Search Results (28)

The increasing occurrence of harmful cyanobacterial blooms in freshwater ecosystems poses important risks to aquatic organisms and human health due to the production of bioactive secondary metabolites such as cyanopeptides. While analytical methods for microcystins are well developed, there is a notable lack of validated protocols for the broader spectrum of cyanopeptides in biota. This study presents the development and validation of a robust UHPLC-QqQ-MS method for the simultaneous extraction, cleanup, and quantification of 27 cyanopeptides, including microcystins, anabaenopeptins, microginins, aeruginosins, aeruginoguanidine, and nodularin, in fish muscle, liver, and whole fish tissues. Comprehensive optimization was conducted to minimize matrix effects and analyte losses during every step of sample preparation. The method demonstrated generally high recoveries (28–98%), good precision (RSD < 20%), and sensitivity, with MQLs below 0.5 ng g⁻¹ for most analytes. Microginins posed analytical challenges due to their amphiphilic structure, which contributed to significant losses during filtration and extraction; the reasoning is discussed. Application to wild fish collected after a mass mortality event revealed no detectable cyanopeptide contamination but confirmed the method’s suitability for comprehensive detection. This represents an important advancement in cyanopeptide analysis, offering a valuable tool for environmental risk assessment and food safety evaluation related to harmful cyanobacteria. Full article

Over the past 15 years, Nodularia spumigena blooms in the Gulf of Gdańsk (southern Baltic Sea) have reached exceptional intensity, accounting for up to 90% of phytoplankton biomass during peak summer periods. Our long-term observations revealed oscillations in blooms intensity, with peak nodularin concentrations (up to 45,000 μg/L) recorded in 2012, 2015, and 2018—the highest levels of this toxin documented to date in both the Baltic Sea and worldwide. An extreme cyanobacterial bloom in 2018, caused by unusually high air and water temperatures, covered almost the whole surface of the Gulf of Gdańsk, causing multi-day closures of bathing areas. During this bloom, high levels of microcystins (up to 6640 μg/L MC-LR) were also detected, as well as the presence of 42 cyanopeptides, mainly anabaenopeptides and spumigins, which were present at concentrations 5–10 times higher than hepatotoxins. Full article

In non-ribosomal peptide synthesis of cyanobacteria, promiscuous adenylation domains allow the incorporation of clickable non-natural amino acids into peptide products—namely into microcystins (MCs) or into anabaenopeptins (APs): 4-azidophenylalanine (Phe-Az), N-propargyloxy-carbonyl-L-lysine (Prop-Lys), or O-propargyl-L-tyrosine (Prop-Tyr). Subsequently, chemo-selective labeling is used to visualize the clickable cyanopeptides using Alexa Fluor 488 (A488). In this study, the time-lapse build up or decline of azide- or alkyne-modified MCs or APs was visualized during maximum growth, specifically MC biosynthesis in Microcystis aeruginosa and AP biosynthesis in Planktothrix agardhii. Throughout the time-lapse build up or decline, the A488 signal occurred with heterogeneous intracellular distribution. There was a fast increase or decrease in the A488 signal for either Prop-Tyr or Prop-Lys, while a delayed or unobservable A488 signal for Phe-Az was related to increased cell size as well as a reduction in growth and autofluorescence. The proportion of clickable MC/AP in peptide extracts as recorded by a chemical–analytical technique correlated positively with A488 labeling intensity quantified via laser-scanning confocal microscopy for individual cells or via flow cytometry at the population level. It is concluded that chemical modification of MC/AP can be used to track intracellular dynamics in biosynthesis using both analytical chemistry and high-resolution imaging. Full article

Anthropogenic influences have increased global warming and eutrophication, escalating the frequency and severity of harmful cyanobacterial blooms (cHABs) in freshwater ecosystems. These blooms release cyanopeptides, a diverse class of bioactive compounds with varying acute and chronic toxicities upon ingestion. To date, research has prioritized acutely toxic cyanopeptides like microcystins. As a result, significantly less is known about other freshwater cyanopeptides. This review highlights six understudied cyanopeptide classes, anabaenopeptins, cyanopeptolins, aeruginosamides, aeruginosins, microginins, and cyclamides, and provides a comprehensive overview of their molecular structures, toxicological profiles, environmental concentrations, and known degradation pathways. Given the potential toxicity, increased environmental abundance, and environmental stability of many cyanopeptides in freshwater sources, further research is needed to understand if degraded cyanopeptides are still biologically active prior to entering drinking water to ensure public health. Full article

Cyanobacteria are an ancient group of prokaryotes capable of oxygenic photosynthesis. Recently, thermal crises symptoms in hot springs have been associated with acute cyanopeptides poisoning. The aim of this work is to develop a fast, easy and reliable method to monitor the presence of toxic cyanopeptides in geothermal waters. The analytical method based on capillary zone electrophoresis coupled with tandem mass spectrometry (CZE-MS/MS) was developed for the simultaneous determination of 14 cyanopeptides in less than 7.5 min. A basic 50 mM ammonium acetate buffer at pH 10.2 was selected as the background electrolyte, positive electrospray ionization (ESI+) was employed for all compounds, and a salting-out assisted liquid–liquid extraction (SALLE) protocol with acetonitrile as an extraction solvent and MgSO₄ as an auxiliary salting-out agent was optimized as sample treatment. Six natural hot springs in the province of Granada (Andalucía, Spain) were sampled at the beginning of the summer season (June) and at the end (September). Biomass collected at two sample points (Santa Fe and Zújar) contained cyanobacteria cells from the genera Phormidium, Leptolyngbya, and Spirulina. Nevertheless, cyanotoxins covered by this work were not found in any of the water samples analyzed. The greenness and transferability of the method was evaluated highlighting its sustainability and applicability. Full article

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In this study, time-lapse experiments have been performed using pulsed feeding of three different non-AAs in order to observe the synthesis or decline of azide- or alkyne-modified microcystins (MCs) or anabaenopeptins (APs). The cyanobacteria Microcystis aeruginosa and Planktothrix agardhii were grown under maximum growth rate conditions (r = 0.35–0.6 and 0.2–0.4 (day⁻¹), respectively) in the presence of non-AAs for 12–168 h. The decline of the azide- or alkyne-modified MC or AP was observed via pulse-feeding. In general, the increase in clickable MC/AP in peptide content reached a plateau after 24–48 h and was related to growth rate, i.e., faster-growing cells also produced more clickable MC/AP. Overall, the proportion of clickable MC/AP in the intracellular fraction correlated with the proportion observed in the dissolved fraction. Conversely, the overall linear decrease in clickable MC/AP points to a rather constant decline via dilution by growth instead of a regulated or induced release in the course of the synthesis process. Full article

Common bloom-forming cyanobacteria produce complex strain-specific mixtures of secondary metabolites. The beneficial and toxic properties of these metabolite mixtures have attracted both research and public health interest. The advancement of mass spectrometry-based platforms and metabolomics data processing has accelerated the identification of new metabolites and feature dereplication from microbial sources. The objective of this study was to use metabolomics data processing to decipher the intracellular cyanopeptide diversity of six Planktothrix strains collected from Canadian lakes. Data-dependent acquisition experiments were used to collect a non-targeted high-resolution mass spectrometry dataset. Principal component analysis and factor loadings were used to visualize cyanopeptide variation between strains and identified features contributing to the observed variation. GNPS molecular networking was subsequently used to show the diversity of cyanopeptides produced by the Planktothrix strains. Each strain produced a unique mixture of cyanopeptides, and a total of 225 cyanopeptides were detected. Planktothrix sp. CPCC 735 produced the most (n = 68) cyanopeptides, and P. rubescens CPCC 732 produced the fewest (n = 27). Microcystins and anabaenopeptins were detected from all strains. Cyanopeptolins, microviridins and aeruginosins were detected from five, four and two strains, respectively. Cyanopeptolin (n = 80) and anabaenopeptin (n = 61) diversity was the greatest, whereas microcystins (n = 21) were the least diverse. Interestingly, three of the P. rubescens strains had different cyanopeptide profiles, despite being collected from the same lake at the same time. This study highlights the diversity of cyanopeptides produced by Planktothrix and further hints at the underestimated cyanopeptide diversity from subpopulations of chemotypic cyanobacteria in freshwater lakes. Full article

A seasonal plethora of cyanobacteria in the plankton community can have severe implications, not only for water ecosystems but also for the availability of treated water. The catchment of the Obrzyca River (a source of drinking water) is seasonally exposed to harmful cyanobacterial bloom. Previous studies (2008–2012; 2019) revealed that the most polluted water of the Obrzyca River was Uście, close to the outlet of Rudno Lake (at the sampling point). Therefore, the effect on this lake was specifically examined in this study. Sampling was performed from May to September at that site and from July to September 2020 at Rudno Lake. The conducted analysis revealed a massive growth of Aphanizomenon gracile, especially in Rudno Lake. The results showed not only the distinct impact of cyanobacterial bloom on phytoplankton biodiversity but also the presence of microcystins and other cyanopeptides in both sampling points. The maximal total concentration of microcystins (dmMC-RR, MC-RR, dmMC-LR, MC-LR, MC-LY, MC-YR) equaled 57.3 μg/L and the presence of cyanopeptides (aeruginosin, anabaenopeptin) was originally determined in Rudno Lake, August 2021. The presence of these toxins was highlighted in our results for the first time. The same samples from the lake were the most toxic in biotoxicological investigations using the planarian Dugesia tigrina. The performed bioassays proved that D. tigrina is a sensitive bioindicator for cyanotoxins. The physical and chemical indicators of water quality, i.e., color, temperature, total suspended solids, and total nitrogen and phosphorus, showed a significant correlation among each other and towards cyanobacterial abundance and microcystin concentrations. Full article

Cyanopeptolins (CPs) are one of the most commonly occurring class of cyanobacterial nonribosomal peptides. For the majority of these compounds, protease inhibition has been reported. In the current work, the structural diversity of cyanopeptolins produced by Nostoc edaphicum CCNP1411 was explored. As a result, 93 CPs, including 79 new variants, were detected and structurally characterized based on their mass fragmentation spectra. CPs isolated in higher amounts were additionally characterized by NMR. To the best of our knowledge, this is the highest number of cyanopeptides found in one strain. The biological assays performed with the 34 isolated CPs confirmed the significance of the amino acid located between Thr and the unique 3-amino-6-hydroxy-2-piperidone (Ahp) on the activity of the compounds against serine protease and HeLa cancer cells. Full article

Neuroblastoma is the most prevalent extracranial solid tumor in pediatric patients, originating from sympathetic nervous system cells. Metastasis can be observed in approximately 70% of individuals after diagnosis, and the prognosis is poor. The current care methods used, which include surgical removal as well as radio and chemotherapy, are largely unsuccessful, with high mortality and relapse rates. Therefore, attempts have been made to incorporate natural compounds as new alternative treatments. Marine cyanobacteria are a key source of physiologically active metabolites, which have recently received attention owing to their anticancer potential. This review addresses cyanobacterial peptides’ anticancer efficacy against neuroblastoma. Numerous prospective studies have been carried out with marine peptides for pharmaceutical development including in research for anticancer potential. Marine peptides possess several advantages over proteins or antibodies, including small size, simple manufacturing, cell membrane crossing capabilities, minimal drug–drug interactions, minimal changes in blood–brain barrier (BBB) integrity, selective targeting, chemical and biological diversities, and effects on liver and kidney functions. We discussed the significance of cyanobacterial peptides in generating cytotoxic effects and their potential to prevent cancer cell proliferation via apoptosis, the activation of caspases, cell cycle arrest, sodium channel blocking, autophagy, and anti-metastasis behavior. Full article

Cyanobacterial blooms that release biologically active metabolites into the environment are increasing in frequency as a result of the degradation of freshwater ecosystems globally. The microcystins are one group of cyanopeptides that are extensively studied and included in water quality risk management frameworks. Common bloom-forming cyanobacteria produce incredibly diverse mixtures of other cyanopeptides; however, data on the abundance, distribution, and biological activities of non-microcystin cyanopeptides are limited. We used non-targeted LC-MS/MS metabolomics to study the cyanopeptide profiles of five Microcystis strains: four M. aeruginosa and one M. flos-aquae. Multivariate analysis and GNPS molecular networking demonstrated that each Microcystis strain produced a unique mixture of cyanopeptides. In total, 82 cyanopeptides from the cyanopeptolin (n = 23), microviridin (n = 18), microginin (n = 12), cyanobactin (n = 14), anabaenopeptin (n = 6), aeruginosin (n = 5), and microcystin (n = 4) classes were detected. Microcystin diversity was low compared with the other detected cyanopeptide classes. Based on surveys of the literature and spectral databases, most cyanopeptides represented new structures. To identify growth conditions yielding high amounts of multiple cyanopeptide groups, we next examined strain-specific cyanopeptide co-production dynamics for four of the studied Microcystis strains. When strains were cultivated in two common Microcystis growth media (BG-11 and MA), the qualitative cyanopeptides profiles remained unchanged throughout the growth cycle. For each of the cyanopeptide groups considered, the highest relative cyanopeptide amounts were observed in the mid-exponential growth phase. The outcomes of this study will guide the cultivation of strains producing common and abundant cyanopeptides contaminating freshwater ecosystems. The synchronous production of each cyanopeptide group by Microcystis highlights the need to make more cyanopeptide reference materials available to investigate their distributions and biological functions. Full article

Cyanobacteria produce a wide range of structurally diverse cyanotoxins and bioactive cyanopeptides in freshwater, marine, and terrestrial ecosystems. The health significance of these metabolites, which include genotoxic- and neurotoxic agents, is confirmed by continued associations between the occurrence of animal and human acute toxic events and, in the long term, by associations between cyanobacteria and neurodegenerative diseases. Major mechanisms related to the neurotoxicity of cyanobacteria compounds include (1) blocking of key proteins and channels; (2) inhibition of essential enzymes in mammalian cells such as protein phosphatases and phosphoprotein phosphatases as well as new molecular targets such as toll-like receptors 4 and 8. One of the widely discussed implicated mechanisms includes a misincorporation of cyanobacterial non-proteogenic amino acids. Recent research provides evidence that non-proteinogenic amino acid BMAA produced by cyanobacteria have multiple effects on translation process and bypasses the proof-reading ability of the aminoacyl-tRNA-synthetase. Aberrant proteins generated by non-canonical translation may be a factor in neuronal death and neurodegeneration. We hypothesize that the production of cyanopeptides and non-canonical amino acids is a more general mechanism, leading to mistranslation, affecting protein homeostasis, and targeting mitochondria in eukaryotic cells. It can be evolutionarily ancient and initially developed to control phytoplankton communities during algal blooms. Outcompeting gut symbiotic microorganisms may lead to dysbiosis, increased gut permeability, a shift in blood-brain-barrier functionality, and eventually, mitochondrial dysfunction in high-energy demanding neurons. A better understanding of the interaction between cyanopeptides metabolism and the nervous system will be crucial to target or to prevent neurodegenerative diseases. Full article

The genus of cyanobacteria Microcystis is one of the most recurrent in blooms and is associated with the hepatotoxin microcystin production. In addition to cyanotoxins, these bacteria produce a wide range of secondary metabolites with a wide repertoire of activities. The co-occurrence of cyanotoxins and other cyanopeptides during blooming is quite common, and the negative effects are not always limited to one class of toxins, which makes it essential to investigate the toxicity of the other compounds individually. The objective of this study was to isolate the cyanopeptide microcyclamide produced by the strain Microcystis aeruginosa LTPNA 08 by liquid chromatography coupled to high-resolution mass spectrometry with a quadrupole-time-of-flight analyzer (LC-HR-QTOF-MS/MS) and to evaluate its acute toxicity in embryos of Danio rerio through the Fish Embryo Acute Toxicity (FET) assay. The fraction containing microcyclamide (95% purity) caused lethality in 62% of the embryos after 96 h exposure (50 µg mL⁻¹), with evidence of cardiotoxicity (cardiac edema). The calculated LC₅₀ value was 42.98 µg mL⁻¹ (with a concentration range of 37.79–48.89 µg mL⁻¹). The characterization of the secondary metabolites produced by cyanobacteria and the investigation of the toxicity of these compounds individually are essential for the identification of the substances responsible for negative effects on living organisms and on the ecosystem, in addition to assisting in the development of risk management policies. Full article

Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins. Full article

Balneotherapy can cause adverse reactions to the usual doses of application of treatments, and consists of a nonspecific clinical picture, the so-called “thermal crisis” or “balneointoxication”. Despite its clinical similarity (gastric discomfort, hepatic congestive outbreaks, cutaneous reactions, etc.) with that observed in acute cyanotoxin poisonings, a thermal crisis has never been associated with the abundant growth of potentially toxic cyanobacteria in mineral water sources. The aim of this work was to verify the hypothetical involvement of cyanotoxins in this clinical picture. Several samples (21) of 18 sources, representative of the different mineral–medicinal waters present in Galicia (northwest Spain) and mostly used for balneotherapy, were collected during September 2018. Samples were filtered and the algae retained were extracted with sonication using acidified methanol and analysed with LC-ESI-HRMS. The target analysis of the cellular matrix samples (limit of detection = 0.01–0.05 µg L⁻¹) did not show nodularin or any of the microcystins (MCs) for which standards are available (MC-dmRR, RR, dmLR, YR, LR, WR, LA, LY, LW and LF). The presence of other MCs, nodularins and related cyanobacterial peptides (CPs) was observed with a suspect screening analysis of cyanotoxins, based on an HRMS home-made database of 157 MCs, 10 nodularins, cylindrospermopsin and 29 CPs previously described in the literature. Signals not referenced in the literature were identified as CPs. Based on HRMS and restrictive criteria (accuracy, isotopic pattern, diagnostic fragments, elements considered, charge, ring plus double bond equivalents and nitrogen rule), the signals were confirmed. In summary, 12 MCs, 2 nodularins and 2 CPs were qualitatively detected. A list of all tentatively identified cyanopeptides in each sample was reported, including the retention times, ion signal type, proposed molecular formula, theoretical m/z, samples where each signal was detected, mass accuracy of measures and their isotopic pattern scores. CP-2 was detected in 14 samples, and it was confirmed as a signal from a cyanobacterial peptide, but with more unsaturations than analogous MCs. The presence of MC-OiaA and MC-OiaAba in three samples was also noteworthy. In addition, [seco-2/3]NOD-R was detected in five samples. Full article