Regulatory Networks Controlling Neurotoxin Synthesis in Clostridium botulinum and Clostridium tetani

Clostridium botulinum and Clostridium tetani are Gram-positive, spore-forming, and anaerobic bacteria that produce the most potent neurotoxins, botulinum toxin (BoNT) and tetanus toxin (TeNT), responsible for flaccid and spastic paralysis, respectively. The main habitat of these toxigenic bacteria is the environment (soil, sediments, cadavers, decayed plants, intestinal content of healthy carrier animals). C. botulinum can grow and produce BoNT in food, leading to food-borne botulism, and in some circumstances, C. botulinum can colonize the intestinal tract and induce infant botulism or adult intestinal toxemia botulism. More rarely, C. botulinum colonizes wounds, whereas tetanus is always a result of wound contamination by C. tetani. The synthesis of neurotoxins is strictly regulated by complex regulatory networks. The highest levels of neurotoxins are produced at the end of the exponential growth and in the early stationary growth phase. Both microorganisms, except C. botulinum E, share an alternative sigma factor, BotR and TetR, respectively, the genes of which are located upstream of the neurotoxin genes. These factors are essential for neurotoxin gene expression. C. botulinum and C. tetani share also a two-component system (TCS) that negatively regulates neurotoxin synthesis, but each microorganism uses additional distinct sets of TCSs. Neurotoxin synthesis is interlocked with the general metabolism, and CodY, a master regulator of metabolism in Gram-positive bacteria, is involved in both clostridial species. The environmental and nutritional factors controlling neurotoxin synthesis are still poorly understood. The transition from amino acid to peptide metabolism seems to be an important factor. Moreover, a small non-coding RNA in C. tetani, and quorum-sensing systems in C. botulinum and possibly in C. tetani, also control toxin synthesis. However, both species use also distinct regulatory pathways; this reflects the adaptation of C. botulinum and C. tetani to different ecological niches.     

The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity

The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain H_N, and the C-terminal receptor binding domain H_CC are largely resolved, but that of the H_CN domain sandwiched between H_N and H_CC has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their H_CN domains or swapping H_CN domains between each other. Both deletion and replacement of TeNT H_CN domain by H_CNA and H_CNB reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping H_CN domains between BoNT/A and B hardly impaired their biological activity, but substitution with H_CNT did. Binding assays revealed that in the absence of H_CN, not all receptor binding sites are equally well accessible. In conclusion, the presence of H_CN is vital for CNTs to exert their neurotoxicity. Although structurally similar, the H_CN domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that H_CNT plays a different role in the intoxication mechanism of TeNT.     

An in vitro assay for detection of tetanus neurotoxin activity: Using antibodies for recognizing the proteolytically generated cleavage product

Tetanus neurotoxin (TeNT¹) is a bacterial protease which specifically cleaves the vesicle protein synaptobrevin-2 (vesicle associated membrane protein-2, VAMP-2). This proteolytic feature of the toxin has been used to develop a sensitive endopeptidase assay for the detection of TeNT activity as an alternative to the in vivo assay for TeNT toxicity. Recombinant synaptobrevin-2 (rSyb2) is immobilized onto a microtiter plate, and the cleavage of immobilized rSyb2 by TeNT is detected with a polyclonal antibody directed against the newly generated C-terminus of the cleavage product. This antibody is shown to be a highly specific tool for detecting rSyb2 proteolysis by TeNT. The method reaches a detection limit of less than 1 pg TeNT/ml. To our knowledge, this is the most sensitive in vitro assay for the detection of TeNT activity, and it is easy to perform. Besides, the assay can also detect the activity of botulinum neurotoxin type B (BoNT/B). The method can be applied to examine the toxicity of TeNT or BoNT/B preparations as well as the influence of chemicals on TeNT and BoNT/B activity. In the future, the assay may also serve as a basis for the replacement of the in vivo safety control of tetanus vaccines.     

Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain

Summary Inhibition of neurotransmitter release by tetanus toxin and botulinum neurotoxin A can be mimicked by intracellular application of the corresponding toxin light chains. The aim of this study was to determine whether the two-chain toxins are reduced by brain preparations to yield free light chains which would represent the ultimate toxins.The interchain disulfide of two-chain tetanus toxin was cleaved by rat cortex homogenate fortified with NADPH. Reduction was promoted further by addition of thioredoxin. Thioredoxin reductase was demonstrated in and purified from porcine brain cortex. The thioredoxin system which consisted of purified enzyme, thioredoxin and NADPH reduced both toxins. The resulting light chains appeared homogeneous in SDS gel electrophoresis. The complementary heavy chain of tetanus but not of botulinum toxin migrated in two bands, the faster one with the velocity of heavy chain obtained by chemical reduction. The major, slower form was converted into the faster by chemical but not by enzymatic reduction. Tetanus toxin, whether in its single-chain or two-chain version also occurred in two forms which differed by their electrophoretic mobility. The two forms of single-chain toxin were interconverted by chemical reduction or oxidation but not by the thioredoxin system.It is concluded that a) a thioredoxin system in brain tissue reduces the interchain disulfide of two-chain tetanus toxin and botulinum neurotoxin A, b) tetanus toxin but not botulinum neurotoxin A consists of two electrophoretically distinct forms which differ by the thiol-disulfide status of their heavy chains, c) the disulfide loop within the heavy chain of tetanus toxin is resistant to the thioredoxin system. Send offprint requests to E. Habermann at the above address     

In vitro determination of tetanus toxicity by an endopeptidase assay linked to a ganglioside-binding step

Assays for the detection of tetanus neurotoxin (TeNT) are relevant for research applications as well as for the safety testing of tetanus vaccines. So far, these assays are usually performed as toxicity tests in guinea pigs or mice. The alternative methods described to date were mostly based on the detection of the toxin’s proteolytic activity. However, these endopeptidase assays turned out to be unreliable because they only measure the enzymatic activity as sole determinant of tetanus toxicity, while not taking into account other parameters like the toxin’s capacity to bind to target cells. In order to better reflect the in vivo situation of a tetanus infection, we have linked an endopeptidase assay to a ganglioside-binding step. The resulting method, which offers a unique combination of two functionally linked assays, detects those TeNT molecules only which possess both a functional binding domain as well as an active enzymatic domain. Our results demonstrate that this assay is able to reliably detect TeNT, and therefore might provide a basis for the replacement of the animal tests for detection of tetanus toxicity. Moreover, the assay concept could also be useful for in vitro toxicity measurements of other toxins with similar subunit structures.     

Structural analysis of the catalytic domain of tetanus neurotoxin.

Clostridium neurotoxins, comprising the tetanus neurotoxin and the seven antigenically distinct botulinum neurotoxins (BoNT/A-G), are among the known most potent bacterial protein toxins to humans. Although they have similar function, sequences and three-dimensional structures, the substrate specificity and the selectivity of peptide bond cleavage are different and unique. Tetanus and botulinum type B neurotoxins enzymatically cleave the same substrate, vesicle-associated membrane protein, at the same peptide bond though the optimum length of substrate peptide required for cleavage by them is different. Here, we present the first experimentally determined three-dimensional structure of the catalytic domain of tetanus neurotoxin and analyze its active site. The structure provides insight into the active site of tetanus toxin's proteolytic activity and the importance of the nucleophilic water and the role of the zinc ion. The probable reason for different modes of binding of vesicle-associated membrane protein to botulinum neurotoxin type B and the tetanus toxin is discussed. The structure provides a basis for designing a novel recombinant vaccine or structure-based drugs for tetanus.     

Angiopep-2修饰载神经毒素介孔二氧化硅脂质囊纳米粒的制备及其体内外评价

目的 制备Angiopep-2（ANG）修饰的载神经毒素（neurotoxin,NT）介孔二氧化硅脂质囊纳米粒（mesoporous silica nanoparticles,MSN）（ANG-LP-MSN-NT）,并进行体内外评价。方法 利用改进的Stober法制备介孔二氧化硅纳米粒,然后运用薄膜水化法制备ANG-LP-MSN-NT。考察其形态、粒径、Zeta电位、载药量和包封率;通过小角粉末衍射、氮气吸-脱附法等技术对其进行表征;透析袋法考察其体外释药特性;热板法和醋酸扭体法考察其镇痛效果。结果 制备的MSN比表面积为557 m²·g^-1,孔径和孔容积（V_p）分别为2.94 nm和0.58 cm³·g^-1。ANG-LP-MSN-NT分布均一,无团聚现象,粒径为（123.37±3.76）nm（PDI 0.20±0.02）,Zeta电位为（-16.57±1.59）mV,载药量与包封率分别为（10.75±0.54）%与（91.82±3.12）%。ANG-LP-MSN-NT较MSN-NT体外突释降低,缓释特性明显;药效学实验结果表明ANG-LP-MSN-NT起效快、最大镇痛效应优于其他组别。结论 ANG-LP-MSN-NT解决了二氧化硅易团聚、易突释的问题,且更有利于NT在脑部富集,发挥更好的镇痛效果,该纳米递药系统作为神经毒素载体在镇痛方面具有较好的应用前景。     

Morphological, genetic, and chemical polymorphism of Curcuma kwangsiensis

Previously, Chinese Gajutsu available in Japan was identified, from the chloroplast trnK gene sequence, to be the rhizomes of Curcuma phaeocaulis and two genotypes of C. kwangsiensis. Although we defined the two genotypes, the pl and gl types, on the basis of the nucleotide difference, their external features did not correspond to the two phenotypes described in the literature. In this paper, to investigate the relationship between genotype and phenotype of C. kwangsiensis, a field investigation was carried out in its main cultivation areas of Guangxi Zhuangzu Autonomous Region and Guangdong Province, China, and sequence analysis of the trnK gene and single-nucleotide polymorphism (SNP) analysis of the nuclear 18S rRNA gene were performed on the collected specimens. Four genotypes of C. kwangsiensis were recognized from the combined 18S rRNA gene–trnK gene sequences: homozygote-K(gl)Wtk type, homozygote-K(pl)Ztk type, heterozygote-K(gl)Wtk type, and heterozygote-Ltk type. Among the four genotypes, C. kwangsiensis in a field used for cultivation of Gajutsu was of heterozygote-K(gl)Wtk type. Formation of a heterozygote in the 18S rRNA gene might be a result of crossbreeding of C. kwangsiensis with several Curcuma species which had cytosine at nucleotide position 234. GC analysis of the rhizomes revealed that C. kwangsiensis was characterized by camphor and β-elemene, and by detecting additional components such as curdione and curcumenol Curcuma species involved in the formation of the heterozygote might be speculated upon.     

Botulinum versus tetanus neurotoxins: Why is botulinum neurotoxin but not tetanus neurotoxin a food poison?

Botulinum and tetanus neurotoxins, produced by Clostridium botulinum and Clostridium tetani, respectively, are the most poisonous poisons known to mankind. Although botulinum and tetanus neurotoxins share several characteristics, such as similar mol. wts, similar macrostructure, virtually identical mode of action, and a strong amino acid sequence homology, the two neurotoxins differ in one very significant way; only botulinum neurotoxin is a food poison. Factors responsible for the food poisoning potential of botulinum neurotoxins seem to be a group of complexing proteins that are also produced by C. botulinum, and are known to associate with the neurotoxin. Translation products of nucleotide sequences upstream to the neurotoxin genes of serotypes A, B, C, D, E and F botulinum neurotoxin reveal the location of genes for one of the complexing proteins that could be transcribed as polycistronic mRNA to include neurotoxin sequences. No such protein seems to be present in C. tetani, suggesting that the lack of complexing proteins might be responsible for tetanus not being a food poison.     

Influence and related mechanism of Retn gene expression on glucose uptake in 3T3-L1 cells

The aim of this article was to investigate the influence and the related mechanism of the Retn gene on glucose uptake and insulin resistance in 3T3-L1 cells. Radioimmuno-assay was used to determine glucose uptake in 3T3-L1 cells with different Retn gene expression levels, whether cells were stimulated by insulin or not. RT-PCR and real-time RT-PCR analysis were used to determine the mRNA levels of several glucose transport proteins in 3T3-L1 cells with different Retn gene expression levels, including insulin receptor substrate-1(IRS-1), phosphatidylinositol 3-kinase (PI-3K), AKT-2, glucose transporter-4 (GLUT-4), p38 mitogen-activated protein kinase (p38MAPK) and glycogen synthase kinase-3β (GSK-3β). The glucose uptake decreased with the increase in Retn gene expression in 3T3-L1 cells, which was independent of whether the cells were stimulated by insulin or not. The mRNA expression of two signal proteins PI-3K and AKT-2 decreased and the other two signal proteins, GSK-3β and p38MAPK, increased with Retn overexpression in 3T3-L1 cells. Resistin could induce insulin resistance in adipocytes, which might be related to the changes of some proteins in PI-3K and Ras pathways. Translated from Academic Journal of Second Military Medical University, 2006, 27(10): 1,067–1,071 [译自: 第二军医大学学报] The authors contributed equally to the study.     

In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA

In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91 → Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91 → Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87 → Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species.     

(<Emphasis Type="Italic">Z</Emphasis>)-3-Hexenyl diglycosides from <Emphasis Type="Italic">Spermacoce laevis</Emphasis> Roxb.

A new (Z)-3-hexenyl O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside was isolated from the aerial part of Spermacoce laevis, along with 17 known compounds: (6S,9R)-roseoside, (Z)-3-hexenyl O-β-d-glucopyranoside, (Z)-3-hexenyl O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside, (Z)-3-hexenyl O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside, phenyethyl O-β-d-glucopyranoside, phenyethyl O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside, phenyethyl O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside, benzyl O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside, benzyl O-β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside, asperuloside, 6α-hydroxyadoxoside, asperulosidic acid, kaempferol 3-O-β-d-glucopyranoside, kaempferol 3-O-rutinoside, quercetin 3-O-β-d-galactopyranoside, quercetin 3-O-α-l-rhamnopyranosyl-(1→6)-β-d-galactopyranoside, and rutin. The structure determinations were based on physical data and spectroscopic evidence.     

New triterpenoid saponins from <Emphasis Type="Italic">Argania spinosa</Emphasis>

Five new triterpene saponins, arganine L (1), O (2), P (3), Q (4) and R (5), were isolated from the barks of Argania spinosa (L.) Skeels. Arganines L-P and R are bidesmosidic saponins. The structures of 1–5 were elucidated as 3-O-[β-d-xylopyranosyl-(1–4)-β-d-glucuronopyranosyl]-28-O-[β-d-apiofuranosyl-(1–3)-β-d-xylopyranosyl-(1–4)-α-l-rhamnopyranosyl-(1–2)-α-l-arabinopyranosyl] bayogenin, 3-O-[β-d-xylopyranosyl-(1–4)-β-d-glucuronopyranosyl]-28-O-[β-d-xylopyranosyl-(1–4)-α-l-arabinopyranosyl] bayogenin, 3-O-[β-d-xylopyranosyl-(1–4)-β-d-glucuronopyranosyl]-28-O-[α-l-arabinopyranosyl] bayogenin, 3-O-[β-d-xylopyranosyl-(1–4)-β-d-glucuronopyranosyl] bayogenin, and 3-O-[β-d-apiofuranosyl-(1–4)-β-d-glucuronopyranosyl]-28-O-[β-d-xylopyranosyl-(1–4)-α-l-rhamnopyranosyl-(1–2)-α-l-arabinopyranosyl] bayogenin, respectively, mainly on the basis of their spectroscopic data.     

Effects of hydroxamate metalloendoprotease inhibitors on botulinum neurotoxin A poisoned mouse neuromuscular junctions

Currently the only therapy for botulinum neurotoxin A (BoNT/A) poisoning is antitoxin. Antidotes that are effective after BoNT/A has entered the motor nerve terminals would dramatically benefit BoNT/A therapy. Inhibition of proteolytic activity of BoNT/A light chain by metalloendoprotease inhibitors (MEIs) is under development. We tested the effects of MEIs on in vitro as well as in vivo BoNT/A poisoned mouse nerve-muscle preparations (NMPs). The K_i for inhibition of BoNT/A metalloendoprotease was 0.40 and 0.36 μM, respectively, for 2,4-dichlorocinnamic acid hydroxamate (DCH) and its methyl derivative, ABS 130. Acute treatment of nerve-muscle preparations with 10 pM BoNT/A inhibited nerve-evoked muscle twitches, reduced mean quantal content, and induced failures of endplate currents (EPCs). Bath application of 10 μM DCH or 5 μM ABS 130 reduced failures, increased the quantal content of EPCs, and partially restored muscle twitches after a delay of 40–90 min. The restorative effects of DCH and ABS 130, as well as 3,4 diaminopyridine (DAP) on twitch tension were greater at 22 °C compared to 37 °C. Unlike DAP, neither DCH nor ABS 130 increased Ca²⁺ levels in cholinergic Neuro 2a cells. Injection of MEIs into mouse hind limbs before or after BoNT/A injection neither prevented the toe spread reflex inhibition nor improved muscle functions. We suggest that hydroxamate MEIs partially restore neurotransmission of acutely BoNT/A poisoned nerve-muscle preparations in vitro in a temperature dependent manner without increasing the Ca²⁺ levels within motor nerve endings.     

Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo

The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), β-naphthoflavone (β-NF), indirubin, indigo, 3,3′-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and β-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr ^−/−). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.     

Chemical constituents of the Thai medicinal plant, <Emphasis Type="Italic">Dioecrescis erythroclada</Emphasis> (Kurz) Tirveng

Six compounds were isolated from the leaves and branches of Dioecrescis erythroclada and identified as apodanthoside, mussaenoside, gardenoside, benzyl alcohol O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside, phenethyl alcohol O-β-d-apiofuranosyl-(1→6)-β-d-glucopyranoside, and oct-1-en-3-ol α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside. The structures were determined based on physical data and spectroscopic evidence.     

Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.     

Five new glycosides from <Emphasis Type="Italic">Hypericum erectum</Emphasis> Thunb.

Five new glycosides, quercetin 3′-O-β-d-galactopyranoside (1), quercetin 3-O-(2″-acetyl)-β-d-glucopyranoside (2), 4,6-dihydroxy-2-methoxyphenyl 1-O-β-d-glucopyranoside (3), 4-hydroxy-2,6-dimethoxyphenyl 1-O-α-l-rhamnopyranosyl (1 → 6)-β-d-glucopyranoside (4) and 3-methyl-but-2-en-1-yl β-d-glucopyranosyl (1 → 6)-β-d-glucopyranoside (5), were isolated from Hypericum erectum Thunb. Their structures were established on the basis of spectral and chemical data.