The Marine Neurotoxin Brevetoxin (PbTx-2) Inhibits Karenia brevis and Mammalian Thioredoxin Reductases by Targeting Different Residues.

The brevetoxins, neurotoxins produced by Karenia brevis, the Florida red tide dinoflagellate, effect fish and wildlife mortalities and adverse public health and economic impacts during recurrent blooms. Knowledge of the biochemical consequences of toxin production for K. brevis could provide insights into an endogenous role of the toxins, yet this aspect has not been thoroughly explored. In addition to neurotoxicity, the most abundant of the brevetoxins, PbTx-2, inhibits mammalian thioredoxin reductase (TrxR). The thioredoxin system, composed of the enzymes TrxR and thioredoxin (Trx), is present in all living organisms and is responsible in part for maintaining cellular redox homeostasis. Herein, we describe the cloning, expression, and semisynthesis of the selenoprotein TrxR from K. brevis (KbTrxR) and reductase activity toward a variety of substrates. Unlike mammalian TrxR, KbTrxR reduces oxidized glutathione (GSSG). We further demonstrate that PbTx-2 is an inhibitor of KbTrxR. Covalent adducts between KbTrxR and rat TrxR were detected by mass spectrometry. While both enzymes are adducted at or near the catalytic centers, the specific residues are distinct. Biochemical differences reported for high and low toxin producing strains of K. brevis are consistent with the inhibition of KbTrxR and suggest that PbTx-2 is an endogenous regulator of this critical enzyme.

Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, ß-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, ß-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light-Harvesting Complex II (LHCII) of Photosystem II.

The brevetoxins are neurotoxins that are produced by the "Florida red tide" dinoflagellate Karenia brevis. They bind to and activate the voltage-gated sodium channels in higher organisms, specifically the Nav 1.4 and Nav 1.5 channel subtypes. However, the native physiological function that the brevetoxins perform for K. brevis is unknown. By using fluorescent and photoactivatable derivatives, brevetoxin was shown to localize to the chloroplast of K. brevis where it binds to the light-harvesting complex II (LHCII) and thioredoxin. The LHCII is essential to non-photochemical quenching (NPQ), whereas thioredoxins are critical to the maintenance of redox homeostasis within the chloroplast and contribute to the scavenging of reactive oxygen. A culture of K. brevis producing low levels of toxin was shown to be deficient in NPQ and produced reactive oxygen species at twice the rate of the toxic culture, implicating a role in NPQ for the brevetoxins.

Synthesis, receptor binding and activity of iso and azakainoids.

Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycloaddition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced directly to the pyrazolidine. An unexpected trans-cis isomerization is observed during Hg/Al reduction of the 1-pyrazoline NN bond. Alternatively, when TMS diazomethane is used as the dipole, the resulting 2-pyrazoline obtained after desilylation may be reduced with NaCNBH3 to provide the trans azakainate analog exclusively. The synthesis of an unsubstituted isokainoid via Michael addition is also described. Glutamate receptor binding assays revealed that the azakaniod has a moderate affinity for unspecified glutamate receptors. Membrane depolarization of Aplysia neurons upon application of the azakainoid demonstrates that it is an ionotropic glutamate receptor agonist.

Brevetoxin induces a shift in the redox state of the proteome and unfolded protein response in human lymphoblast cells that can be alleviated with the acrolein scavenger MESNA.

Human lymphoblast cells were treated with the marine algal toxin, brevetoxin-2 (PbTx-2), and its effects on the proteome were assessed by redox proteomics using cysteine reactive tandem mass tags (TMT). Additionally, cells were simultaneously treated with PbTx-2 and the antioxidant and acrolein scavenger sodium 2-mercaptoethylsulfonate (MESNA) to determine if MESNA could prevent the proteomic effects of brevetoxin-2. A massive shift in the redox state of the proteome of brevetoxin-2 treated cells was observed. The main pathway affected was genetic information processing. Significantly oxidized proteins included Trx-1, peroxyredoxins (Prxs), ribosomal proteins, and the eukaryotic initiation factor 2 ß subunit (eIF2ß). Proteins that were overexpressed in brevetoxin-treated cells included four folding chaperones. These effects were diminished in the presence of MESNA indicating that MESNA may act through its antioxidant properties or as a brevetoxin scavenger. These studies provide novel insights into new prophylactics for brevetoxicosis in humans and wildlife.

The structures of three metabolites of the algal hepatotoxin okadaic acid produced by oxidation with human cytochrome P450.

Four metabolites of okadaic acid were generated by incubation with human recombinant cytochrome P450 3A4. The structures of two of the four metabolites have been determined by MS/MS experiments and 1D and 2D NMR methods using 94 and 133 µg of each metabolite. The structure of a third metabolite was determined by oxidation to a metabolite of known structure. Like okadaic acid, the metabolites are inhibitors of protein phosphatase PP2A. Although one of the metabolites does have an α,ß unsaturated carbonyl with the potential to form adducts with an active site cysteine, all of the metabolites are reversible inhibitors of PP2A.

Biosynthesis of Athmu, a α,γ-hydroxy-ß-amino acid of pahayokolides A-B.

Pahayokolides A-B are cyanobacteria derived non-ribosomal peptides which exhibit cytotoxicity against a number of cancer cell lines. The biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu) moiety has been investigated using stable isotope incorporation experiments. While α-ketoisocaproic acid (α-KIC), α-hydroxyisocaproic acid (α-HIC) and leucine all serve as precursors to Athmu, the feeding of [1-(13)C] α-KIC results in more than threefold greater (13)C enrichment than the other precursors. This result suggests that α-KIC is the immediate precursor which is selected and activated by the adenylation domain of the loading NRPS module and subsequently reduced in a fashion similar to that of the recently identified pathways for cryptophycins A-B, cereulide and valinomycin.

Biosynthetic origin of the 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid moiety and absolute configuration of pahayokolides A and B.

Pahayokolides A and B are cyclic undecapeptides that were isolated from the cyanobacterium Lyngbya sp. They contain the unusual α-hydroxy-ß-amino acid 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configurations of the amino acids of the pahayokolides, except for the four oxygen-bearing stereocenters of Athmu, have been determined by Marphy's method. Incorporation of labeled leucine and acetate precursors into the pahayokolides has established that Athmu is derived from a leucine or α-keto isocaproic acid starter unit, which is further extended with three acetate units.

Essential components of the xanthophyll cycle differ in high and low toxin Karenia brevis.

The dinoflagellate Karenia brevis, blooms annually in the Gulf of Mexico, producing a suite of neurotoxins known as the brevetoxins. The cellular toxin content of K. brevis, however, is highly variable between or even within strains. Herein, we investigate physiological differences between high (KbHT) and low (KbLT) toxin producing cultures both derived from the Wilson strain, related to energy-dependent quenching (qE) by photosystem II, and reduced thiol content of the proteome. We demonstrate that gene and protein expression of the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (Dde) and monogalactosyldiacylglycerol (MGDG) synthase are not significantly different in the two cultures. Using redox proteomics, we report a significantly higher reduced cysteine content in the low toxin proteome, including plastid localized thioredoxin reductase (Trx) which can result in inactivation of Dde and activation of MGDG synthase. We also report significant differences in the lipidomes of KbHT and KbLT with respect to MGDG, which facilitates the xanthophyll cycle.

New peptides isolated from Lyngbya species: a review.

Cyanobacteria of the genus Lyngbya have proven to be prodigious producers of secondary metabolites. Many of these compounds are bioactive and show potential for therapeutic use. This review covers peptides and hybrid polyketide-non-ribosomal peptides isolated from Lyngbya species. The structures and bioactivities of 50 Lyngbya peptides which were reported since 2007 are presented.

Biodegradation of polyether algal toxins--isolation of potential marine bacteria.

Marine algal toxins such as brevetoxins, okadaic acid, yessotoxin, and ciguatoxin are polyether compounds. The fate of polyether toxins in the aqueous phase, particularly bacterial biotransformation of the toxins, is poorly understood. An inexpensive and easily available polyether structural analog salinomycin was used for enrichment and isolation of potential polyether toxin degrading aquatic marine bacteria from Florida bay area, and from red tide endemic sites in the South Florida Gulf coast. Bacterial growth on salinomycin was observed in most of the enrichment cultures from both regions with colony forming units ranging from 0 to 6×10(7) per mL. The salinomycin biodegradation efficiency of bacterial isolates determined using LC-MS ranged from 22% to 94%. Selected bacterial isolates were grown in media with brevetoxin as the sole carbon source to screen for brevetoxin biodegradation capability using ELISA. Out of the two efficient salinomycin biodegrading isolates MB-2 and MB-4, maximum brevetoxin biodegradation efficiency of 45% was observed with MB-4, while MB-2 was unable to biodegrade brevetoxin. Based on 16S rRNA sequence similarity MB-4 was found have a match with Chromohalobacter sp.

Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Effectors of thioredoxin reductase: Brevetoxins and manumycin-A.

The activities of two effectors, brevetoxin (PbTx) and manumycin-A (Man-A), of thioredoxin reductase (TrxR) have been evaluated against a series of fourteen TrxR orthologs originating from mammals, insects and protists and several mutants. Man-A, a molecule with numerous electrophilic sites, forms a covalent adduct with most selenocystine (Sec)-containing TrxR enzymes. The evidence also demonstrates that Man-A can form covalent adducts with some non-Sec-containing enzymes. The activities of TrxR enzymes towards various substrates are moderated by Man-A either positively or negatively depending on the enzyme. In general, the reduction of substrates by Sec-containing TrxR is inhibited and NADPH oxidase activity is activated. For non-Sec-containing TrxR the effect of Man-A on the reduction of substrates is variable, but NADPH oxidase activity can be activated even in the absence of covalent modification of TrxR. The effect of PbTx is less pronounced. A smaller subset of enzymes is affected by PbTx. With a single exception, the activities of most of this subset are activated. Although both PbTx variants can react with selenocysteine, a stable covalent adduct is not formed with any of the TrxR enzymes. The key findings from this work are (i) the identification of an alternate mechanism of toxicity for the algal toxin brevetoxin (ii) the demonstration that covalent modification of TrxR is not a prerequisite for the activation of NADPH oxidase activity of TrxR and (iii) the identification of an inhibitor which can discriminate between cytosolic and mitochondrial TrxR.

Allelopathic activity among Cyanobacteria and microalgae isolated from Florida freshwater habitats.

We evaluated allelopathic interactions between strains of Cyanobacteria and green algae isolated from south and central Florida. Allelopathy, including inhibition or stimulation of growth, was assessed by cocultivation of each of the isolated strains, as well as by evaluation of extracts prepared from the isolates. All of the strains of Cyanobacteria, and four of the six isolates of green algae, showed some allelopathic activity (i.e. inhibition or stimulation of the growth of other strains). Of these, the most pronounced activity was observed for the cyanobacterial isolate Fischerella sp. strain 52-1. In the cocultivation experiments this cyanobacterium inhibited the growth of all tested green algae and Cyanobacteria. The crude lipophilic extracts from Fischerella sp. strain 52-1 isolated from both the biomass and the culture liquid inhibited photosynthesis of the green alga Chlamydomonas sp. in a concentration- and time-dependent manner and caused extensive loss of ultrastructural cell organization. Preliminary chemical characterization of compounds extracted from Fischerella sp. strain 52-1 indicated the presence of indole alkaloids, and further characterization has confirmed that these compounds belong to the hapalindoles previously isolated from other species of Fischerella and related genera. Further chemical characterization of these compounds, and further investigation of their apparent role in allelopathy is ongoing.

Diverse bacterial PKS sequences derived from okadaic acid-producing dinoflagellates.

Okadaic acid (OA) and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS) genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.

Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O2-•).

Brevetoxin (PbTx-2) influences the redox status and NPQ of Karenia brevis by way of thioredoxin reductase.

The Florida red tide dinoflagellate, Karenia brevis, is the major harmful algal bloom dinoflagellate of the Gulf of Mexico and plays a destructive role in the region. Blooms of K. brevis can produce brevetoxins: ladder-shaped polyether (LSP) compounds, which can lead to adverse human health effects, such as reduced respiratory function through inhalation exposure, or neurotoxic shellfish poisoning through consumption of contaminated shellfish. The endogenous role of the brevetoxins remains uncertain. Recent work has shown that some forms of NADPH dependent thioredoxin reductase (NTR) are inhibited by brevetoxin-2 (PbTx-2). The study presented herein reveals that high toxin and low toxin K. brevis, which have a ten-fold difference in toxin content, also show a significant difference in their ability, not only to produce brevetoxin, but also in their cellular redox status and distribution of xanthophyll cycle pigments. These differences are likely due to the inhibition of NTR by brevetoxin. The work could shed light on the physiological role that brevetoxin fills for K. brevis.

Toxin composition of the 2016 Microcystis aeruginosa bloom in the St. Lucie Estuary, Florida.

A bloom of the cyanobacteria, Microcystis aeruginosa occurred in the St. Lucie Estuary during the summer of 2016, stimulated by the release of waters from Lake Okeechobee. This cyanobacterium produces the microcystins, a suite of heptapeptide hepatotoxins. The toxin composition of the bloom was analyzed and was compared to an archived bloom sample from 2005. Microcystin-LR was the most abundant toxin with lesser amounts of microcystin variants. Nodularin, cylindrospermopsin and anatoxin-a were not detected.

Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis.

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis.

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.