Marine Toxins - American Chemical Society

Chapter 5 A Bacterial Source of Tetrodotoxins and Saxitoxins 1

Mark L. Tamplin

Downloaded by MONASH UNIV on April 11, 2016 | http://pubs.acs.org Publication Date: January 29, 1990 | doi: 10.1021/bk-1990-0418.ch005

Center of Marine Biotechnology, Maryland Biotechnology Institute, University of Maryland, Baltimore, MD 21202

Tetrodotoxin (TTX) and saxitoxin (STX) are potent sodium channel blockers that are found in phylogenetically diverse species o f marine life. T h e wide distribution o f T T X and S T X has resulted i n speculation that bacteria are the source o f these toxins. Recently, investigators have reported isolation o f marine bacteria, including Vibrio, Alteromonas, Plesiomonas, and Pseudomonas species, that produce T T X and STX. This chapter details the methods and results o f research to define bacterial sources o f T T X and S T X .

T h e tetrodotoxins ( T T X s ) and saxitoxins ( S T X s ) have i n c o m m o n the ability to block sodium channels o f excitable membranes (1—5). Saxitoxin and tetrodotoxin are some o f the most potent non-proteinaceous neurotoxins k n o w n and are responsible for significant human morbidity and mortality (6, 7). A l t h o u g h for many years the biosynthetic origin(s) o f T T X s and S T X s has not been identified, recent evidence indicates that bacteria may be a source. Tetrodotoxin poisoning has been recognized for more than two thousand years. Japanese historical records show that the c o n s u m p t i o n o f certain species o f pufferfish (Tetraodon spp.) resulted in paralytic intoxication (8). This problem continues i n modern times i n various A s i a n countries, especially Japan, where pufferfish are still regarded as a delicacy. C l i n i c a l symptoms o f T T X intoxication include numbness, paralysis, and i n some instances death. In fact, the "zombie" state described i n the V o o d o o religion has been attributed to T T X i n potions derived from pufferfish (9). Numerous species o f edible shellfish and finfish are k n o w n to contain natural toxins during all or part o f their life cycle. T T X s and S T X s have been isolated from species o f fish, starfish, crab, octopus, frog, newt, salamander, goby, gastropod, mollusk, flatworm, annelid, z o o p l a n k t o n , and algae (10-16). For example, the A u s t r a l i a n blue-ringed octopus (Hapalochlaena maculosa) contains tetrodotoxin i n its posterior salivary gland and can k i l l an adult human with a single bite (17).

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Current address: U.S. Food and Drug Administration, Fishery Research Branch, P.O. Box 158, Dauphin Island, AL 36528 0097-6156/90/0418-0078$06.00/0 o 1990 American Chemical Society

Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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A Bacterial Source of Tetrodotoxins and Saxitoxins


Concentrations o f T T X s i n fish and other marine organisms vary among species, individual, and tissues o f animals (18). Liver and ovary o f fish usually contain the highest concentrations o f T T X s , although relatively high quantities can be found i n other tissues (18). Terrestrial animals have also been shown to contain T T X s , including brightly-colored frogs, newts, and their egg clusters (19, 20). There are two documented cases o f newt-associated T T X h u m a n poisonings, o n e o f which resulted i n death (21). T h e S T X s have been purified from many marine species, including fish, crabs, annelids, and algae (4, 16, 22—26). S T X and its derivatives are the toxic agents o f paralytic shellfish poisoning (PSP), an illness caused by ingestion o f contaminated shellfish, such as mussels and clams, which accumulate S T X by feeding o n toxic dinoflagellates (e.g. Alexandrium spp.) (27, 28). Reports indicate that concentrations o f S T X i n dinoflagellates vary between species and clones (23, 29). F o r many years the source(s) o f T T X s and S T X s has been a controversial subject. T h e discovery that T T X s were i n tissues o f animals that were phylogenetically distinct resulted i n speculation that T T X s were bioaccumulated and/or originated from symbiotic microorganisms, such as bacteria. Since 1986, a growing number o f reports describe bacteria that produce T T X s (30—35). Bacteria have also been proposed as the source o f S T X i n marine dinoflagellates. Sousa y Silva (36) provided early suggestions o f a bacterial source o f S T X . Subsequently, S T X and n e o - S T X were isolated from strains o f a cyanobacterium, Aphanizomenon flos-aquae, a procaryotic organism (22). Recently, K o d a m a (37) has reported that a bacterium cultured from Gonyaulax (= Alemndrium) tamarensis produces S T X . This chapter reviews recent experimental evidence o f a bacterial source o f sodium channel blockers, principly T T X s . These findings support the hypothesis that procaryotic organisms produce T T X s which contaminate oceanic food chains.

Materials and Methods Organisms. T T X , a n h y d r o - T T X , and S T X have been reported from various procaryotic species (Table I). A l l o f these species, with the exception o f Escherichia coli, are marine organisms. Extraction of Sodium Channel Blockers.

A review o f published reports shows that methods for purification o f sodium channel blockers from bacterial cultures are similar to techniques for isolation o f T T X and S T X from pufferfish and dinoflagellates (30, 31, 38, 39). Typically, cell pellets o f bacterial cultures are extracted w i t h hot 0.1% acetic acid, the resulting supernatant ultra-filtered, lyophilized, and reconstituted i n a m i n i m a l v o l u m e o f 0.1% acetic acid. C u l t u r e media can also be extracted for T T X by a similar procedure (31). B o t h cell and supernatant extracts are analyzed further by gel filtration chromatography and other biological, chemical, and i m m u n o l o g i c a l methods. F e w reports describe purification schemes that include extraction o f c o n t r o l samples o f bacteriological media (e.g., broths and agars) which may be derived from marine plant and animal tissues.

Mouse Bioassay.

T h e mouse is the traditional animal o f choice for detecting biological activity due to S T X and T T X . M i c e receive an intraperitoneal inject i o n o f sample and are observed for symptoms o f intoxication, i.e., dypsnea, convulsions, and death. This method is effective for detecting biological activity o f S T X and T T X i n numerous samples. F o r the standard S T X assay, o n e mouse unit is defined as that quantity o f S T X injected i.p. i n 1 m l s o l u t i o n that w i l l


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Table I. Procaryotic Organisms that Produce Sodium Channel Blockers Species

Source

Reference


TTXs 35, 31

Vibrio fischeri-like

X a n t h i d crab (Atergatis floridus)

Pseudomonas sp.

Pufferfish (Fugu poecilonotus)

Alteromonas sp.

R e d calcareous alga (Jania sp.)

Vibrio alginolyticus

Pufferfish (Fugu vermicularis vermicularis)

Vibrio anguillarum

ATCC,

NCMB

Vibrio costicola

ATCC,

NCMB

34 34

Vibrio cholerae

ATCC,

NCMB

54

Vibrio fischeri

ATCC,

NCMB

34

Vibrio harveyi

ATCC,

NCMB

34

Vibrio marinus

ATCC,

NCMB

34

Vibrio parahaemofyticus

ATCC,

NCMB

34

Photobacterium phosphoreum

ATCC,

NCMB

34

Aeromonas hydrophila

ATCC,

NCMB

34, 54

Aeromonas salmonicida

ATCC,

NCMB

34

Plesiomonas shigelloides

ATCC,

NCMB

34

Escherichia coli

ATCC,

NCMB

34

Alteromonas communis

ATCC,

NCMB

34

Alteromonas haloplankas

ATCC,

NCMB

34

Alteromonas nigrifaciens

ATCC,

NCMB

34

Alteromonas undina

ATCC,

NCMB

34

Alteromonas vaga

ATCC,

NCMB

34

a

33 53 32, 34

STXs Vibrio-like sp.

Protogonyaulax tamarensis

Aphnizomenon flos-aquae A T C C = A m e r i c a n Type C u l t u r e C o l l e c t i o n ; NCMB

= N a t i o n a l C o l l e c t i o n o f M a r i n e Bacteria


37 4, 22

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k i l l a 20 g mouse i n 15 m i n (40). This amount is close t o the detection limit and under standard conditions corresponds to about 200 n g o f S T X , a concentrat i o n o f about 500 n M i n the injected s o l u t i o n . H i e corresponding definition for T T X uses similar conditions and a 30 m i n death time.


Immunological Assays.

Carlson et a l . (41) and C h u et a l . (42) describe i m m u nological assays for detecting S T X , at a level o f sensitivity o f approximately 1 ng S T X / m l (ca. 3 n M ) . A s with most i m m u n o l o g i c a l assays, antigenic, n o t biologic, activity is measured. T h e potential for sample matrix a n d non-specific binding to affect antibody-antigen reactions strictly necessitates that b o t h positive and negative controls be r u n for each sample. D a v i o et a l . (43) report efforts to obtain m o n o c l o n a l antibodies (mAbs) to S T X . Because S T X is a small molecule o f approximately 300 daltons, well below the size necessary for immunogenicity, a carrier molecule must be conjugated to the hapten ( S T X ) . This technique must m i n i m i z e alterations o f the antigenic form. F o r the a n t i - S T X antibodies tested to date, the ratios o f immunoassay response factor to pharmacological potency for various S T X derivatives differ substantially, the immunoassay being virtually unresponsive to some o f the c o m m o n natural derivatives (44).

Displacement Assay for T T X and

S T X . D a v i o a n d F o n t e l o (45) a n d R i c h i e et al. (46) describe methods for detecting S T X by measuring displacement o f radiolabelled S T X from brain membranes. T h e sensitivity o f this assay is approximately 1 n g S T X / m l . T T X can also be detected since S T X and T T X share the same biological receptor o n the sodium channel.

Blocking Events of Sodium Currents Applied to Rat Sarcolemmal Sodium Channels in Planar Lipid Bilayers. T h e physiological effects o f S T X and T T X o n the movement o f sodium ions through membrane sodium channels can be detected by a method first described by Krueger et a l . (47). I n this procedure, the voltage-dependent gating o f sodium channels is m o n i t o r e d i n artifical l i p i d bilayers containing purified sodium channels. Specific gating patterns can be used to identify different sodium channel-blocking toxins.

Tissue Culture Assay.

K o g u r e et al. (48) report a novel tissue culture assay for

detecting several types o f sodium channel blockers. T h e mouse neuroblastoma cell l i n e A T C C C C L 131 is grown i n R P M I 1640 supplemented w i t h 13.5% fetal bovine serum and 100 jig/ml gentamycin, i n an atmosphere o f 5 % C 0 ~ 9 5 % air at 37 ° C . Ninety-six well plates are seeded w i t h 1 x 1 0 cells i n 200 u\ o f medium containing 1 m M ouabain and 0.075 m M veratridine. V e r a t r i d i n e and ouabain cause neuroblastoma cells to round-up and die. I n the presence o f sodium channel blockers (e.g., T T X s o r S T X s ) , the lethal action o f veratridine is obviated and cells retain n o r m a l morphology and viability. A n important feature o f this assay is that a positive test for sodium channel blockers results i n n o r m a l cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. T h e m i n i m u m detectable level o f T T X is approximately 3 n M , o r approximately 1/1000 mouse unit. 2

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Chemical Methods. Chemical purification a n d structural analyses o f T T X s and S T X s include t h i n layer chromatography ( T L C ) , high performance liquid chromatography ( H P L C ) , i o n exchange chromatography, size exclusion chromatography, gas chromatography ( G C ) , mass spectroscopy ( M S ) , and nuclear magnetic resonance spectroscopy ( N M R ) (33, 39, 49-52). Several laboratories have designed continuous T T X analyzers that use H P L C , and i n many instances, G C - M S instrumentation.


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Results Biological Characterization of Bacterial Sodium Channel Blockers. A variety o f bacteria are reported to produce sodium channel blockers (Table I). Y a s u m o t o et al. (30) and N o g u c h i et al. (31) have isolated bacteria that produce T T X and its less toxic derivative, a n h y d r o - T T X from tissues o f alga and toxic crabs, respectively. I n subsequent reports, T T X , a n h y d r o - T T X , and 4 - e p i - T T X were shown to be produced by bacteria isolated from various toxic marine organisms. These include a Vibrio sp. cultured from the gut o f a xanthid crab (Atergatis floridus sp.) (31), V. alginolyticus isolated from the intestines o f a pufferfish (Fugu vermicularus vermicularis) (32), an Alteromonas sp. isolated from reef animals (53), and a Pseudomonas sp. cultured from the surface o f pufferfish (33). S i m i d u et al. (34) describe a variety o f marine bacteria, including Vibrio sp., Photobacterium sp., Aeromonas sp., Plesiomonas sp., and Alteromonas sp. that produce T T X and/or anhydro-TTX. Interestingly, they report that E. coli, a n o r m a l inhabitant o f the mammalian gastrointestinal tract, produces a n h y d r o - T T X U n i d e n t i f i e d sodium channel blockers have also been detected i n cultures o f Vibrio cholerae, an estuarine bacterium and h u m a n enteropathogen (54). Y a s u m o t o et al. (30) describe two components o f a Pseudomonas sp. culture w i t h identical H P L C retention times to T T X and a n h y d r o - T T X . These fractions produced typical signs o f T T X intoxication i n mice, with median death times similar to standard T T X and a n h y d r o - T T X . N o g u c h i et al. (32) demonstrate by H P L C and G C - M S analyses that 7 biotypes o f Vibrio sp. produced substances w i t h retention times and molecular weights similar to T T X and a n h y d r o - T T X . However, they observed mouse toxicity i n only 1 biotype. Likewise, S i m i d u et al. (34) report that extracts o f V. alginolyticus A T C C 17749 cultures displayed T T X - l i k e toxicity i n mice. T h e latter study shows that a variety o f marine bacteria, plus E. coli, produced substances that, by H P L C analysis, were identical to T T X and a n h y d r o - T T X . It is emphasized that some investigations show that bacterial cultures contain T T X - l i k e substances which are not detected by mouse bioassay and are "difficult to detect" by H P L C and G C - M S analyses. Structural analyses o f these substances w i t h other techniques was not reported. T a m p l i n et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with T T X - l i k e biological activity i n tissue culture assay, counteracting the lethal effect o f veratridine o n ouabain-treated mouse neuroblastoma cells. Concentrations o f T T X - l i k e activity ranged from 5 to 100 n g / L o f culture when compared to standard T T X . T h e same bacterial extracts also displaced radiolabelled S T X from rat brain membrane sodium channel receptors and inhibited the c o m p o u n d action potential o f frog sciatic nerve. However, the same extracts did not show T T X - l i k e blocking events o f sodium current when applied to rat sarcolemmal sodium channels i n planar l i p i d bilayers. Chromatographic Characterization of

T T X s . T h e vast majority o f reports have identified T T X and a n h y d r o - T T X i n bacterial cultures using H P L C , T L C , and G C - M S . Y a s u m o t o et al. (30) showed that T T X - l i k e substances extracted from a Pseudomonas sp. culture could bind to activated charcoal at p H 5.5 and be eluted with 2 0 % ethanol i n 1% acetic acid. In addition, H P L C analysis demonstrated T T X and a n h y d r o - T T X - l i k e fluorophors following strong base treatment. These compounds migrated o n silica gel comparably to T T X and a n h y d r o - T T X . Furthermore, when analyzed by electron i o n i z a t i o n ( E I ) - M S and fast atom


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83

bombardment ( F A B ) - M S , they yielded chromatograms identical to C bases produced by alkali treatment o f T T X and a n h y d r o - T T X . Similar findings have been reported by others (30-35). 9


Discussion Experimental evidence indicates that many marine bacteria produce T T X s . H o w ever, T T X production by some bacteria has not been validated since T T X and anhydro-like T T X are described as "difficult to detect" by using H P L C and G C M S methods, and show n o activity i n the mouse bioassay. If c o m m o n marine bacteria, such as Vibrio sp. and Pseudomonas sp., indeed produce T T X s , it might be expected that more animals, particularly those living in aquatic environments, w o u l d be toxic. However, apparently only specific animals can concentrate T T X and/or provide a niche for T T X - p r o d u c i n g bacteria. T h e mechanisms involved i n transfer o f bacterial T T X s to animal tissues (e.g., liver, skin, intestines, and gonads o f fish) are u n k n o w n . Tetrodotoxin could originate from bacteria o n skin, intestines, o r other internal fish tissues. Indeed, evidence indicates that Vibrio sp. may be normal flora o f fish tissues (55). D i e t may also be a potential source o f toxin, since algae and other animals i n the food chain are k n o w n to contain T T X and S T X (23, 29, 30). This has particular relevance since K o g u r e et a l . (56) have shown that relatively high concentrations o f T T X can be found i n marine sediments, potentially affecting benthic-feeding animals. Hypothetically, animals that concentrate T T X may have evolved unique proteins with the binding properties o f the T T X binding site, sequestering T T X , and reducing its toxicity for host tissues. Indeed, it has been reported that the m i n i m u m lethal dose o f T T X is greater for toxic versus non-toxic species o f pufferfish (57). Furthermore, experiments measuring the fate o f intraperitoneally injected, tritiated T T X demonstrate that T T X accumulates i n pufferfish tissues (i.e., skin, liver, intestines, muscle) similar to wild pufferfish stocks (58). It has been suggested that T T X and chemically related compounds are part o f an anti-predatory mechanism o f animals and offspring (59-61). Toxic starfish (Astropecten polyacanthus) are k n o w n to be eaten by the trumpet shell (Charonia sauliae) which accumulates large quantities o f T T X (59, 60). Furthermore, the role o f T T X as a defense agent o f pufferfish was supported by studies showing that m i l d handling causes pufferfish skin to release 5 - 8 0 mouse units o f T T X (61). Such evidence w o u l d indicate that T T X - p r o d u c i n g bacteria have evolved important relationships with marine animals. T h e conditions for production o f T T X and S T X by bacteria are u n k n o w n . T h e l o w levels o f T T X and S T X observed i n laboratory cultures may indicate that the host environment has not been duplicated. Likely, the c o m p o s i t i o n o f culture medium and other physicochemical parameters for T T X and S T X product i o n have not yet been defined i n vitro. Conversely, bacteria may actually produce only small amounts o f T T X and S T X i n vivo that accumulate i n host tissues over l o n g time intervals. The experimental evidence described above has lead to new hypotheses o n the natural source o f T T X s and S T X s , offering strong support for a bacterial origin. Independent studies indicate that marine and estuarine bacteria residing o n o r i n tissues o f marine organisms are potential sources o f T T X s and S T X s (30-35). F u t u r e research will likely determine the distribution o f bacteria producing sodium channel blockers, as well as factors that influence the production and distribution o f these toxins i n tissues o f marine organisms.


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MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY Acknowledgment The author wishes to thank Dr. K . Kogure and Dr. S. Hall for their helpful discussions in preparing this manuscript.


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