Effects of Saxitoxin (STX) and Veratridine on Bacterial Na+-K+ Fluxes: a Prokaryote-Based STX Bioassay

Saxitoxin (STX) is a potent natural sodium channel blocker andrepresents a significant health concern worldwide. We describehere the antagonistic effects of STX and veratridine (VTD),an Na⁺ channel activator, on three gram-negative bacteria andtheir application to an STX bioassay. STX reduced the totalcellular levels of both Na⁺ and K⁺, as measured by flame photometry,whereas VTD increased the cellular concentrations relative tocontrol ion fluxes in the cyanobacterium Cylindrospermopsis raciborskiiAWT205. Endogenous STX production in toxic cyanobacterial strainsof C. raciborskii and Anabaena circinalis prevented cell lysisinduced by VTD stress. Microscopic cell counts showed that non-STXproducing cyanobacteria displayed complete cell lysis and trichomefragmentation 5 to 8 h after addition of VTD and vanadate (VAN),an inhibitor of sodium pumps. The addition of STX, or its analogueneoSTX, prior to treatment with VTD plus VAN prevented completelysis in non-STX-producing cyanobacteria. VTD also affectedcyanobacterial metabolism, and the presence of exogenous STXin the sample also ameliorated this decrease in metabolic activity,as measured by the cellular conversion of tetrazolium into formazan.Reduced primary metabolism was also recorded as a decrease in thelight emissions of Vibrio fischeri exposed to VTD. Additionof STX prior to VTD resulted in a rapid and dose-dependent responseto the presence of the channel blocker, with samples exhibitingresistance to the VTD effect. Our findings demonstrate that STXand VTD influence bacterial Na⁺ and K⁺ fluxes in opposite ways,and these principles can be applied to the development of aprokaryote-based STX bioassay.

INTRODUCTION

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Introduction

Paralytic shellfish poisoning (PSP) is a deadly affliction thatresults from the accidental consumption of some potent naturalneurotoxins, typically via contaminated seafood. These molecules,of which the most potent representative is saxitoxin (STX),are highly selective blockers of Na⁺ channels in excitable cells,thereby affecting nerve impulse generation in animals (6). Thissyndrome can lead, in extreme cases, to death (31). STX andanalogue compounds, known collectively as the PSP toxins, havebeen reported to occur in both marine and freshwater microorganisms.Three genera of marine dinoflagellates have been described tosynthesize PSP toxins (15, 24, 28), a limited number of bacterialstrains (10), and several species of freshwater filamentouscyanobacteria (1, 5, 26), including strains of Anabaena circinalisand Cylindrospermopsis raciborskii (17, 20). Occurrences of harmfulalgal blooms associated with PSP toxins represent a serious healthconcern worldwide and have been increasingly reported (18, 25, 31).All nonanalytical detection systems for these neurotoxins relyon bioassays based on whole animals or animal cell lines (14, 21),since much is known regarding the pharmacological effect ofSTX on eukaryotes. On the other hand, PSP toxins have rarelybeen studied according to their effect on prokaryotic cells(30). For economic, practical, and ethical reasons, alternativesto the standard animal biological tests are desired. Here wedescribe our findings, which represent the first investigationof the feasibility of a prokaryote-based bioassay for STX andits analogues.

In a previous study we reported that, in the STX-producing cyanobacterium C.raciborskii T3, STX accumulation was directly correlated to variationsin intracellular Na⁺ levels (F. Pomati, C. Rossetti, G. Manarolla,and B. A. Neilan, unpublished data). These results suggesteda possible role of this toxin in the maintenance of cyanobacterialhomeostasis under Na⁺ stress. Questions regarding whether STX-producingcyanobacteria have a potential advantage over other nonneurotoxicstrains under conditions of critical Na⁺ levels or whether STXinterferes with bacterial Na⁺ fluxes as it does in eukaryotic cellsarose from that study (Pomati et al., unpublished).

In the present study we first investigated the effect of STXand veratridine (VTD), a sodium channel activator that increasesNa⁺ permeability in eukaryotic cells (8), on the cyanobacterial totalNa⁺ and K⁺ cellular contents measured by flame photometry. C.raciborskii AWT205, a cyanobacterium known not to produce anyneurotoxin, including STX (16), was exposed to STX and VTD.Further, by using VTD to induce intracellular Na⁺ stress, weassayed PSP toxins producing and nontoxic strains of C. raciborskiiand A. circinalis in a "cyanolytic" test similar to those usedwith eukaryotic cells (21, 29). In the assay developed herecyanobacteria were stressed with VTD and o-vanadate (VAN), aninhibitor of bacterial ion pumps (ion translocating P-type ATPases) (9, 13).The detection of cell lysis was used as the endpoint. Two nonneurotoxicstrains (C. raciborskii AWT205 and A. circinalis 271C) werethen used in the same test to assay the presence of channelblockers such as lidocaine, amiloride, STX, and neoSTX. In orderto investigate whether the demonstrated sensitivity of C. raciborskiiAWT205 to VTD and STX was also related to changes in the cell'smetabolic activity, the test was applied to a cell titer cytotoxicityassay. Subsequently, the method based on VTD-induced Na⁺ stresswas used to assay the presence of STX with the commercially availableand standardized toxicity test LUMIStox by using the bioluminescentbacterium Vibrio fischeri.

MATERIALS AND METHODS

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Materials and METHODS

Cyanobacterial strains and culture conditions.
C. raciborskii AWT205, a non-PSP-toxin producer (16), was obtainedfrom Peter R. Hawkins (Australia Water Technologies, EnSight,West Ryde, New South Wales, Australia). C. raciborskii T3, anSTX producer (20), was obtained from Sandra Azevedo (FederalUniversity of Rio de Janeiro, Rio de Janeiro, Brazil). StrainsAWT205 and T3 were maintained in ASM-1 medium (12). PSP toxin-producing strainsof A. circinalis, 344B, 134C, 150A, and 131C and the nontoxicstrains 271C, 306A, and 332H were obtained from the Australian WaterQuality Centre (Adelaide, South Australia) and maintained in Jaworski'smedium (17). All cyanobacterial cultures were grown in 250-mlglass flasks at a constant temperature of 26°C under continuousirradiance of cool white light at an intensity of 15 µmolof photons m^-2 s^-1. Cyanobacterial growth was monitored by recordingthe optical density at 750 nm with a Lambda 10 UV/VS spectrometer(Perkin-Elmer, Inc., Shalton, Conn.). Mid-exponential-phasecultures were chosen for the tests described below.

Chemicals.
All reagents and chemicals were obtained from Sigma-Aldrich(Dorset, United Kingdom). Lidocaine hydrochloride, amiloride,and VAN solutions (100 µM, 100 mM, and 10 mM, respectively)were prepared in Milli-Q water, stored at 4°C (protectedfrom light), and diluted into the culture medium to obtain thedesired concentration. VTD was dissolved to a final concentration10 mM in acidic Milli-Q water (pH 2) and stored at -20°C.Certified standard solutions of PSP toxins (PSP-1C and STXdiHCl-C)were obtained from the Institute of Marine Bioscience, NationalResearch Council of Canada, Halifax, Nova Scotia. PSP toxinstandards were stored at -20°C with the stock solutionsdiluted in culture medium to obtain the final test concentrations.

Total cellular Na⁺ and K⁺ content and flame photometry.
To evaluate the effect of 1 µM STX and 100 µM VTDon total Na⁺ and K⁺ cellular levels, aliquots of the same culture(20 ml) of C. raciborskii AWT205 were adjusted to pH 8.1 byadding HEPES buffer to a final concentration of 10 mM. Samples(2 ml) were harvested before exposure (-5 min), immediatelyafter exposure (0 min), and at 10, 30, and 60 min postexposure.Experimental replicates included negative controls (unexposedculture sample) and positive controls (10 mM NaCl). Aliquots ofchallenged AWT205 cultures were collected by centrifugationin 2-ml plastic tubes at 11,000 x g for 15 min. All sampled pelletswere resuspended in 0.5 ml of diluent flame solution (3 mM lithiumin MilliQ water) and analyzed for total Na⁺ and K⁺ cellularcontent by using a FLM3 flame photometer (Radiometer, Copenhagen,Denmark). All experiments were performed in quadruplicate. Controltraces were subtracted from the tested samples for thresholdcorrection, and the data were normalized by expressing the valuesas the percentile variation over samples at -5 min.

Cyanobacterial cell lysis test.
The cyanobacterial cell lysis assay was based on the same principlesas the animal neuroblastoma and red blood cell culture assaysdescribed elsewhere (21, 29), except for the use of VAN to inhibitNa⁺/ K⁺ ATPase activity instead of ouabain since ouabain isknown to be ineffective against algal Na⁺ pumps (11). Briefly,96-well microtiter plates were used for the cyanolytic assay,in which 100 µl of the cyanobacterial cultures was inoculatedand exposed to the agents. Controls consisted of untreated aliquotsof cyanobacterial cultures and samples with 4 µl of either10 mM VTD or 10 mM VAN. Toxic and nontoxic cyanobacteria werealso assayed after the addition of a combination of 4 µlof 10 mM VTD and 4 µl of 10 mM VAN. The final concentrationof VTD and VAN, 400 µM, was chosen based on previous studies (13, 29).For the treatment of C. raciborskii AWT205 and A. circinalis271C with STX and neoSTX at 1 µM, the PSP toxins wereadded to the tested wells 30 min prior to the addition of VTDand VAN to simulate natural the conditions of toxic cultures.In these experiments, the positive controls consisted of samplesexposed to VTD-VAN added with lidocaine hydrochloride at 1 µM.Replicates dosed with amiloride at 10 mM were used as negativecontrols. In a previous study, lidocaine hydrochloride was demonstratedto induce an increase in total cellular Na⁺, whereas amiloridereduced the cellular ion levels in cyanobacteria (Pomati etal., unpublished).

In both assays, the microtiter plates were incubated at roomtemperature (25°C) for 5 to 8 h (minimum time for completecell lysis observed in the VTD-VAN samples). Determination ofcell lysis was performed by light microscopic inspection every30 to 60 min from the onset of the test, and cells were countedby using a Neubauer improved counting chamber (0.1 mm deep).Complete cyanobacterial lysis in the VTD-VAN samples was utilizedas the endpoint of the assay. If no cyanolysis was observedin the inoculated wells, the presence of a channel-blockingagent, including PSP toxins, was indicated. Three to five trialswere performed for each strain or treatment to test the reproducibilityof the results.

Cell titer assay for metabolic activity.
Microtiter plate cytotoxicity assays on cyanobacterial cellswere performed by using the CellTiter 96 nonradioactive cellproliferation assay kit (Promega Corp., Madison, Wis.). Thismethod utilizes, as an indicator of a cell's metabolic activity,the cellular conversion of a tetrazolium salt into a formazan productthat can be quantified with a spectrophotometer plate reader. Assayswere performed essentially, as suggested in the standard protocolprovided with the kit. C. raciborskii cells in mid-exponentialgrowth were centrifuged (15 min at 4,000 x g) and concentratedto reach approximately an optical density at 750 nm of 1. Subsequently,100-µl aliquots of concentrated cyanobacterial suspensionwere inoculated in 96-well microtiter plates and then exposedto the test agents. For comparison between the metabolic responsesof C. raciborskii strains AWT205 and T3, the culture sampleswere tested with a combination of 4 µl of 10 mM VTD and4 µl of 10 mM VAN, yielding a final concentration of 400µM for each compound. Controls consisted of untreated cyanobacteria.In the evaluation of the effect of combined VTD and STX on C.raciborskii AWT205, cyanobacterial cells were exposed to 1 µMSTX, incubated at room temperature for 30 min, and then addedto VTD at 100 µM. Controls consisted, for each sample,of untreated cells and cyanobacteria exposed to VTD at 100 µM.The dye solution was added at different times, and the stopsolution after 4 h of incubation at room temperature (25°C).Plates were allowed to rest overnight, and then the absorbanceat 600 nm was determined with a Metertech ${Sigma}$ 960 microplate reader(Metertech, Inc., Taipei, Taiwan). All experiments were performedin quadruplicate, and the data were expressed as an averagepercent variation of sample values versus levels in untreated control.

Luminescent bacteria test.
Inhibition of bioluminescence in cultures of V. fischeri NRRL-B-11177was performed by using a commercially available standard luminescent bacteriumLUMIStox test kit (Dr Bruno Lange GmbH & Co., Dusseldorf, Germany)and specific analytical equipment, including the LUMIStox 300measuring station and a Lumistherm thermostat. Reactivationof freeze-dried bacteria and preparation of samples was doneaccording to the instructions provided. Light emission of reactivatedbacteria was adjusted to a relative intensity of $~$ 1,000 by dilutionwith sterile 2% NaCl. For the test, 0.5 ml of luminescent bacterialsuspension was combined with 0.5 ml of the test solutions. Testsolutions were prepared in sterile saline medium (2% NaCl) andadjusted to pH 7 with 10 mM phosphate buffer. When needed, STXsupplemented the solutions to the desired final concentration.Bacterial suspensions and samples were maintained at 15°C,combined, and monitored with the luminometer, while allowingbacteria to adapt for 5 to 10 min. Subsequently, 100 µMVTD was added, and the light emission was measured over time.Positive and negative controls were included for each test and consistedof bacteria exposed to only VTD and unexposed samples, respectively.The effect on bioluminescence was monitored and was expressedas the percent inhibition relative to the untreated controls. Valueswere calculated by using, as a correction factor, the changesin intensity of the controls. This was achieved by subtractingthe trace negative control reading from the test samples overthe duration of the experiment.

Statistical analyzes.
All graphical and statistical analyses were performed by usingPC Origin 5.0 software (Microcal Software, Inc., Northampton, Mass.).

RESULTS

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Results

Effect of Na⁺ stress, VTD, and STX on the total cellular Na⁺ and K⁺ content.
In C. raciborskii AWT205, the stress induced by 10 mM NaCl increasedthe total cellular Na⁺ levels compared to the untreated controls(Fig. 1A). Na⁺ uptake by the cells was shown to be very rapid, andthe total cyanobacterial sodium content remained stable overthe 60-min course of the experiment. The total K⁺ content ofcells was only slightly affected by 10 mM NaCl, indicating thatthe homeostasis of K⁺ is of marginal consequence in the overallcyanobacterial Na⁺ stress response. On the other hand, bothNa⁺ and K⁺ cellular levels in C. raciborskii AWT205 were altereddue to the effects of STX at 1 µM and VTD at 100 µM(Fig. 1B). The addition of VTD dramatically stimulated cyanobacterial Na⁺and K⁺ accumulation, whereas STX markedly inhibited the cellularuptake and thus the intracellular levels of both ions. Theseresults suggest that Na⁺ flux is not the only cellular responseelicited by these two compounds in cyanobacteria.

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FIG. 1. (A) Time course of total cellular Na⁺ and K⁺ levels in C. raciborskii AWT205 cultures exposed to 10 mM NaCl (Na⁺ ${blacksquare}$ and K⁺ ${square}$ ) compared to untreated control samples (Na⁺ • and K⁺ ${circ}$ ). (B) Effects of STX at 1 µM (Na⁺ ${blacktriangledown}$ and K⁺ ${triangledown}$ ) and VTD at 100 µM (Na⁺ ${blacktriangleup}$ and K⁺ ${triangleup}$ ) on total cellular Na⁺ and K⁺ concentrations in C. raciborskii AWT205. All values are the mean of four experimental replicates and are expressed as the percent variation over time.

Lysis test with toxic and nontoxic cyanobacteria.
The effects of VTD and VAN each at 400 µM and in combinationwere monitored by microscopically counting the cells of thetwo strains of C. raciborskii: AWT205 and T3. Cell densitieswere recorded over a 180-min time period (Fig. 2). VAN had nosignificant effect on both strains, whereas culture samples ofAWT205 were manifestly more sensitive to VTD and the combinationof both VTD and VAN compared to the STX-producing strain T3.For AWT205, the first indications of cyanobacterial lysis occurredafter 60 min of exposure to VTD or VTD-VAN, whereas cell lysiswas evident for both T3 and AWT205 at 120 min after the onsetof the experiment. The combination of VTD and VAN was more effectivethan VTD alone in both strains as indicated by the decreasein the number of intact cells (Fig. 2). In AWT205 cultures,180-min exposure to VTD-VAN resulted in almost complete lysis ofcells. By microscopic examination, cyanobacterial lysis occurred afterevident enlargement of the cells, which subsequently burst.This indicated that lysis could have been caused by a dramaticincrease in the intracellular osmotic pressure due to excessiveion uptake.

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FIG. 2. Effects of VTD and VAN at 400 µM and their combination on cell numbers in samples of C. raciborskii AWT205 and T3. All values represent the average of five experimental replicates and are expressed as the percent variation over time. Symbols for C. raciborskii AWT205: ${blacksquare}$ , VAN; •, VTD; and ${blacktriangledown}$ , VAN-VTD. Symbols for C. raciborskii T3: ${square}$ , VAN; ${circ}$ , VTD; and ${triangledown}$ , VAN-VTD.

PSP toxin-producing and nonneurotoxic strains of the cyanobacterialspecies C. raciborskii and A. circinalis were assayed for resistanceto VTD-VAN by the method described above, and the results aresummarized in Table 1. All of the non-PSP-toxin-producing strainstested showed complete cell lysis after 3 to 8 h, with no filaments recorded,whereas the neurotoxic strains were characterized by intact filamentsand a lack of complete cell lysis even after overnight exposureto these agents.

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TABLE 1. Cyanobacterial strains used in this study and their relative resistance to treatment with VTD (400 µM) plus VAN (400) µM

Two non-PSP toxins producing cyanobacterial strains, C. raciborskiiAWT205 and A. circinalis 271C, were also chosen to evaluatethe effect of STX and neoSTX at 1 µM on the stress inducedby exposure to VTD-VAN. The results, summarized in Table 2,demonstrated that the production of PSP toxins prevented completecell lysis caused by VTD-VAN in culture samples, as seen forthe positive controls and in the cyanobacteria added with lidocainehydrochloride at 1 µM. In cyanobacteria exposed to lidocaine,cell lysis was observed at the same time as the untreated controls.On the other hand, the effects of amiloride at 1 mM, STX at1 µM, and neoSTX at 1 µM were comparable with regardto the level of inhibition of cyanobacterial lysis.

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TABLE 2. Results of the application of channel-blocking agents to two non-PSP-toxin-producing cyanobacterium strains over a 5-h exposure time to VTD (400 µM) plus VAN (400 µM)

Effect of VTD and STX on cyanobacterial metabolic activity.
In order to investigate whether the increased Na⁺ uptake incyanobacterial cells was coupled to a consequent measurabledecrease in general metabolic activity, cultures of C. raciborskiiAWT205 were treated with 400 µM VTD-400 µM VAN,100 µM VTD, or 100 µM VTD-1 µM STX and thenmonitored by using a CellTiter 96 cell proliferation assay.As a control, cultures of the STX-producing C. raciborskii T3were also exposed to 400 µM VTD-400 µM VAN, andthe metabolic activity was measured over time. The treatmentswith VTD and VTD-VAN both resulted in a reduced metabolic activityof C. raciborskii strains over a 90-min exposure; however, thetoxic strain T3 was affected less than AWT205 (Fig. 3). Treatmentwith STX at 1 µM alone did not result in any adverse effecton cyanobacterial metabolism (data not shown). The additionof 1 µM STX to cultures of AWT205, followed by 100 µMVTD, induced a less dramatic decrease in metabolic activitycompared to exposure to 100 µM VTD alone (Fig. 3).

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FIG. 3. Time course of metabolic activity in culture samples of C. raciborskii AWT205 treated with 100 µM VTD ( ${blacktriangleup}$ ), 400 µM VTD-400 µM VAN ( ${blacktriangledown}$ ), and 100 µM VTD-1 µM STX ( ${circ}$ ). Control culture samples of C. raciborskii T3 were also tested with 400 µM VTD-400 µM VAN (x). All values are the average of 4 experimental replicates and are expressed as the percent variation over time.

Effect on bioluminescence by V. fischeri.
In the standard toxicity test LUMIStox, no significant effectof varied concentrations of STX (from 100 nM to 1 µM)on the luminescence of V. fischeri was observed. On the otherhand, treatment with VTD at 100 µM reduced bacterial lightemission over time, as shown in Fig. 4A. The addition of STX(1 µM) to the Vibrio cultures after exposure to 100 mMVTD resulted in a slight recovery of bioluminescence (maximumof 4.4% after 70 min) compared to samples with no STX added(Fig. 4A). Exposure of the bioluminescent bacteria to STX priorto the addition of 100 µM VTD, however, led to differentresponses over time for the test samples compared to the controlswith no STX added. Figure 4B shows the time course of the percentlight emission for the various treatments, with values correctedby subtracting the trace levels of unexposed control samples toeliminate threshold variations in bioluminescence. In the first2 min after the addition of 100 µM VTD, samples not exposedto STX drastically decreased their light emission compared tobacterial solutions treated with the channel-blocking toxin.STX at 600 nM was the most effective treatment for preventingthe VTD effect (21% compared to VTD controls), followed by STXat 300 nM (19.5%) and at 1.2 µM (18.3%). By 5 min afterthe addition of VTD, samples with no STX added reached a levelof bioluminescence that were, on average, 6% higher than theuntreated controls. Bacterial solutions with STX at 300 and600 nM showed no significant variation over control levels.Samples with STX added to 1.2 µM did not completely recovercontrol levels of light emission, attaining an average of -3.8%of the luminescence in unexposed bacteria.

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FIG. 4. (A) Time course of bioluminescence in V. fischeri. VTD (100 µM) was added 52 min after the onset of the experiment. Subsequently, half of the experimental replicates were supplemented with 1 µM STX (•), whereas the other half were supplemented with physiological saline solution ( ${square}$ ). (B) Effects of 100 µM VTD on light emission by samples of V. fischeri exposed to STX at 0 ( ${blacksquare}$ ), 300 ( ${circ}$ ), 600 (shaded triangles), and 1,200 ( ${blacktriangledown}$ ) nM, expressed as the percent variation over time and corrected for the control levels. All values are the average of four experimental replicates.

DISCUSSION

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Discussion

During the present study, we observed opposing effects of STXand VTD on cyanobacterial Na⁺ and K⁺ ion fluxes, as measuredby flame photometry analysis. The effects detected were rapid butnot Na⁺ specific, as predicted by the interaction of these compoundswith eukaryotic cells. Our results suggested either that STXand VTD have less specific effects on prokaryotic cells thanthose reported in the literature for eukaryotic sodium fluxesor that the target of these two agents on cyanobacterial cellsis a binding protein involved in both Na⁺ and K⁺ homeostasis.The latter hypothesis is consistent with several reports inthe literature demonstrating the presence, in cyanobacterialcells, of channel proteins that are permeable to both sodiumand potassium ions (19, 22, 23).

Based on these observations, PSP toxin-producing and nonneurotoxiccyanobacterial strains were assayed to investigate whether,in vivo, the production of STX would have prevented the excessiveion uptake and subsequent cell lysis induced by VTD stress.As noted from microscopic observations, non-PSP-toxin-producingcyanobacteria under VTD-VAN stress were swollen, probably dueto an increase in the internal osmotic pressure, and subsequentlycollapsed within 5 to 8 h. Similar findings were reported inprevious studies with animal cells subjected to the activityof the sodium channel activator and the ion pump inhibitor ouabain(29). In contrast, PSP toxin-producing cultures exhibited lowerrates of cell lysis. This differential sensitivity to VTD andVAN exposure is proposed to be due to the presence of channel-blockingcompounds in the cultures, which interfere with the action ofthe channel-activating agents. To verify this hypothesis andto exclude the possibility of a variable intrinsic sensitivityof the toxic strains to the chemicals used, the two nonneurotoxiccyanobacteria C. raciborskii AWT205 and A. circinalis 271C wereexposed to VTD-VAN in the presence of STX and neoSTX at 1 µM.This experiment clearly revealed the acquired resistance tolysis of nonneurotoxic strains after the addition of PSP toxinsto the cultures. Therefore, in vivo, a direct antagonism ofSTX and VTD was demonstrated in a prokaryotic microorganism,a result similar to what has been noted in the eukaryotic cell-basedassays for channel-blocking toxins (21).

Consistent with our hypothesis that the main effect of VTD oncyanobacterial cells was due to the increased uptake of Na⁺and K⁺ ions, we investigated whether such stress was correlatedwith a decrease in the metabolic activity of the cyanobacteria.Sodium, above a certain critical concentration, represents athreat to normal cellular functions. This ion, if in excesscompared to normal physiological levels, can disrupt several crucialbiological functions, such as photosynthetic and electron transportactivities in cyanobacterial cells (2). The cell titer toxicityassays, applied in the present study with the addition of VTD-VAN,demonstrated that antagonistic effects of STX and VTD can also bedetected via the monitoring of bacterial metabolic activity.

Since measurements of metabolic activity, compared to cell lysis,represent a more precise, easy-to-quantify, and standardizedmeans of investigation, we applied the principle of inducingion uptake by using VTD in the bioluminescent bacterium V. fischeri.Such stress, resulting in decreased primary metabolism, canbe measured in this microorganism as variations in light emission bya commercially standardized method. In suspensions of V. fischeri,VTD exposure reduced bioluminescence. Light emission also followeda similar pattern after the post-VTD addition of STX (Fig. 4A).This effect could be explained by the occurrence of nonreversiblecell damage caused by either the initial VTD stress or a criticalincrease in cytoplasmic Na⁺ levels (2). Alternatively, these datacould indicate a difference in the affinity of VTD and STX fora putative binding site on the bacterial cells. STX may haveless specificity than VTD for the receptor molecule in V. fischeri, orthe binding protein(s) on the bacterial cell membranes couldhave a completely different structure compared to the definedtargets of these two agents, i.e., the eukaryotic voltage-gatedNa⁺ channels.

On the other hand, exposure to STX prior to the addition ofVTD to bioluminescent bacteria resulted in the observed differencein the time course of cellular metabolic activity. As expectedfor changes in membrane ion fluxes, the effect displayed by V.fischeri was rapid and dose dependent. A 1.2 µM concentrationof STX was shown to have a minor level of toxicity to bioluminescentbacteria. This effect could be a result of the prolonged inhibitionof basal bacterial Na⁺ and K⁺ activity by such high concentrationsof the channel-blocking toxin. In general, however, during thecourse of the present study, STX alone did not have any particulareffect on bacterial (i.e., Vibrio sp. or cyanobacteria) growthor metabolism. Accordingly, exposure to STX could not be useddirectly in a bacterial bioassay. These data were consistentwith reports that show low to no toxicity of STX on other microorganisms (14, 30).In contrast, no investigations of the effects of STX on prokaryoticion fluxes have been reported. Bacterial ion channels are single-domainproteins (for reviews, see references 3 and 7) that have been reportedto be insensitive to both STX and TTX and confirmed by recent findingsregarding the voltage-gated prokaryotic Na⁺ channel in the halophilicbacterium Bacillus halodurans (27).Further comprehensive investigationsof the effect of STX on microorganisms may lead to importantevolutionary findings regarding an ancestral ion channel sensitiveto neurotoxins.

The application of the bioluminescent bacterium method describedhere for toxicity assays showed a nanomolar order of magnitudedetection range for STX and a limit of <300 nM. Preliminarydata also confirmed (29) that varying the concentrations ofVTD or VAN or the number of cells in the assay can affect thesensitivity of the test. In the present study, the parameterswere selected to provide the best results in a short time scale,as required for a rapid bioassay. We predict that additional developmentand standardization of this test would afford a novel and accuratemethod for the detection and quantification of PSP toxins. All threegram-negative bacteria tested with VTD showed a lower affinityto STX compared to their potency against eukaryotic cells. However,the use of microorganisms represents an easy, economic, andethical alternative to animal tests for screening environmental,clinical, and industrial samples for neurotoxins, includingPSP toxins, tetrodotoxin, ciguatoxins, and brevetoxins.

In conclusion, the present study presents the first evidenceof the effect of the Na⁺ channel blocker STX, as well as thesodium channel activator VTD, on the Na⁺ and K⁺ ion fluxes and metabolismof bacterial cells. Previously, these two agents were thoughtto act almost exclusively on eukaryotic membrane channels. In addition,we demonstrated the applicability of VTD and STX antagonism inprokaryotic cells for the development of a novel PSP toxin bioassay.

ACKNOWLEDGMENTS

We thank G. Manarolla for helpful experimental assistance andL. Llwellyn for advice.

F.P. is the recipient of research scholarships from the Universityof New South Wales and the School of Biotechnology and BiomolecularSciences, together with an A. Lee Travel Scholarship kindlygranted by the School of Microbiology and Immunology.

FOOTNOTES

* Corresponding author. Mailing address: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney 2052, NSW, Australia. Phone: 61-2-9385-3235. Fax: 61-2-9385-1591. E-mail: b.neilan{at}unsw.edu.au.

REFERENCES

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