RESUMEN
Major advances over the past decade in the field of ancient DNA are providing access to past paleogenomic diversity, but the diverse functions and biosynthetic capabilities of this growing paleome remain largely elusive. We investigated the dental calculus of 12 Neanderthals and 52 anatomically modern humans ranging from 100,000 years ago to the present and reconstructed 459 bacterial metagenome-assembled genomes. We identified a biosynthetic gene cluster shared by seven Middle and Upper Paleolithic individuals that allows for the heterologous production of a class of previously unknown metabolites that we name "paleofurans." This paleobiotechnological approach demonstrates that viable biosynthetic machinery can be produced from the preserved genetic material of ancient organisms, allowing access to natural products from the Pleistocene and providing a promising area for natural product exploration.
Asunto(s)
Productos Biológicos , Furanos , Genoma Bacteriano , Hominidae , Hombre de Neandertal , Animales , Humanos , Productos Biológicos/metabolismo , Hominidae/genética , Metagenoma , Hombre de Neandertal/genética , Furanos/metabolismo , ADN AntiguoRESUMEN
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.
Asunto(s)
Amoeba , Productos Biológicos , Dictyostelium , Policétidos , Amoeba/genética , Productos Biológicos/metabolismo , Dictyostelium/fisiología , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismoRESUMEN
Chemical and biochemical analyses of one of the most basic nonribosomal peptide synthetases (NRPS) from a Pseudomonas fluorescens strain revealed its striking plasticity. Determination of the potential substrate scope enabled us to anticipate novel secondary metabolites that could subsequently be isolated and tested for their bioactivities. Detailed analyses of the monomodular pyreudione synthetase showed that the biosynthesis of the bacterial pyreudione alkaloids does not require additional biosynthetic enzymes. Heterologous expression of a similar and functional, yet cryptic, NRPS of Pseudomonas entomophila was successful and allowed us to perform a phylogenetic analysis of their thioesterase domains.
Asunto(s)
Alcaloides/biosíntesis , Escherichia coli/metabolismo , Péptido Sintasas/metabolismo , Pseudomonas/metabolismo , Alcaloides/química , Dictyostelium/química , Dictyostelium/metabolismo , Escherichia coli/química , Estructura Molecular , Pseudomonas/químicaRESUMEN
Human pathogens often produce soluble protein toxins that generate pores inside membranes, resulting in the death of target cells and tissue damage. In pathogenic amoebae, this has been exemplified with amoebapores of the enteric protozoan parasite Entamoeba histolytica. Here we characterize acanthaporin, to our knowledge the first pore-forming toxin to be described from acanthamoebae, which are free-living, bacteria-feeding, unicellular organisms that are opportunistic pathogens of increasing importance and cause severe and often fatal diseases. We isolated acanthaporin from extracts of virulent Acanthamoeba culbertsoni by tracking its pore-forming activity, molecularly cloned the gene of its precursor and recombinantly expressed the mature protein in bacteria. Acanthaporin was cytotoxic for human neuronal cells and exerted antimicrobial activity against a variety of bacterial strains by permeabilizing their membranes. The tertiary structures of acanthaporin's active monomeric form and inactive dimeric form, both solved by NMR spectroscopy, revealed a currently unknown protein fold and a pH-dependent trigger mechanism of activation.
Asunto(s)
Acanthamoeba/química , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Acanthamoeba/patogenicidad , Secuencia de Aminoácidos , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad , VirulenciaRESUMEN
Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals. In free-living protozoa, lysozyme-like proteins are involved in the digestion of phagocytosed bacteria. Here, we purified a protein with lysozyme activity from Dictyostelium amoebae, which constitutes the founding member, a novel class of lysozymes. By tagging the protein with green fluorescent protein or the Myc epitope, a new type of lysozyme-containing vesicle was identified that was devoid of other known lysosomal enzymes. The most highly expressed isoform, encoded by the alyA gene, was knocked out by homologous recombination. The mutant cells had greatly reduced enzymatic activity and grew inefficiently when bacteria were the sole food source. Over time the mutant gained the ability to internalize bacteria more efficiently, so that the defect in digestion was compensated by increased uptake of food particles.
Asunto(s)
Dictyostelium/metabolismo , Muramidasa/química , Muramidasa/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Bases de Datos como Asunto , Endocitosis , Retículo Endoplásmico/metabolismo , Epítopos/química , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Datos de Secuencia Molecular , Muramidasa/fisiología , Mutagénesis , Mutación , Fagocitosis , Fenotipo , Filogenia , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Recombinación Genética , Homología de Secuencia de AminoácidoRESUMEN
Pore-forming polypeptides have been purified from several amoeboid protozoans that are well-known human pathogens. Obligate enteric parasites, such as Entamoeba histolytica, and free-living but potentially highly pathogenic species, such as Naegleria fowleri, contain these cytolytic molecules inside cytoplasmic granules. Comprehensive functional and structural studies have been conducted that include isolation of the proteins from their natural sources, monitoring of their biological activity towards different targets, and molecular cloning of the genes of their precursors. In the case of the most prominent member of the protein family, with respect to protozoans, the three-dimensional structure of amoebapore A was solved recently. The amoebic pore-forming polypeptides can rapidly perforate human cells. The antibacterial activity of amoebapores and of related polypetides from free-living protozoa points to a more vital function of these molecules: inside the digestive vacuoles they combat growth of phagocytosed bacteria which are killed when their cytoplasmic membranes are permeabilized. The concommitant activity of these proteins towards host cells may be due to a coincidental selection for an efficient effector molecule. Nonetheless, several lines of evidence indicate that these factors are involved in pathogenesis of fatal diseases induced by amoeboid protozoa.
Asunto(s)
Entamoeba histolytica/metabolismo , Naegleria fowleri/metabolismo , Péptidos/metabolismo , Fagosomas/fisiología , Proteínas Protozoarias/metabolismo , Animales , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/genética , Entamoeba histolytica/genética , Entamoeba histolytica/inmunología , Entamoeba histolytica/ultraestructura , Naegleria fowleri/genética , Naegleria fowleri/inmunología , Péptidos/química , Proteínas Protozoarias/genéticaRESUMEN
The pore-forming polypeptides of Naegleria fowleri, naegleriapores A and B, are processed from separate multipeptide precursor structures. According to their transcripts, each precursor molecule appears to contain additional naegleriapore-like polypeptides, all of which share a structural motif of six invariant cysteine residues within their amino acid sequence. To identify the putative pronaegleriapore-derived peptides at the protein level, amoebic extracts were screened for small cysteine-rich polypeptides by fluorescently labeling their cysteine residues. Three novel naegleriapore isoforms derived from the precursor molecule of naegleriapore B were identified. Two of the isoforms were purified to homogeneity and tested for their biological activity. The pore-forming activity of the novel peptides was remarkably lower than that of the originally isolated naegleriapores, but both peptides killed bacteria by permeabilizing their cytoplasmic membranes. Collectively, these results indicate that naegleriapore isoforms with antibacterial and pore-forming activity are proteolytically released from the same precursor protein, presumably to generate a phylogenetically ancient complementary antimicrobial arsenal.
Asunto(s)
Antiinfecciosos/farmacología , Naegleria fowleri/metabolismo , Péptidos/química , Péptidos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Cisteína/química , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Antimicrobial peptides are widespread in animal species and their function as defensive molecules may even have appeared before the evolution of metazoa. The amoeboid protozoon Entamoeba histolytica discharge membrane-permeabilizing polypeptides named amoebapores into the phagosome in which engulfed bacteria are situated as evidenced here by confocal laser microscopy and electron microscopy using specific antibodies. We demonstrate that the purified three isoforms of the amoebic polypeptides exhibit complementary antibacterial activities in vitro. The potency of amoebapores were compared with that of antimicrobial peptides of phylogenetically widespread species by monitoring in parallel their activities against representatives of gram-positive and gram-negative bacteria and liposomes in various assays, and differences in the mechanism of membrane permeabilization became apparent. Northern blot analysis revealed that expression of genes coding for amoebapores and amoebic lysozymes is not dramatically changed upon co-culture of amoebae with bacteria indicating that the antimicrobial arsenal is rather constitutively expressed than induced in these primitive phagocytes.
Asunto(s)
Bacterias/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Entamoeba histolytica/inmunología , Canales Iónicos , Proteínas de la Membrana/fisiología , Fagocitosis , Fagosomas/fisiología , Proteínas Protozoarias/fisiología , Animales , Bacterias/metabolismo , Entamoeba histolytica/ultraestructuraRESUMEN
The free-living amoeboflagellate and potential human pathogen Naegleria fowleri causes the often fatal disease primary amoebic meningoencephalitis. The molecular repertoire responsible for the cytolytic and tissue-destructive activity of this amoeboid protozoon is largely unknown. We isolated two pore-forming polypeptides from extracts of highly virulent trophozoites of N. fowleri by measuring their membrane-permeabilizing activity. N-terminal sequencing and subsequent molecular cloning yielded the complete primary structures and revealed that the two polypeptides are isoforms. Both polypeptides share similar structural properties with antimicrobial and cytolytic polypeptides of the protozoon Entamoeba histolytica (amoebapores) and of cytotoxic natural killer (NK) and T cells of human (granulysin) and pig (NK-lysin), all characterized by a structure of amphipathic alpha-helices and an invariant framework of cysteine residues involved in disulfide bonds. In contrast to the aforementioned proteins, the Naegleria polypeptides both are processed from large precursor molecules containing additional isoforms of substantial sequence divergence. Moreover, biochemical characterization of the isolated polypeptides in combination with mass determination showed that they are N-glycosylated and variably processed at the C terminus. The biological activity of the purified polypeptides of Naegleria was examined toward human cells and bacteria, and it was found that these factors, named naegleriapores, are active against both types of target cells, which is in good agreement with their proposed biological role as a broad-spectrum effector molecule.