Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels

Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the α subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either α-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2 + influx.

Premature Polyadenylation at Multiple Sites within a Bacillus thuringiensis Toxin Gene-Coding Region1

Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter. Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t. toxins. As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells. Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts. Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

Direct Evidence for Rapid Degradation of Bacillus thuringiensis Toxin mRNA as a Cause of Poor Expression in Plants1

It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial. Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms. To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5′ and 3′ flanking sequences. Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays. In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene. Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum). These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants. Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. Binding of cholera toxin to the cell surface and transport of toxin to the Golgi apparatus were not changed in thapsigargin-treated cells, suggesting increased retrograde transport of cholera toxin from the Golgi apparatus to the ER. The data demonstrate that retrograde transport of cholera toxin can take place and that the transport is under regulation. The results are consistent with the idea that retrograde transport can be important for the action of cholera toxin.

Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo.

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.

Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.

Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.

Transcytosis of cholera toxin subunits across model human intestinal epithelia.

Cholera toxin (CT) elicits a massive secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and ultimately activating basolateral effectors (adenylate cyclase). The mechanism of signal transduction from apical to basolateral membrane, however, remains undefined. We have previously shown that CT action on the polarized human intestinal epithelial cell line T84 requires endocytosis and processing in multiple intracellular compartments. Our aim in the present study was to test the hypothesis that CT may actually move to its site of action on the basolateral membrane by vesicular traffic. After binding apical receptors, CT entered basolaterally directed transcytotic vesicles. Both CT B subunits and to a lesser extent CT A subunits were delivered intact to the serosal surface of the basolateral membrane. The toxin did not traverse the monolayer by diffusion through intercellular junctions. Transcytosis of CT B subunits displayed nearly identical time course and temperature dependency with that of CT-induced Cl- secretion--suggesting the two may be related. These data identify a mechanism that may explain the link between the toxin's apical receptor and basolateral effector.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.