Browsing by Subject "natural products"

Antibiotic resistance is a worldwide problem and it threatens the prevention and treatment of infections caused by different pathogens. All living organisms produce natural products including ribosomal peptides with great variety. They are widely distributed in nature and they are playing more significant role in the search of new antimicrobial compounds used as therapeutical agents. Bacteria are a prolific source of peptides many of which are antimicrobial and microbial genomes are widely believed to encode new antimicrobial peptides. Genome mining has expanded the number of families of ribosomally synthesized natural products in recent years. These In silico approaches together with molecular biology and chemical analysis aim to identify novel compounds. In this study an unknown cyanobactin-like gene cluster was discovered by genome mining from genomes of cyanobacteria and also other bacteria. The aim of this work was to study the occurrence of the gene clusters in various bacterial genomes and the structures of novel peptides. The active biosynthesis of these peptides was tested by LCMS- and Q-TOF -analyses based on bioinformatic predictions. The production of the predicted peptides was also tested with stable sulphur isotope labelling. The aim was also to clone the genes needed for peptide biosynthesis into E. coli and to study antimicrobial activities of these peptides. Bioinformatic analyses suggested that the gene clusters encoded 1–8 precursor peptides together with protease. The precursor peptides had conserved leader sequence (LPxQxxPVxR) and a highly variable core sequences, often encoding an even number of cysteines. The mature peptide is eventually formed from core sequence through post-translational changes in the precursor peptide. The gene cluster was present in 38 bacterial genomes representing a diverse selection of bacterial phyla including cyanobacteria, proteobacteria, actinobacteria, bacteroidetes, firmicutes and planctomycetes. Analyses of the precursor peptide core regions suggested that the products are 8–131 amino acids in length. These peptides could be divided into two groups based on their structures: They form a selection of disulphide-bridge stabilized peptides with 2–5 disulphide-bridges as well as short cationic peptides with an ?-helical structure. Surprisingly, these types of peptides are common in eukaryotes and part of the innate immune system displaying potent antimicrobial properties but very rarely reported for bacteria. The peptides predicted from bioinformatic analysis were detected from Pseudanabaena sp. PCC 6802 using a combination of molecular biology and structural chemistry. Heterologous expression of the gene cluster from Pseudanabaena sp. PCC 6802 in E. coli confirmed that the gene cluster is active. A set of short cationic synthetic peptides with ?-helical structure predicted from Oscillatoria sp. PCC 10802, Dickeya zeae Ech1591, Vibrio nigripulchritudo SOn1, Agarivorans albus MKT 106, Roseibium sp. TrichSKD4 and Yersinia frederiksenii ATCC 33641 were shown to have potent antimicrobial activity between 0.8–100 ?g/ml. These findings prove that predicted cysteine containing peptides are produced by bacteria and some peptides from this novel family have antimicrobial activity, which might pave the way for new possible drugs derived from natural products.

Natural products have enormous structural and chemical diversity and are either the source or direct inspiration for many drugs in use today. Cyanobacteria are prolific producers of complex natural products with serine protease inhibiting activity. Many of these natural products are the product of non-ribosomal peptide synthetase (NRPS) modular enzyme complexes. Suomilide is a complex tetrapeptide produced by strains of the benthic cyanobacterium Nodularia sphaerocarpa. It has a highly complicated structure and contains an unusual azabicyclononane moiety, a methylglyceric acid, a xylose unit with hexanoic acids and a terminal 1-amidino-3-(2-aminoethyl)-3-pyrroline moiety. Suomilide inhibits thrombin, plasmin and trypsin in low micro-molar concentrations. The biosynthetic of this unusual glycoside remain unclear. However, suomilide is long predicted to be part to the aeruginosin family of protease inhibitors. A 5.4 Mb draft genome of Nodularia sphaerocarpa HKVV was obtained in order to identify the suomilide biosynthetic. The 43.7 kb suomilide gene cluster was identified on a single contig by performing tBLASTn searches on the draft genome of Nodularia HKVV using aerDEF genes from aeruginosins gene cluster as query. This gene cluster encodes 27 genes including two complex NRPS enzymes and a set of tailoring enzymes for the assembly of suomilide. The suomilide gene cluster shares extensive homology to known aeruginosin gene clusters including two aerB and aerG genes encoding NRPS enzymes, 12 genes (aerC, aerD, aerE, aerF, aerI, aerK, two copies of aerN and four copies of aerH) encoding for the enzymes responsible for synthesis of precursor non-proteinogenic amino acids and 13 other tailoring enzymes. The suomilide gene cluster was much larger and encoded a greater number of biosynthetic enzymes reflecting the structural complexity of suomilide. We identified 10 aeruginosin gene clusters and 2 suomilide gene clusters from 12 strains of cyanobacteria by genome mining. Bioinformatics analyses suggested these gene clusters encoded an unanticipated chemical diversity of aeruginosins and suomilides. LC-MS and Q-TOF analysis detected aeruginosins or suomilide variants from 12 of the 15 strains. Surprisingly, inhibition assays with the crude extracts using all three isoforms of human trypsin suggest that these compounds may have potent and selective inhibition of human trypsin isoforms. Further work is required to prove that suomilide alone can carry out selective inhibition of trypsin isoforms or is it a result of synergism between the compounds produce by cyanobacteria. Phylogenetic analysis demonstrated that the aeruginosin evolved through the acquisition of multiple loading mechanisms and tailoring enzymes through horizontal gene transfers. Our results support the hypothesis that suomilides are a part of aeruginosin family as they are made through the same genetic pathway, however have gained a greater degree of structural diversity due to the acquisition of tailoring enzymes. These results together suggest that cyanobacteria produce an unexpected wealth of complex natural products belonging to the aeruginosin family and that some of these may be potent and selective inhibitors of isoforms of human trypsin.