Puwainaphycins (PUW) and minutissamides (MIN) are structurally homologous cyclic lipopeptides that exhibit high structural variability and possess antifungal and cytotoxic activities. While only a minor variation can be found in the amino acid composition of the peptide cycle, the fatty acid (FA) moiety varies largely. The effect of FA functionalization on the bioactivity of PUW/MIN chemical variants is poorly understood. A rapid and selective liquid chromatography-mass spectrometry-based method led us to identify 13 PUW/MIN (1-13) chemical variants from the benthic cyanobacterium Nodularia harveyana strain UHCC-0300 from the Baltic Sea. Five new variants identified were designated as PUW H (1), PUW I (2), PUW J (4), PUW K (10), and PUW L (13) and varied slightly in the peptidic core composition, but a larger variation was observed in the oxo-, chloro-, and hydroxy-substitutions on the FA moiety. To address the effect of FA substitution on the cytotoxic effect, the major variants (3 and 5-11) together with four other PUW/MIN variants (14-17) previously isolated were included in the study. The data obtained showed that hydroxylation of the FA moiety abolishes the cytotoxicity or significantly reduces it when compared with the oxo-substituted C18-FA (compounds 5-8). The oxo-substitution had only a minor effect on the cytotoxicity of the compound when compared to variants bearing no substitution. The activity of PUW/MIN variants with chlorinated FA moieties varied depending on the position of the chlorine atom on the FA chain. This study also shows that variation in the amino acids distant from the FA moiety (position 4-8 of the peptide cycle) does not play an important role in determining the cytotoxicity of the compound. These findings confirmed that the lipophilicity of FA is essential to maintain the cytotoxicity of PUW/MIN lipopeptides. Further, a 63 kb puwainaphycin biosynthetic gene cluster from a draft genome of the N. harveyana strain UHCC-0300 was identified. This pathway encoded two specific lipoinitiation mechanisms as well as enzymes needed for the modification of the FA moiety. Examination on biosynthetic gene clusters and the structural variability of the produced PUW/MIN suggested different mechanisms of fatty-acyl-AMP ligase cooperation with accessory enzymes leading to a new set of PUW/MIN variants bearing differently substituted FA.

• Publication type
• Journal Article MeSH

Microbial cyclic lipopeptides are an important class of antifungal compounds with applications in pharmacology and biotechnology. However, the cytotoxicity of many cyclic lipopeptides limits their potential as antifungal drugs. Here we present a structure-activity relationship study on the puwainaphycin/minutissamide (PUW/MIN) family of cyclic lipopeptides isolated from cyanobacteria. PUWs/MINs with variable fatty acid chain lengths differed in the dynamic of their cytotoxic effect despite their similar IC50 after 48 hours (2.8 μM for MIN A and 3.2 μM for PUW F). Furthermore, they exhibited different antifungal potency with the lowest MIC values obtained for MIN A and PUW F against the facultative human pathogen Aspergillus fumigatus (37 μM) and the plant pathogen Alternaria alternata (0.6 μM), respectively. We used a Grignard-reaction with alkylmagnesium halides to lengthen the lipopeptide FA moiety as well as the Steglich esterification on the free hydroxyl substituents to prepare semi-synthetic lipopeptide variants possessing multiple fatty acid tails. Cyclic lipopeptides with extended and branched FA tails showed improved strain-specific antifungal activity against A. fumigatus (MIC = 0.5-3.8 μM) and A. alternata (MIC = 0.1-0.5 μM), but with partial retention of the cytotoxic effect (∼10-20 μM). However, lipopeptides with esterified free hydroxyl groups possessed substantially higher antifungal potencies, especially against A. alternata (MIC = 0.2-0.6 μM), and greatly reduced or abolished cytotoxic activity (>20 μM). Our findings pave the way for a generation of semi-synthetic variants of lipopeptides with improved and selective antifungal activities.

• Publication type
• Journal Article MeSH

Man-made shallow fishponds in the Czech Republic have been facing high eutrophication since the 1950s. Anthropogenic eutrophication and feeding of fish have strongly affected the physicochemical properties of water and its aquatic community composition, leading to harmful algal bloom formation. In our current study, we characterized the phytoplankton community across three eutrophic ponds to assess the phytoplankton dynamics during the vegetation season. We microscopically identified and quantified 29 cyanobacterial taxa comprising non-toxigenic and toxigenic species. Further, a detailed cyanopeptides (CNPs) profiling was performed using molecular networking analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) data coupled with a dereplication strategy. This MS networking approach, coupled with dereplication, on the online global natural product social networking (GNPS) web platform led us to putatively identify forty CNPs: fourteen anabaenopeptins, ten microcystins, five cyanopeptolins, six microginins, two cyanobactins, a dipeptide radiosumin, a cyclooctapeptide planktocyclin, and epidolastatin 12. We applied the binary logistic regression to estimate the CNPs producers by correlating the GNPS data with the species abundance. The usage of the GNPS web platform proved a valuable approach for the rapid and simultaneous detection of a large number of peptides and rapid risk assessments for harmful blooms.

• Keywords
• cyanobacteria, cyanopeptides, dereplication strategy, global natural product social networking (GNPS), harmful bloom, liquid chromatography-tandem mass spectrometry,
• MeSH
• Bacterial Toxins analysis   toxicity   MeSH
• Spectrometry, Mass, Electrospray Ionization * MeSH
• Risk Assessment MeSH
• Metabolomics MeSH
• Water Microbiology MeSH
• Microbiota MeSH
• Environmental Monitoring * MeSH
• Marine Toxins analysis   toxicity   MeSH
• Online Social Networking * MeSH
• Population Dynamics MeSH
• Seasons MeSH
• Ponds microbiology   MeSH
• Cyanobacteria classification   growth & development   metabolism   MeSH
• Harmful Algal Bloom * MeSH
• Tandem Mass Spectrometry * MeSH
• Chromatography, High Pressure Liquid * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Bacterial Toxins MeSH
• Marine Toxins MeSH

Heterocytous cyanobacteria are among the most prolific sources of bioactive secondary metabolites, including anabaenopeptins (APTs). A terrestrial filamentous Brasilonema sp. CT11 collected in Costa Rica bamboo forest as a black mat, was studied using a multidisciplinary approach: genome mining and HPLC-HRMS/MS coupled with bioinformatic analyses. Herein, we report the nearly complete genome consisting of 8.79 Mbp with a GC content of 42.4%. Moreover, we report on three novel tryptophan-containing APTs; anabaenopeptin 788 (1), anabaenopeptin 802 (2), and anabaenopeptin 816 (3). Furthermore, the structure of two homologues, i.e., anabaenopeptin 802 (2a) and anabaenopeptin 802 (2b), was determined by spectroscopic analysis (NMR and MS). Both compounds were shown to exert weak to moderate antiproliferative activity against HeLa cell lines. This study also provides the unique and diverse potential of biosynthetic gene clusters and an assessment of the predicted chemical space yet to be discovered from this genus.

• Keywords
• Brasilonema, anabaenopeptins, antiproliferative activity, hexapeptides, molecular networking, tryptophan-containing peptides,
• MeSH
• Peptides, Cyclic * chemistry   genetics   isolation & purification   pharmacology   MeSH
• HeLa Cells MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Cell Proliferation drug effects   MeSH
• Cyanobacteria * chemistry   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides, Cyclic * MeSH

The following series of articles form a special issue organized by the Algatech Center of the Institute of Microbiology CAS dedicated to the memory of Dr. Ivan Šetlík.

• MeSH
• Biofuels history   MeSH
• History, 20th Century MeSH
• History, 21st Century MeSH
• Photosynthesis MeSH
• Microbiology history   MeSH
• Cyanobacteria genetics   metabolism   radiation effects   MeSH
• Check Tag
• History, 20th Century MeSH
• History, 21st Century MeSH
• Publication type
• Biography MeSH
• Historical Article MeSH
• Festschrift MeSH
• Geographicals
• Czechoslovakia MeSH
• Names of Substances
• Biofuels MeSH

• About
• Šetlík, Ivan