Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Cell immunolocalization of ciguatoxin-like compounds in the benthic dinoflagellate Gambierdiscus australes M. Chinain & M.A. Faust by confocal microscopy.

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are able to produce potent neurotoxins like ciguatoxins (CTXs), which, after biooxidation in fish, are responsible for ciguatera intoxication. An isolate of G. australes from the Canary Islands, that revealed the presence of CTX-like compounds by immunosensing tools, was studied by immunocytochemistry to localize intracellular CTX-like compounds, using 8H4 monoclonal antibody that specifically recognizes the right wing of CTX1B and CTX3C analogues. Confocal microscopy observations of immunostained whole cells revealed a strong positive reaction on cell surface and all along the cell outline, while no reaction was detected inside the cells, probably because the antibody was not able to pass through thecal plates. Cell sections showed a positive antibody staining not only on thecal plates, but also inside cytoplasm, with numerous small dots and larger tubule-like reticulate structures. Small fluorescent dots were detected also on the nuclear surface. These observations indicate that CTX-like compounds are present in G. australes cytoplasm, and then are, at least in part, released to cover the cell surface.

Electrochemical biosensor for the dual detection of Gambierdiscus australes and Gambierdiscus excentricus in field samples. First report of G. excentricus in the Balearic Islands.

Several genera of marine dinoflagellates are known to produce bioactive compounds that affect human health. Among them, Gambierdiscus and Fukuyoa stand out for their ability to produce several toxins, including the potent neurotoxic ciguatoxins (CTXs), which accumulate through the food web. Once fishes contaminated with CTXs are ingested by humans, it can result in an intoxication named ciguatera. Within the two genera, only some species are able to produce toxins, and G. australes and G. excentricus have been highlighted to be the most abundant and toxic. Although the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, their presence in subtropical and temperate regions has been recently recorded. In this work, the combined use of species-specific PCR primers for G. australes and G. excentricus modified with short oligonucleotide tails allowed the development of a multiplex detection system for these two toxin-producing species. Simultaneous detection was achieved using capture probes specific for G. australes and G. excentricus immobilized on maleimide-coated magnetic beads (MBs), separately placed on the working electrodes of a dual electrode array. Additionally, a rapid DNA extraction technique based on a portable bead beater system and MBs was developed, significantly reducing the extraction time (from several hours to 30 min). The developed technique was able to detect as low as 10 cells of both Gambierdiscus species and allowed the first detection of G. excentricus in the Balearic Islands in 8 out of the 12 samples analyzed. Finally, field samples were screened for CTXs with an immunosensor, successfully reporting 13.35 ± 0.5 pg CTX1B equiv. cell-1 in one sample and traces of toxins in 3 out of the 9 samples analyzed. These developments provide rapid and cost-effective strategies for ciguatera risk assessment, with the aim of guaranteeing seafood safety.

Nucleic acid lateral flow dipstick assay for the duplex detection of Gambierdiscus australes and Gambierdiscus excentricus.

The proliferation of harmful microalgae endangers aquatic ecosystems and can have serious economic implications on a global level. Harmful microalgae and their associated toxins also pose a threat to human health since they can cause seafood-borne diseases such as ciguatera. Implementation of DNA-based molecular methods together with appropriate detection strategies in monitoring programs can support the efforts for effective prevention of potential outbreaks. A PCR-lateral flow assay (PCR-LFA) in dipstick format was developed in this work for the detection of two Gambierdiscus species, G. australes and G. excentricus, which are known to produce highly potent neurotoxins known as ciguatoxins and have been associated with ciguatera outbreaks. Duplex PCR amplification of genomic DNA from strains of these species utilizing species-specific ssDNA tailed primers and a common primer containing the binding sequence of scCro DNA binding protein resulted in the generation of hybrid ssDNA-dsDNA amplicons. These were captured on the dipsticks via hybridization with complementary probes and detected with a scCro/carbon nanoparticle (scCro/CNPs) conjugate. The two different test zones on the dipsticks allowed the discrimination of the two species and the assay exhibited high sensitivity, 6.3 pg/µL of genomic DNA from both G. australes and G. excentricus. The specificity of the approach was also demonstrated using genomic DNA from non-target Gambierdiscus species and other microalgae genera which did not produce any signals. The possibility to use cells directly for amplification instead of purified genomic DNA suggested the compatibility of the approach with field sample testing. Future work is required to further explore the potential use of the strategy for on-site analysis and its applicability to other toxic species.

Further advance of Gambierdiscus Species in the Canary Islands, with the First Report of Gambierdiscus belizeanus.

Ciguatera Poisoning (CP) is a human food-borne poisoning that has been known since ancient times to be found mainly in tropical and subtropical areas, which occurs when fish or very rarely invertebrates contaminated with ciguatoxins (CTXs) are consumed. The genus of marine benthic dinoflagellates Gambierdiscus produces CTX precursors. The presence of Gambierdiscus species in a region is one indicator of CP risk. The Canary Islands (North Eastern Atlantic Ocean) is an area where CP cases have been reported since 2004. In the present study, samplings for Gambierdiscus cells were conducted in this area during 2016 and 2017. Gambierdiscus cells were isolated and identified as G. australes, G. excentricus, G. caribaeus, and G. belizeanus by molecular analysis. In this study, G. belizeanus is reported for the first time in the Canary Islands. Gambierdiscus isolates were cultured, and the CTX-like toxicity of forty-one strains was evaluated with the neuroblastoma cell-based assay (neuro-2a CBA). G. excentricus exhibited the highest CTX-like toxicity (9.5-2566.7 fg CTX1B equiv. cell-1) followed by G. australes (1.7-452.6.2 fg CTX1B equiv. cell-1). By contrast, the toxicity of G. belizeanus was low (5.6 fg CTX1B equiv. cell-1), and G. caribaeus did not exhibit CTX-like toxicity. In addition, for the G. belizeanus strain, the production of CTXs was evaluated with a colorimetric immunoassay and an electrochemical immunosensor resulting in G. belizeanus producing two types of CTX congeners (CTX1B and CTX3C series congeners) and can contribute to CP in the Canary Islands.

Addressing the Analytical Challenges for the Detection of Ciguatoxins Using an Electrochemical Biosensor.

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 µg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

Maitotoxin-4, a Novel MTX Analog Produced by Gambierdiscus excentricus.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.