropanol-substituted chain and a long hydrophobic alkyl chain. The biosynthetic origin of the latter has remained unknown. Here, we report the discovery of novel overlooked malleicyprol congeners with varied chain lengths and identify medium-sized fatty acids as polyketide synthase (PKS) starter units that constitute the hydrophobic carbon tails. Mutational and biochemical analyses show that a designated coenzyme A-independent fatty acyl-adenylate ligase (FAAL, BurM) is essential for recruiting and activating fatty acids in malleicyprol biosynthesis. In vitro reconstitution of the BurM-catalyzed PKS priming reaction and analysis of ACP-bound building blocks reveal a key role of BurM in the toxin assembly. Insights into the function and role of BurM hold promise for the development of enzyme inhibitors as novel antivirulence therapeutics to combat infections with BP pathogens.
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Ácidos Graxos , Policetídeo Sintases , Animais , Humanos , Antibacterianos/farmacologia , Bactérias/metabolismo , Policetídeo Sintases/metabolismoRESUMO
Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved â¼5-fold more effective than the canonical nitroreductase NfsB.
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Metronidazol , Peixe-Zebra , Animais , Metronidazol/farmacologia , Metronidazol/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Metagenoma , Clonagem Molecular , Nitrorredutases/genéticaRESUMO
Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.
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The type I polyketide SF2487/A80577 (herein referred to as tetromadurin) is a polyether tetronate ionophore antibiotic produced by the terrestrial Gram-positive bacterium Actinomadura verrucosospora. Tetromadurin is closely related to the polyether tetronates tetronasin (M139603) and tetronomycin, all of which are characterised by containing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. We have sequenced the genome of Actinomadura verrucosospora to identify the biosynthetic gene cluster responsible for tetromadurin biosynthesis (the mad gene cluster). Based on bioinformatic analysis of the 32 genes present within the cluster a plausible biosynthetic pathway for tetromadurin biosynthesis is proposed. Functional confirmation of the mad gene cluster is obtained by performing in-frame deletions in each of the genes mad10 and mad31, which encode putative cyclase enzymes responsible for cyclohexane and tetrahydropyran formation, respectively. Furthermore, the A. verrucosospora Δmad10 mutant produces a novel tetromadurin metabolite that according to mass spectrometry analysis is analogous to the recently characterised partially cyclised tetronasin intermediate lacking its cyclohexane and tetrahydropyran rings. Our results therefore elucidate the biosynthetic machinery of tetromadurin biosynthesis and lend support for a conserved mechanism of cyclohexane and tetrahydropyran biosynthesis across polyether tetronates.
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Macrolídeos/química , Policetídeo Sintases/genética , Policetídeos/metabolismo , Actinobacteria/enzimologia , Actinobacteria/metabolismo , Actinomadura , Sequência de Aminoácidos/genética , Antibacterianos/química , Sequência de Bases/genética , Vias Biossintéticas , Clonagem Molecular , Éteres/metabolismo , Furanos/metabolismo , Família Multigênica/genética , Alinhamento de SequênciaRESUMO
Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.
In the cell, most tasks are performed by big molecules called proteins, which behave like molecular machines. Although proteins are often described as having one job each, this is not always true, and many proteins can perform different roles. Enzymes are a type of protein that facilitate chemical reactions. They are often specialised to one reaction, but they can also accelerate other side-reactions. During evolution, these side-reactions can become more useful and, as a result, the role of the enzyme may change over time. The main role of the enzyme called NfsA in Escherichia coli bacteria is thought to be to convert molecules called quinones into hydroquinones, which can protect the cell from toxic molecules produced in oxidation reactions. As a side-reaction, NfsA has the potential to protect bacteria from an antibiotic called chloramphenicol, but it generally does this with such low efficacy that the effects are negligible. Producing hydroquinones is helpful to the cell in some situations, but if bacteria are regularly exposed to chloramphenicol, NfsA's role aiding antibiotic resistance could become more important. Over time, the enzyme could evolve to become better at neutralising chloramphenicol. Therefore, NfsA provides an opportunity to study the evolution of proteins and how bacteria adapt to antibiotics. To see how evolution might affect the activity of NfsA, Hall et al. generated 250 million E. coli with either random or targeted changes to the gene that codes for the NfsA enzyme. The resulting variants of NfsA that were most effective against chloramphenicol all had a change that eliminated the enzyme's ability to convert quinones. This result demonstrates a key trade-off between roles for NfsA, where one must be lost for the other to improve. These results demonstrate the interplay between a protein's different roles and provide insight into bacterial drug resistance. Additionally, the experiments showed that the bacteria with improved resistance to chloramphenicol also became more sensitive to another antibiotic, metronidazole. These findings could inform the fight against drug-resistant bacterial infections and may also be helpful in guiding the design of proteins with different roles.
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Cloranfenicol/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Nitrorredutases/metabolismo , Domínio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Evolução Molecular , Inativação Metabólica , Mutação , Nitrorredutases/química , Nitrorredutases/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.