Ion channel and membrane translocation of diphtheria toxin

Abstract Diphtheria toxin is the best studied member of a family of bacterial protein toxins which act inside cells. To reach their cytoplasmic targets, these toxins, which include tetanus and botulinum neurotoxins and anthrax toxin, have to cross the hydrophobic membrane barrier. All of them have been shown to form ion channels across planar lipid bilayer and, in the case of diphtheria toxin, also in the plasma membrane of cells. A relation between the ion channel and the process of membrane translocation has been suggested and two different models have been put forward to account for these phenomena. The two models are discussed on the basis of the available experimental evidence and in terms of the focal points of difference, amenable to further experimental investigations.     

Production of diphtheria toxin CRM228 in B. subtilis

The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.     

Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with β-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.     

白喉毒素无毒突变体CRM197基因的克隆与表达

目的构建白喉毒素(Diphtheria toxin)无毒突变体CRM197(Cross-reacting materials 197)的原核表达载体,并在大肠杆菌中表达重组蛋白。方法以白喉杆菌(ATCC39255)基因组DNA为模版,采用聚合酶链式反应(Polymerase chain reaction,PCR)扩增CRM197基因,插入表达载体pET11b中,构建重组原核表达质粒pET11b-CRM197。经双酶切及测序鉴定正确后,重组质粒被转化入大肠杆菌Rosetta 2(DE3)pLysS,IPTG诱导表达,表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒经双酶切及测序鉴定,结果表明与预期一致;表达的重组蛋白相对分子质量约58 000,并可与鼠抗CRM197单克隆抗体特异性结合。结论已成功构建了重组原核表达载体pET11b-CRM197,重组的CRM197蛋白在大肠杆菌中得到了表达,为以该重组突变体作蛋白载体制备结合疫苗奠定了基础。     

Computer modelling of the transmembrane channel formed by a CNBr peptide of diphtheria toxin B fragment

Abstract Diphtheria toxin (DT) forms transmembrane, voltage-dependent channels in a planar lipid bilayer. Channels with similar characteristics were obtained with CB1, a cyanogen bromide peptide of diphtheria toxin B fragment (DTB) (res 340–459). Tryptophan 398 is in interaction with the hydrophobic core of the lipid bilayer. Using the Eisenberg method in association with the Shiffer-Edmunson wheel representation, we have identified two amphipathic α-helices within CB1 (res 346–364 and 389–406) that could be involved in the interaction with lipids. Bearing this information in mind, we are providing a model for the structure of the CB1 channel.     

Enhancement of diphtheria toxin potency by replacement of the receptor binding domain with tetanus toxin C-fragment: a potential vector for delivering heterologous proteins to neurons

This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB(389)TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB(389)) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB(389)TTC was approximately 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB(389)MSH. The cytotoxic effect of DAB(389)TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB(389)TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB(389) catalytic fragment. These results suggest that a catalytically inactive form of DAB(389)TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.     

Characterization of production processes for tetanus and diphtheria anatoxins

Tetanus and diphtheria are diseases that still cause significant morbidity and mortality. Clostridium tetani produces the tetanus toxin, a 150-kDa protein. The diphtheria toxin is synthesized by Corynebacterium diphtheriae as a protein of 58 kDa. The objective of this study was to carry out a chemical characterization of the tetanus and diphtheria toxin forms in the several production process stages, and thus to establish an affordable alternative in vitro quality control to aggregate to the classical tests. The 150 kDa band of the tetanus toxin and approximately 58 kDa band of the diphtheria toxin were observed by electrophoresis similar as that described in the literature. The same band of 58 KDa was detected in Western blotting reactions. The results obtained for diphtheria toxin showed very similar protein profiles between distinct lots. For the tetanus toxin, the profiles of the initial stage showed some variability, but the ones of the following stages were similar. The similarity of the electrophoresis results indicated reproduction and consistency of the production processes in Butantan Institute and correlated with the yield and antigenic purity classical data. The establishment of alternative in vitro quality control tests can significantly contribute to achieve the consistency approach supported by WHO.     

Conformation and model membrane interactions of diphtheria toxin fragment A

Low pH is believed to play a critical role in the penetration of membranes by diphtheria toxin in vivo. In this report, the pH dependence of the conformation of fragment A of diphtheria toxin has been studied using fluorescence techniques. As pH is decreased, fragment A in solution undergoes a reversible conformational change beginning below pH 5. The conformational change occurs rapidly upon exposure to low pH. It involves both an increase in the exposure of tryptophanyl residues to solution and a switch from a hydrophilic state to a hydrophobic state as judged by fragment A binding to micelles of a mild detergent (Brij 96). At low pH fragment A also rapidly and tightly binds to and penetrates model membranes. Binding is reversed when pH is neutralized. The transition pH, the apparent midpoint of the change between the hydrophilic state and the membrane-penetrating hydrophobic state, occurs at about pH 3.5 in the presence of Brij 96 micelles, pH 4 in the presence of small unilamellar vesicles (SUV) composed of zwitterionic phosphatidylcholine, and pH 5 in the presence of SUV composed of 25 mol % anionic phosphatidylglycerol and 75% phosphatidylcholine. The effects of high temperature provide an important clue as to the nature of the changes at low pH. At neutral pH and high temperature, i.e. in the thermally denatured state, a conformational change similar to that observed at low pH occurs, although fragment A does not become hydrophobic. In addition, the effects of low pH and high temperature on the stability of the native state are cumulative. This indicates that the changes in fragment A both at high temperature and at low pH involve denaturation, although there appears to be only partial unfolding under these conditions. Based on the results of this study, the role of fragment A in diphtheria toxin membrane penetration and translocation is evaluated.     

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes -helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish

In order to study lens-retina relationships during development, we cloned the zebrafish alphaA-crystallin cDNA and its promoter region. Using a 2.8-kb fragment of the zebrafish alphaA-crystallin promoter (z(alpha)Acry), we expressed the diphtheria toxin A fragment (DTA) in zebrafish embryos in a lens-specific manner. Injection of the z(alpha)Acry-DTA plasmid into eggs at the one-or two-cell stage resulted in the formation of small eyes, in which both lens and retina were reduced in size. In the DTA-expressing lenses, their fiber structure was disorganized, indicating that normal lens development had been abrogated. The neural retina also showed abnormal development, although this tissue did not express DTA. Lamination in the retina did not develop well, and molecular markers for the outer and inner plexiform layers were either abnormally expressed or absent. However, cell type-specific markers of ganglion and bipolar cells, as well as photoreceptors, were expressed in appropriate positions, indicating that initial differentiation of these retinal subpopulations occurred in the DTA-expressing embryos. Cell proliferation also proceeded normally in these embryos, although apoptosis was enhanced. These results suggest that the differentiated lens plays a critical role in the morphogenetic organization of retinal cells during eye development in zebrafish embryos.     

Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an a-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two -helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 m KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Safety evaluation of DT388IL3, a diphtheria toxin/interleukin 3 fusion protein, in the cynomolgus monkey

We developed a fusion toxin, DT₃₈₈IL3, consisting of the catalytic and translocation domains of diphtheria toxin (DT₃₈₈) linked to interleukin 3 (IL3) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to estimate a range for the maximum tolerated dose (MTD) and to evaluate the dose-limiting toxicity (DLT) of DT₃₈₈IL3 in cynomolgus monkeys (Macaca fasicularis), which possess cross-reactive IL3 receptors. In our previous study, we administered up to six infusions of DT₃₈₈IL3 at 40, 60, or 100 g/kg every other day to three pairs (one male monkey and one female monkey) of young adult monkeys. In five of six monkeys, results showed a dose-dependent increase in malaise and anorexia but no consistent abnormalities in serum chemistries or blood counts. There was no evidence of organ damage by blood tests or histopathology. However, the female treated at 100 g/kg, died of moderate to severe vasculitis of multiple tissues. Based on these findings, this study repeated the 100 g/kg group and added a group that received 150 g/kg in an effort to confirm a dose response. Two female monkeys were treated with up to six infusions of DT₃₈₈IL3 at 100 g/kg or 150 g/kg every other day. One additional female monkey was treated as a negative control. Monkeys in the 100 g/kg group showed moderate malaise and anorexia, but no consistent abnormalities in blood counts or serum chemistries. Moderate elevations of liver enzymes were noted in the 150 g/kg group in addition to severe malaise and anorexia. No significant findings were revealed at gross necropsy. The histopathological findings revealed regenerative myeloid hyperplasia and hepatic degeneration and regeneration in the 150 g/kg group. Similar lesions of less severity were detected in the 100 g/kg group. DT₃₈₈IL3 plasma half-life was approximately 20 min with a peak concentration of approximately 2 g/ml (30,000 pM). The IC₅₀ for AML blasts in vitro was 6 pM. Collectively, our results suggest that DT₃₈₈IL3 can be tolerated at doses up to 100 g/kg in a nonhuman primate, which is higher than previously reported for other AML directed diphtheria toxin fusion proteins, and should in principle allow for dose escalation with reduced toxic side effects. Based on these findings a phase I clinical trial has recently been initiated with DT₃₈₈IL3 for the treatment of AML.     

Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance

The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membranes depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes

In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of β-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 °C, no desorption took place at 4 °C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.     

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH

Like diphtheria toxin and the N-terminal (M_r 23 000) region of fragment B, CB1 (M_r 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (M_r 23 000 and 13 000) share a common segment (M_r 6300) containing the predicted amphipathic, -helical, transverse lipid-associating domain (M_r 2750) of fragment B[J. Cell Biol. (1980) 87, 837–840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172–176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C-terminal region of CB1 is proposed.     

Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco

Summary A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm. The mature seed thus consists of a seed coat of one rigid cell layer, and a degenerated embryo. The genetic ablation technique has thus contributed to the establishment of the sequence of events and elucidation of the role of different cell lineages and tissues in seed development.     

Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10^–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH₂)P are able to displace EF2 from blue Sepharose.     

Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain

We have established a transgenic mouse line in which floxed neomycin resistant cassette was inserted between the CAG promoter and EGFP. When these transgenic mice were mated with Cre-expressing transgenic animals, the offspring obtained were fluorescent green. We then established a transgenic mouse line in which EGFP in the above construct was replaced by diphtheria toxin A chain (DT). When the latter transgenic mice were mated with mice expressing Cre restricted to germ cells, we obtained healthy but sterile offspring due to a disruption of germ line cells by DT expression. We predict that this strategy will be useful for the construction of new animal models for human diseases, featuring a variety of missing cell lineages produced by disruption with DT.     

白喉毒素超滤浓缩后膜通透性恢复试验的研究

应用超滤浓缩技术对白喉毒素培养液进行澄清过滤和超滤浓缩,已经早已为生产厂家所使用.但是,超滤膜的通透性下降很快,使膜的使用期限大大缩短,生产成本较高.因此在试验过程中,注重摸索超滤过程的中间控制以及超滤结束后的清洗方法,特别是使用0.5 mol/LNaOH+200ppm次氯酸钠作为清洗剂,在超滤结束以后对滤膜进行彻底的恢复性清洗,可以使澄清和浓缩过滤速度分别达到平均77 L/M2·h和75 L/M2·h,回收率分别达到93.1%和92.8%,使滤膜的滤过性能(NWP)基本恢复到滤前水平.