Puwainaphycins (PUW) and minutissamides (MIN) are cyanobacterial lipopeptides found in various cyanobacterial species. The first possible target of human exposure to them is intestinal epithelium but effect of PUW/MIN on enterocytes is not known at all. Using differentiated Caco-2 cells, PUW F was found to be cytotoxic from 5 µM concentration based on lactate dehydrogenase release assay and total protein concentration. However, it is also able to induce production of interleukin 8 in non-cytotoxic concentrations 1 and 2.5 µM detected by ELISA. Effects of MIN A and C were similar but less pronounced compared to PUW F. On the other hand, MIN D was the least toxic compound with no significant pro-inflammatory effects. Surprisingly, pro-inflammatory activation of the cells by PUW F and MIN C resulted in an increase in tight junction (TJ) protein claudin 4 expression determined by western blot analysis and confirmed by confocal microscopy. Furthermore, decrease in expression of zonula occludens 3, another TJ protein, was observed after the exposure to PUW F. Taken together, these cytotoxic lipopeptides, especially PUW F, are to be studied more deeply due to their capability to activate and/or deregulate human enterocytes in low concentrations.
- MeSH
- Caco-2 buňky MeSH
- lidé MeSH
- lipopeptidy * MeSH
- sinice * MeSH
- střevní sliznice MeSH
- těsný spoj MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The rapid emergence of resistance in pathogenic bacteria together with a steep decline in economic incentives has rendered a new wave in the drug development by the pharmaceutical industry and researchers. Since cyanobacteria are recognized as wide producers of pharmaceutically important compounds, we investigated thirty-four cyanobacterial extracts prepared by solvents of different polarities for their antimicrobial potential. Almost all tested cyanobacterial strains exhibited some degree of antimicrobial bioactivity, with more general effect on fungal strains compared with bacteria. Surprisingly ~50% of cyanobacterial extracts exhibited specific activity against one or few bacterial indicator strains with Gram-positive bacteria being more affected. Extracts of two most promising strains were subjected to activity-guided fractionation and determination of the minimum inhibitory concentration (MIC) against selected bacterial and fungal isolates. Multiple fractions were responsible for their antimicrobial effect with MIC reaching low-micromolar concentrations and in some of them high level of specificity was recorded. Twenty-six bioactive fractions analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) online workflow using dereplication resulted in identification of only forty-nine peptide spectrum matches (PSMs) with eleven unique metabolites spectrum matches (MSMs). Interestingly, only three fractions from Nostoc calcicola Lukešová 3/97 and four fractions from Desmonostoc sp. Cc2 showed the presence of unique MSMs suggesting the presence of unknown antimicrobial metabolites among majority of bioactive fractions from both the strains. Our results highlight potential for isolation and discovery of potential antimicrobial bioactive lead molecules from cyanobacterial extracts.
Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes.IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.
- MeSH
- Anabaena genetika MeSH
- antifungální látky MeSH
- antiinfekční látky MeSH
- bakteriální geny genetika MeSH
- bakteriální proteiny genetika MeSH
- cyklické peptidy biosyntéza chemie genetika MeSH
- lipopeptidy biosyntéza chemie genetika farmakologie MeSH
- multigenová rodina MeSH
- peptidsynthasy genetika MeSH
- polyketidsynthasy genetika MeSH
- sinice genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1⁻3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2⁻3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.
- MeSH
- antiinfekční látky chemie izolace a purifikace farmakologie MeSH
- Ascomycota účinky léků MeSH
- Bacillus subtilis účinky léků MeSH
- bakteriální toxiny chemie izolace a purifikace farmakologie MeSH
- cyklické peptidy chemie farmakologie MeSH
- lipopeptidy chemie farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- sinice metabolismus MeSH
- syntetické pryskyřice chemie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.
- MeSH
- hem chemie metabolismus MeSH
- koproporfyrinogenoxidasa chemie metabolismus MeSH
- molekulární modely MeSH
- oxidace-redukce MeSH
- protoporfyrinogenoxidasa chemie metabolismus MeSH
- Synechocystis enzymologie MeSH
- tetrapyrroly chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The pederin family includes a number of bioactive compounds isolated from symbiotic organisms of diverse evolutionary origin. Pederin is linked to beetle-induced dermatitis in humans, and pederin family members possess potent antitumor activity caused by selective inhibition of the eukaryotic ribosome. Their biosynthesis is accomplished by a polyketide/nonribosomal peptide synthetase machinery employing an unusual trans-acyltransferase mechanism. Here, we report a novel pederin type compound, cusperin, from the free-living cyanobacterium Cuspidothrix issatschenkoi (earlier Aphanizomenon). The chemical structure of cusperin is similar to that of nosperin recently isolated from the lichen cyanobiont Nostoc sharing the tehrahydropyran moiety and major part of the linear backbone. However, the cusperin molecule is extended by a glycine residue and lacks one hydroxyl substituent. Pederins were previously thought to be exclusive to symbiotic relationships. However, C. issatschenkoi is a nonsymbiotic planktonic organism and a frequent component of toxic water blooms. Cusperin is devoid of the cytotoxic activity reported for other pederin family members. Hence, our findings raise questions about the role of pederin analogues in cyanobacteria and broaden the knowledge of ecological distribution of this group of polyketides.
- MeSH
- bakteriální geny MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- magnetická rezonanční spektroskopie MeSH
- multigenová rodina MeSH
- peptidsynthasy metabolismus MeSH
- polyketidsynthasy metabolismus MeSH
- polyketidy izolace a purifikace metabolismus MeSH
- sinice genetika metabolismus MeSH
- symbióza MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method.
- MeSH
- Chlorella vulgaris metabolismus MeSH
- limita detekce MeSH
- lineární modely MeSH
- malonáty MeSH
- methansulfonáty MeSH
- methionin analýza metabolismus MeSH
- pronasa MeSH
- reprodukovatelnost výsledků MeSH
- selenomethionin analýza metabolismus MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
Puwainaphycins are a recently described group of β-amino fatty acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane-ethyl acetate-ethanol-water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min-1and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate-ethanol-water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane-ethyl acetate-ethanol-water (1:5:1:5, v/v/v/v) system, afforded compounds 3-7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min-1with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1-7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC-electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1-7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-amino-2-hydroxy tetradecanoic acid. Ahdoa: 3-amino-2-hydroxy dodecanoic acid.
- MeSH
- biomasa MeSH
- cyklické peptidy izolace a purifikace MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- lipopeptidy izolace a purifikace MeSH
- magnetická rezonanční spektroskopie MeSH
- protiproudá chromatografie metody MeSH
- sinice chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
Oxadiazines are heterocyclic compounds containing N-N-O or N-N-C-O system within a six membered ring. These structures have been up to now exclusively prepared via organic synthesis. Here, we report the discovery of a natural oxadiazine nocuolin A (NoA) that has a unique structure based on 1,2,3-oxadiazine. We have identified this compound in three independent cyanobacterial strains of genera Nostoc, Nodularia, and Anabaena and recognized the putative gene clusters for NoA biosynthesis in their genomes. Its structure was characterized using a combination of NMR, HRMS and FTIR methods. The compound was first isolated as a positive hit during screening for apoptotic inducers in crude cyanobacterial extracts. We demonstrated that NoA-induced cell death has attributes of caspase-dependent apoptosis. Moreover, NoA exhibits a potent anti-proliferative activity (0.7-4.5 μM) against several human cancer lines, with p53-mutated cell lines being even more sensitive. Since cancers bearing p53 mutations are resistant to several conventional anti-cancer drugs, NoA may offer a new scaffold for the development of drugs that have the potential to target tumor cells independent of their p53 status. As no analogous type of compound was previously described in the nature, NoA establishes a novel class of bioactive secondary metabolites.
- MeSH
- apoptóza účinky léků MeSH
- chromatografie kapalinová MeSH
- HeLa buňky MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- multigenová rodina MeSH
- oxaziny chemie farmakologie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sinice chemie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
