RESUMO
Interactions between microorganisms are often mediated by specialized metabolites. Although the structures and biosynthesis of these compounds may have been elucidated, microbes exist within complex microbiomes and chemical signals can thus also be subject to community-dependent modifications. Increasingly powerful chemical and biological tools allow to shed light on this poorly understood aspect of chemical ecology. We provide an overview of loss-of-function and gain-of-function chemical mediator (CM) modifications within microbial multipartner relationships. Although loss-of-function modifications are abundant in the literature, few gain-of-function modifications have been described despite their important role in microbial interactions. Research in this field holds great potential for our understanding of microbial interactions and may also provide novel tools for targeted interference with microbial signaling.
Assuntos
MicrobiotaRESUMO
Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.
Assuntos
Amoeba , Produtos Biológicos , Dictyostelium , Policetídeos , Amoeba/genética , Produtos Biológicos/metabolismo , Dictyostelium/fisiologia , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/metabolismoRESUMO
Bacteria are inherently social organisms whose actions should ideally be studied within an interactive ecological context. We show that the exchange and modification of natural products enables two unrelated bacteria to defend themselves against a common predator. Amoebal predation is a major cause of death in soil bacteria and thus it exerts a strong selective pressure to evolve defensive strategies. A systematic analysis of binary combinations of coisolated bacteria revealed strains that were individually susceptible to predation but together killed their predator. This cooperative defense relies on a Pseudomonas species producing syringafactin, a lipopeptide, which induces the production of peptidases in a Paenibacillus strain. These peptidases then degrade the innocuous syringafactin into compounds, which kill the predator. A combination of bioprospecting, coculture experiments, genome modification, and transcriptomics unravel this novel natural product-based defense strategy.
Assuntos
Bactérias/metabolismo , Lipopeptídeos/metabolismo , Comportamento Predatório/fisiologia , Amoeba/fisiologia , Animais , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Lipopeptídeos/química , Paenibacillus/citologia , Filogenia , Pseudomonas/citologia , Microbiologia do SoloRESUMO
Investigating the ecological context of microbial predator-prey interactions enables the identification of microorganisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.
Assuntos
Anti-Infecciosos , Lipopeptídeos , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Aminoácidos/genética , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Genômica , Família MultigênicaRESUMO
Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the Acore domains, yet domain interfaces and the flexible Asub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.
Assuntos
Evolução Molecular , Modelos Genéticos , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Peptídeo Sintases/genética , Recombinação Genética , Família MultigênicaRESUMO
A total synthesis of the cyclic lipodepsipeptide natural product orfamide A was achieved. By developing a synthesis format using an aminoacid ester building block and SPPS protocol adaptation, a focused library of target compounds was obtained, in high yield and purity. Spectral and LC-HRMS data of all library members with the isolated natural product identified the 5 Leu residue to be d- and the 3'-OH group to be R-configured. The structural correction of orfamide A by chemical synthesis and analysis was confirmed by biological activity comparison in Chlamydomonas reinhardtii, which indicated compound configuration to be important for bioactivity. Acute toxicity was also found against Trypanosoma brucei, the parasite causing African sleeping sickness.
Assuntos
Produtos Biológicos , Trypanosoma brucei brucei , Tripanossomíase Africana , Animais , Lipopeptídeos , Peptídeos Cíclicos/químicaRESUMO
The first total synthesis of the actin-stabilizing marine natural product geodiamolide H was achieved. Solid-phase based peptide assembly paired with scalable stereoselective syntheses of polyketide building blocks and an optimized esterification set the stage for investigating the key ring-closing metathesis. Geodiamolide H and synthetic analogues were characterized for their toxicity and for antiproliferative effects in cellulo, by characterising actin polymerization induction inâ vitro, and by docking on the F-actin target and property computation in silico, for a better understanding of structure-activity relationships (SAR). A non-natural analogue of geodiamolide H was discovered to be most potent in the series, suggesting significant potential for tool compound design.
Assuntos
Produtos Biológicos , Depsipeptídeos , Actinas , Depsipeptídeos/farmacologia , Humanos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.
Assuntos
Produtos Biológicos/farmacologia , Dictyostelium/fisiologia , Sinergismo Farmacológico , Mupirocina/farmacologia , Pseudomonas/metabolismo , Percepção de Quorum/fisiologia , Infecções Estafilocócicas/tratamento farmacológico , 4-Butirolactona/análogos & derivados , 4-Butirolactona/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologiaRESUMO
Bacteria of the genus Pseudomonas are ubiquitous in nature. Pseudomonads display a fascinating metabolic diversity, which correlates with their ability to colonize an extremely wide range of ecological niches. As a result, these bacteria are a prolific source of natural products. Biosynthesis of the latter is often orchestrated by arrays of chemical signals arising from intraspecies communication or interspecies relationships with bacteria, fungi, amoebae, plants, and insects. Especially nonribosomal lipopeptides, which have diverse biological activities, play important roles in the lifestyle of pseudomonads. In this review, we will focus on the molecular structures, properties, biosynthetic pathways, and biological functions of pseudomonal lipopeptides. This review is not only addressed to bio/chemists rather it serves as a comprehensive guide for all researchers (micro/biologists, ecologists, and environmental scientists) working in this multidisciplinary field.
Assuntos
Lipopeptídeos/biossíntese , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Pseudomonadaceae/metabolismo , Interações Hospedeiro-Patógeno , Estrutura Molecular , Plantas/microbiologia , Pseudomonadaceae/fisiologia , Pseudomonas/metabolismo , Pseudomonas/patogenicidade , Rizosfera , Ribossomos/metabolismo , Microbiologia do SoloRESUMO
Natural products are important mediators and effectors in complex microbial communities. This special collection is devoted to the multifaceted roles of these natural products as well as on understanding how, when, and why they are produced. (Picture created with biorender.com.).
RESUMO
Nonribosomal lipopeptides (NRLPs) are complex natural products of bacterial origin that not only fulfill important ecological functions but also serve as lead structures for the development of new pharmaceuticals. In order to carry out detailed structure-activity relationship studies and to decipher the biological activities of NRLPs, the primary structure, including stereochemical assignment, of every new member of this natural product family has to be established first. In this review, we want to focus on analytical techniques and tools that can be employed to elucidate the structure of bacterial NRLPs.
Assuntos
Proteínas de Bactérias/química , Lipopeptídeos/química , Estrutura Molecular , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
LuxR-type transcriptional activator proteins frequently regulate the expression of biosynthetic gene clusters (BGCs). With only a fraction of bacterial BGCs being expressed under standard culturing conditions, modulation of LuxRs would provide a powerful approach to activate silent clusters. We show that by exploiting the modular nature of LuxR proteins, it is possible to construct functional chimeric LuxRs, which enables both the rewiring of quorum sensing systems and the activation of silent BGCs. Importantly, our strategy allowed us to identify the novel natural product pseudomonol from a bacterium of the genus Pseudomonas.
Assuntos
Produtos Biológicos/metabolismo , Engenharia Genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Família Multigênica/genética , Percepção de Quorum , Proteínas Recombinantes/genética , Proteínas Repressoras/genética , Transativadores/genéticaRESUMO
Butenolides are well-known signaling molecules in Gram-positive bacteria. Here, we describe a novel class of butenolides isolated from a Gram-negative Pseudomonas strain, the styrolides. Structure elucidation was aided by the total synthesis of styrolideâ A. Transposon mutagenesis enabled us to identify the styrolide biosynthetic gene cluster, and by using a homology search, we discovered the related and previously unknown acaterin biosynthetic gene cluster in another Pseudomonas species. Mutagenesis, heterologous expression, and identification of key shunt and intermediate products were crucial to propose a biosynthetic pathway for both Pseudomonas-derived butenolides. Comparative transcriptomics suggests a link between styrolide formation and the regulatory networks of the bacterium.
Assuntos
4-Butirolactona/análogos & derivados , Pseudomonas/química , 4-Butirolactona/biossíntese , 4-Butirolactona/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis/genética , Família Multigênica , Mutagênese , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Microbiologia do SoloRESUMO
Jagaricin is a lipopeptide produced by the bacterial mushroom pathogen Janthinobacterium agaricidamnosum, the causative agent of mushroom soft rot disease. Apart from causing lesions in mushrooms, jagaricin is a potent antifungal active against human-pathogenic fungi. We show that jagaricin acts by impairing membrane integrity, resulting in a rapid flux of ions, including Ca2+, into susceptible target cells. Accordingly, the calcineurin pathway is required for jagaricin tolerance in the fungal pathogen Candida albicans Transcriptional profiling of pathogenic yeasts further revealed that jagaricin triggers cell wall strengthening, general shutdown of membrane potential-driven transport, and the upregulation of lipid transporters, linking cell envelope integrity to jagaricin action and resistance. Whereas jagaricin shows hemolytic effects, it exhibited either no or low plant toxicity at concentrations at which the growth of prevalent phytopathogenic fungi is inhibited. Therefore, jagaricin may have potential for agricultural applications. The action of jagaricin as a membrane-disrupting antifungal is promising but would require modifications for use in humans.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Cálcio/metabolismo , Candida albicans/genética , Candida albicans/isolamento & purificação , Candida glabrata/efeitos dos fármacos , Candida glabrata/genética , Candidíase/microbiologia , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
Natural products are invaluable sources of structural diversity and complexity ideally suited for the development of therapeutic agents. The search for novel bioactive molecules has prompted scientists to explore various ecological niches. Microorganisms have been shown to constitute such an important source. Despite their biosynthetic potential, social amoebae, that is, microorganisms with both a uni- and multicellular lifestyle, are underexplored regarding their secreted secondary metabolome. In this review, we present the structural diversity of amoebal natural products and discuss their biological functions as well as their total syntheses.
Assuntos
Amoeba/química , Produtos Biológicos/química , Amoeba/metabolismo , Produtos Biológicos/síntese química , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Humanos , Metaboloma , Estrutura MolecularRESUMO
In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, and systems biology, among others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria which induce architectural changes in biofilm colonies when cocultured with Bacillus subtilis We identified the soil bacterium Lysinibacillus fusiformis M5 as an inducer of wrinkle formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm matrix-related operons epsABCDEFGHIJKLMNO and tasA-sipW-tapA Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression but rather is caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death.IMPORTANCE Biofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be beneficial, for instance, in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they occur outside laboratory conditions. Here, we present a screening method suitable for the identification of multispecies interactions and showcase L. fusiformis as a soil bacterium that is able to live alongside B. subtilis and modify the architecture of its biofilms.
Assuntos
Bacillaceae/metabolismo , Bacillus subtilis/fisiologia , Biofilmes/crescimento & desenvolvimento , Hipoxantina/metabolismo , Interações Microbianas , Microbiologia do Solo , Bacillaceae/isolamento & purificação , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Meios de Cultura/química , Ensaios de Triagem em Larga Escala/métodos , Histidina Quinase/genética , Hipoxantina/isolamento & purificação , Hipoxantina/farmacologia , Hipoxantina/fisiologia , Óperon , Fosforilação , Transdução de SinaisRESUMO
We present a versatile synthesis of the eukaryotic signaling peptide glorin as well as glorinamide, a synthetic analog. The ability of these compounds to activate glorin-induced genes in the social amoeba Polysphondylium pallidum was evaluated by quantitative reverse transcription PCR, whereby both compounds showed bioactivity comparable to a glorin standard. This synthetic route will be useful in conducting detailed structure-activity relationship studies as well as in the design of chemical probes to dissect glorin-mediated signaling pathways.
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Severe forms of pneumococcal meningitis, bacteraemia and pneumonia result in more than 1 million deaths each year despite the widespread introduction of carbohydrate-protein conjugate vaccines against Streptococcus pneumoniae. Here we describe a new and highly efficient antipneumococcal vaccine design based on synthetic conjugation of S. pneumoniae capsule polysaccharides to the potent lipid antigen α-galactosylceramide, which stimulates invariant natural killer T (iNKT) cells when presented by the nonpolymorphic antigen-presenting molecule CD1d. Mice injected with the new lipid-carbohydrate conjugate vaccine produced high-affinity IgG antibodies specific for pneumococcal polysaccharides. Vaccination stimulated germinal center formation; accumulation of iNKT cells with a T follicular helper cell phenotype; and increased frequency of carbohydrate-specific, long-lived memory B cells and plasmablasts. This new lipid-carbohydrate vaccination strategy induced potent antipolysaccharide immunity that protected against pneumococcal disease in mice and may also prove effective for the design of carbohydrate-based vaccines against other major bacterial pathogens.
Assuntos
Carboidratos/química , Lipídeos/química , Vacinas Pneumocócicas/síntese química , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Carboidratos/administração & dosagem , Linhagem Celular , Centro Germinativo/citologia , Centro Germinativo/imunologia , Células HL-60 , Humanos , Imunização Passiva , Memória Imunológica/imunologia , Cinética , Lipídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Vacinas Pneumocócicas/administração & dosagem , Vacinas Pneumocócicas/química , Fatores de Tempo , Vacinas Sintéticas/administração & dosagemAssuntos
Interações entre Hospedeiro e Microrganismos , Microbiologia , Química , Humanos , Pesquisa , TranscriptomaRESUMO
Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria's host is a "farmer" clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer's spore production and depresses a nonfarmer's spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a "domesticated" food source.