Health Effects of Toxin-Producing Cyanobacteria: “The CyanoHABs”

Increasingly, harmful algal blooms (HABs) are being reported worldwide due to several factors, primarily eutrophication, climate change and more scientific monitoring. All but cyanobacteria toxin poisonings (CTPs) are mainly a marine occurrence. CTPs occur in fresh (lakes, ponds, rivers and reservoirs) and brackish (seas, estuaries, and lakes) waters throughout the world. Organisms responsible include an estimated 40 genera but the main ones are Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nostoc, and Oscillatoria (Planktothrix). Cyanobacteria toxins (cyanotoxins) include cytotoxins and biotoxins with biotoxins being responsible for acute lethal, acute, chronic and sub-chronic poisonings of wild/domestic animals and humans. The biotoxins include the neurotoxins; ana-toxin-a, anatoxin-a(s) and saxitoxins plus the hepatotoxins; microcystins, nodularins and cylindrospermopsins. Confirmations of human deaths from cyanotoxins are limited to exposure through renal dialysis at a haemodialysis center in Caruaru, Brazil, in 1996. A major effort to compile all available information on toxic cyanobacteria including issues of human health, safe water practices, management, prevention and remediation have been published by the World Health Organization. This paper will review our current understanding of CTP's including their risk to human health.     

Estimating toxic cyanobacteria in a Brazilian reservoir by quantitative real-time PCR, based on the microcystin synthetase D gene

The production of microcystin toxins by cyanobacteria is an intrapopulation feature and the toxic and nontoxic genotypes can be separated only through molecular analyses targeting the mcy markers. Quantitative real-time PCR (qPCR) is a procedure that has been established, not only to detect but to specifically quantify these genotypes. In the present work, primers were designed for the mcyD region to estimate the number of cyanobacteria that are potential microcystin producers. Laboratory tests to verify the efficiency and the specificity of the primers were performed. The methodology was first established for single strain cultures and thereafter was applied in environmental water samples, from a reservoir located in the Brazilian savannah (“cerrado”). The results were very satisfactory, demonstrating the high efficiency and the specificity of the primers used, and their ability to detect different cyanobacteria genera. Of particular interest were the results showing a high proportion of toxic strains (as high as 100 %) in the environmental samples, as previously reported in another tropical system. Furthermore, the occurrence of a smaller fraction of toxic strains at high cyanobacteria densities, and of more toxic populations when fewer cyanobacteria were present, deserves further investigation. Although records of cyanobacteria blooms are very common in the tropics and suggest an increasing incidence of toxic populations, the present research is one of the few applying qPCR in a tropical environment. The results obtained here, by a technique that allows a more precise quantification and in situ follow-up of changes in toxicity, will make possible new observations of seasonal and spatial dynamics in these environments.     

Suitability of a cytotoxicity assay for detection of potentially harmful compounds produced by freshwater bloom-forming algae

Detecting harmful bioactive compounds produced by bloom-forming pelagic algae is important to assess potential risks to public health. We investigated the application of a cell-based bioassay: the rainbow trout gill-w1 cytotoxicity assay (RCA) that detects changes in cell metabolism. The RCA was used to evaluate the cytotoxic effects of (1) six natural freshwater lake samples from cyanobacteria-rich lakes in central Ontario, Canada; (2) analytical standards of toxins and noxious compounds likely to be produced by the algal communities in these lakes; and (3) complex mixtures of compounds produced by cyanobacterial and chrysophyte cultures. RCA provided a measure of lake water toxicity that could not be reproduced using toxin or noxious compound standards. RCA was not sensitive to toxins and only sensitive to noxious compounds at concentrations higher than reported environmental averages (EC₅₀ ≥ 10³ nM). Cultured algae produced bioactive compounds that had recognizable dose dependent and toxic effects as indicated by RCA. Toxicity of these bioactive compounds depended on taxa (cyanobacteria, not chrysophytes), growth stage (stationary phase more toxic than exponential phase), location (intracellular more toxic than extracellular) and iron status (cells in high-iron treatment more toxic than cells in low-iron treatment). The RCA provides a new avenue of exploration and potential for the detection of natural lake algal toxic and noxious compounds.     

Microalgae as sources of pharmaceuticals and other biologically active compounds

In the last decade the screening of microalgae, especially the cyanobacteria (blue-green algae), for antibiotics and pharmacologically active compounds has received ever increasing interest. A large number of antibiotic compounds, many with novel structures, have been isolated and characterised. Similarly many cyanobacteria have been shown to produce antiviral and antineoplastic compounds. A range of pharmacological activities have also been observed with extracts of microalgae, however the active principles are as yet unknown in most cases. Several of the bioactive compounds may find application in human or veterinary medicine or in agriculture. Others should find application as research tools or as structural models for the development of new drugs. The microalgae are particularly attractive as natural sources of bioactive molecules since these algae have the potential to produce these compounds in culture which enables the production of structurally complex molecules which are difficult or impossible to produce by chemical synthesis.     

Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.     

Methods to detect cyanobacteria and their toxins in the environment

Cyanobacteria blooms are since early times a cause for environmental concern because of their negative impact through the release of odors, water discoloration, and more dangerously through the release of toxic compounds (i.e. the cyanotoxins) that can affect both human and animal welfare. Surveillance of the aquatic ecosystems is therefore obligatory, and methods to achieve such require a prompt answer not only regarding the species that are producing the blooms but also the cyanotoxins that are being produced and/or released. Moreover, besides this well-known source of possible intoxication, it has been demonstrated the existence of several other potential routes of exposure, either for humans or other biota such as through food additives and in terrestrial environments (in plants, lichens, biological soil crusts) and the recognition of their harmful impact on less studied ecosystems (e.g. coral reefs). Nowadays, the most frequent approaches to detect toxic cyanobacteria and/or their toxins are the chemical-, biochemical-, and molecular-based methods. Above their particular characteristics and possible applications, they all bring to the environmental monitoring several aspects that are needed to be discussed and scrutinized. The end outcome of this review will be to provide newer insights and recommendations regarding the methods needed to apply in an environmental risk assessment program. Therefore, a current state of the knowledge concerning the three methodological approaches will be presented, while highlighting positive and negative aspects of each of those methods within the purpose of monitoring or studying cyanobacteria and their toxins in the environment.     

Bioactive polar lipids from Chroococcidiopsis sp. (Cyanobacteria)

Many studies indicate that various bioactive metabolites subsist in cyanobacteria. Glycolipids of cyanobacteria are reported as molecules that exert specific bioactivities. In this study, total lipids of Chroococcidiopsissp., a coccoid cyanobacterium isolated from a Greek cave, were separated into neutral and polar-lipids and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each polar lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of phospho-glyco analog of ceramide. Another fraction that was a potent inhibitor of PAF- as well as of thrombin-induced platelet aggregation was structurally elucidated as a phospho-acetylated glyco-analog of diglyceride. The fraction that induced platelet aggregation was identified as a phospho-acetylated-glyco analog of ceramide. These novel bioactive polar lipids in cyanobacteria in regard to the structure and biological activity may contribute to the allergic character of cyanobacteria.     

Phylogenetic Inferences Reveal a Large Extent of Novel Biodiversity in Chemically Rich Tropical Marine Cyanobacteria

Benthic marine cyanobacteria are known for their prolific biosynthetic capacities to produce structurally diverse secondary metabolites with biomedical application and their ability to form cyanobacterial harmful algal blooms. In an effort to provide taxonomic clarity to better guide future natural product drug discovery investigations and harmful algal bloom monitoring, this study investigated the taxonomy of tropical and subtropical natural product-producing marine cyanobacteria on the basis of their evolutionary relatedness. Our phylogenetic inferences of marine cyanobacterial strains responsible for over 100 bioactive secondary metabolites revealed an uneven taxonomic distribution, with a few groups being responsible for the vast majority of these molecules. Our data also suggest a high degree of novel biodiversity among natural product-producing strains that was previously overlooked by traditional morphology-based taxonomic approaches. This unrecognized biodiversity is primarily due to a lack of proper classification systems since the taxonomy of tropical and subtropical, benthic marine cyanobacteria has only recently been analyzed by phylogenetic methods. This evolutionary study provides a framework for a more robust classification system to better understand the taxonomy of tropical and subtropical marine cyanobacteria and the distribution of natural products in marine cyanobacteria.     

Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades

The genus of filamentous cyanobacteria, Lyngbya, has been found to be a rich source of bioactive metabolites. However, identification of such compounds from Lyngbya has largely focused on a few marine representatives. Here, we report on the pharmacology and toxicology of pahayokolide A from a freshwater isolate, Lyngbya sp. strain 15-2, from the Florida Everglades. Specifically, we investigated inhibition of microbial representatives and mammalian cell lines, as well as toxicity of the compound to both invertebrate and vertebrate models. Pahayokolide A inhibited representatives of Bacillus, as well as the yeast, Saccharomyces cerevisiae. Interestingly, the compound also inhibited several representatives of green algae that were also isolated from the Everglades. Pahayokolide A was shown to inhibit a number of cancer cell lines over a range of concentrations (IC50 varied from 2.13 to 44.57 microM) depending on the cell-type. When tested against brine shrimp, pahayokolide was only marginally toxic at the highest concentrations tested (1 mg/mL). The compound was, however, acutely toxic to zebrafish embryos (LC50=2.15 microM). Possible biomedical and environmental health aspects of the pahayokolides remain to be investigated; however, the identification of bioactive metabolites such as these demonstrates the potential of the Florida Everglades as source of new toxins and drugs.     

Bioprospecting microalgae as potential sources of "green energy"--challenges and perspectives (review)

Microalgae and cyanobacteria are potential foods, feeds, sources of high-value bioactive molecules and biofuels, and find tremendous applications in bioremediation and agriculture. Although few efforts have been undertaken to index the microalgal germplasm available in terms of lipid content, information on suitability of strains for mass multiplication and advances in development of methods for extraction and generating biofuel are scarce. Our review summarizes the potential of microalgae, latest developments in the field and analyzes the "pitfalls" in oversimplification of their promise in the years to come. Microalgae represent "green gold mines" for generating energy; however, the path to success is long and winding and needs tremendous and concerted efforts from science and industry, besides political will and social acceptance for overcoming the limitations. The major advantages of second generation biofuels based on microalgal systems, include their higher photon conversion efficiency, growth all around the year, even in wastewaters, and production of environment friendly biodegradable biofuels.     

Environmental factors associated with toxic cyanobacterial blooms across 20 drinking water reservoirs in a semi-arid region of Brazil

Cyanobacteria are known to produce a wide variety of bioactive, toxic secondary metabolites generally described as hepatotoxins, neurotoxins, cytotoxins, or dermatoxins. In Brazil, the regular monitoring of cyanobacterial toxins has intensified after the death of 65 patients in a hemodialysis clinic in Caruaru in the state of Pernambuco due to microcystin exposure. The primary objective of this study was to use multivariate statistics that incorporated environmental parameters (both biotic and abiotic) to forecast blooms of cyanobacteria and their toxic secondary metabolites in 20 drinking water reservoirs managed by the Water Treatment Company of Ceará (CAGECE) in the semi-arid region of Ceará, Brazil. Across four years (January 2013 to January 2017), 114 different phytoplankton taxa were identified, including 24 cyanobacterial taxa. In general, Ceará reservoirs were dominated by cyanobacteria due to eutrophication but also because of the dry and warm climate found throughout the region. Interestingly, specific cyanobacterial taxa were influenced by different biotic and abiotic factors. For example, nitrogen-to-phosphorus (N:P) and evaporation were positively related to saxitoxin-producing taxa, especially Raphidiopsis raciborskii, while temperature, electrical conductivity, total phosphorus, and transparency (measured as Secchi depth) were positively associated with microcystin-producing taxa, such as Microcystis aeruginosa. Climate forecasts predict higher evaporation and temperatures in the semi-arid Ceará region, which will likely magnify droughts and water scarcity as well as promote toxic cyanobacterial blooms in reservoirs in the future. Therefore, understanding the factors associated with algal blooms dominated by specific taxa is paramount for water resource management.     

BACTIBASE: a new web-accessible database for bacteriocin characterization

Background  

Bacteriocins are very diverse group of antimicrobial peptides produced by a wide range of bacteria and known for their inhibitory activity against various human and animal pathogens. Although many bacteriocins are now well characterized, much information is still missing or is unavailable to potential users. The assembly of such information in one central resource such as a database would therefore be of great benefit to the exploitation of these bioactive molecules in the present context of increasing antibiotic resistance and natural bio-preservation need.     

The expanding role of marine microbes in pharmaceutical development

Marine microbes have received growing attention as sources of bioactive metabolites and offer a unique opportunity to both increase the number of marine natural products in clinical trials as well as expedite their development. This review focuses specifically on those molecules currently in the clinical pipeline that are established or highly likely to be produced by bacteria based on expanding circumstantial evidence. We also include an example of how compounds from harmful algal blooms may yield both tools for measuring environmental change as well as leads for pharmaceutical development. An example of the karlotoxin class of compounds isolated from the dinoflagellate Karlodinium veneficum reveals a significant environmental impact in the form of massive fish kills, but also provides opportunities to construct new molecules for the control of cancer and serum cholesterol assisted by tools associated with rational drug design.     

Photoprotective compounds from marine organisms

The substantial loss in the stratospheric ozone layer and consequent increase in solar ultraviolet radiation on the earth’s surface have augmented the interest in searching for natural photoprotective compounds in organisms of marine as well as freshwater ecosystems. A number of photoprotective compounds such as mycosporine-like amino acids (MAAs), scytonemin, carotenoids and several other UV-absorbing substances of unknown chemical structure have been identified from different organisms. MAAs form the most common class of UV-absorbing compounds known to occur widely in various marine organisms; however, several compounds having UV-screening properties still need to be identified. The synthesis of scytonemin, a predominant UV-A-photoprotective pigment, is exclusively reported in cyanobacteria. Carotenoids are important components of the photosynthetic apparatus that serve both light-harvesting and photoprotective functions, either by direct quenching of the singlet oxygen or other toxic reactive oxygen species or by dissipating the excess energy in the photosynthetic apparatus. The production of photoprotective compounds is affected by several environmental factors such as different wavelengths of UVR, desiccation, nutrients, salt concentration, light as well as dark period, and still there is controversy about the biosynthesis of various photoprotective compounds. Recent studies have focused on marine organisms as a source of natural bioactive molecules having a photoprotective role, their biosynthesis and commercial application. However, there is a need for extensive work to explore the photoprotective role of various UV-absorbing compounds from marine habitats so that a range of biotechnological and pharmaceutical applications can be found.     

Detection of Anatoxin-a(s) in Environmental Samples of Cyanobacteria by Using a Biosensor with Engineered Acetylcholinesterases

Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.     

Underestimated biodiversity as a major explanation for the perceived rich secondary metabolite capacity of the cyanobacterial genus Lyngbya

Marine cyanobacteria are prolific producers of bioactive secondary metabolites responsible for harmful algal blooms as well as rich sources of promising biomedical lead compounds. The current study focused on obtaining a clearer understanding of the remarkable chemical richness of the cyanobacterial genus Lyngbya. Specimens of Lyngbya from various environmental habitats around Curaçao were analysed for their capacity to produce secondary metabolites by genetic screening of their biosynthetic pathways. The presence of biosynthetic pathways was compared with the production of corresponding metabolites by LC‐ESI‐MS² and MALDI‐TOF‐MS. The comparison of biosynthetic capacity and actual metabolite production revealed no evidence of genetic silencing in response to environmental conditions. On a cellular level, the metabolic origin of the detected metabolites was pinpointed to the cyanobacteria, rather than the sheath‐associated heterotrophic bacteria, by MALDI‐TOF‐MS and multiple displacement amplification of single cells. Finally, the traditional morphology‐based taxonomic identifications of these Lyngbya populations were combined with their phylogenetic relationships. As a result, polyphyly of morphologically similar cyanobacteria was identified as the major explanation for the perceived chemical richness of the genus Lyngbya, a result which further underscores the need to revise the taxonomy of this group of biomedically important cyanobacteria.     

Detection of anatoxin-a(s) in environmental samples of cyanobacteria by using a biosensor with engineered acetylcholinesterases

Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.     

Maternal consumption of non‐toxic Microcystis by Daphnia magna induces tolerance to toxic Microcystis in offspring

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Bioprospecting microalgae as potential sources of “Green Energy”—challenges and perspectives (Review)

Microalgae and cyanobacteria are potential food and feed, sources of high-value bioactive molecules and biofuels, and find tremendous applications in bioremediation and agriculture. Although few efforts have been undertaken to index the microalgal germplasm available in terms of lipid content, information on suitability of strains for mass multiplication and advances in development of methods for extraction and generating biofuel are scarce. Our review summarizes the potential of microalgae, latest developments in the field and analyzes the “pitfalls” in oversimplification of their promise in the years to come. Microalgae represent “green gold mines” for generating energy; however, the path to success is long and winding and needs tremendous and concerted efforts from science and industry, besides political will and social acceptance for overcoming the limitations. The major advantages of second generation biofuels based on microalgal systems, include their higher photon conversion efficiency, growth all around the year, even in wastewaters, and production of environment friendly biodegradable biofuels.     

Cadmium transport, resistance, and toxicity in bacteria, algae, and fungi

Cadmium is an important environmental pollutant and a potent toxicant to bacteria, algae, and fungi. Mechanisms of Cd toxicity and resistance are variable, depending on the organism. It is very clear that the form of the metal and the environment it is studied in, play an important role in how Cd exerts its effect and how the organism(s) responds. A wide range of Cd concentrations have been used to designate resistance in organisms. To date, no concentration has been specified that is applicable to all species studied under standardized conditions. Cadmium exerts its toxic effect(s) over a wide range of concentrations. In most cases, algae and cyanobacteria are the most sensitive organisms, whereas bacteria and fungi appear to be more resistant. In some bacteria, plasmid-encoded resistance can lead to reduced Cd2+ uptake. However, some Gram-negative bacteria without plasmids are just as resistant to Cd as are bacteria containing plasmids encoding for Cd resistance. According to Silver and Misra (1984), there is no evidence for enzymatic or chemical transformations associated with Cd resistance. Insufficient information is available on the genetics of Cd uptake and resistance in cyanobacteria and algae. Mechanisms remain largely unknown at this point in time. Cadmium is toxic to these organisms, causing severe inhibition of such physiological processes as growth, photosynthesis, and nitrogen fixation at concentrations less than 2 ppm, and often in the ppb range (Tables 2 and 3). Cadmium also causes pronounced morphological aberrations in these organisms, which are probably related to deleterious effects on cell division. This may be direct or indirect, as a result of Cd effects on protein synthesis and cellular organelles such as mitochondria and chloroplasts. Cadmium is accumulated internally in algae (Table 4) as a result of a two-phase uptake process. The first phase involves a rapid physicochemical adsorption of Cd onto cell wall binding sites, which are probably proteins and (or) polysaccharides. This is followed by a lag period and then a slow, steady intracellular uptake. This latter phase is energy dependent and may involve transport systems used to accumulate other divalent cations, such as Mn2+ and Ca2+. Some data indicate that Cd resistance, and possibly uptake, in algae and cyanobacteria is controlled by a plasmid-encoded gene(s). Although considerable information is available on Cd toxicity to, and uptake in fungi, further work is clearly needed in several areas. There is little information about Cd uptake by filamentous fungi, and even in yeasts, information on the specificity, kinetics, and mechanisms of Cd uptake is limited.(ABSTRACT TRUNCATED AT 400 WORDS)