Crystallization and preliminary X-ray analysis of botulinum neurotoxin type A.

Botulinum neurotoxin serotype A was isolated from liquid culture of Clostridium botulinum. The pure Mr approximately 150,000 neurotoxin, composed of Mr approximately 50,000 light and Mr approximately 100,000 heavy chains, has been crystallized in three different crystal morphologies; all three have the same crystal form. The most suitable crystal form for X-ray analysis are bipyrimidal and crystallize in the hexagonal space group P3(1)21 (or P3(2)21) with one dimer per asymmetric unit. The unit cell dimensions are a = b = 170.5 A, c = 161.7 A. The crystals diffract to 3 A resolution.     

抗A型肉毒神经毒素中和抗体间接ELISA的建立

目的建立间接ELISA,检测血清中抗A型肉毒神经毒素(botulinum neurotoxin type A, BoNT-A)中和抗体。方法以纯化的BoNT-A作为包被抗原,抗BoNT-A兔血清作为一抗,辣根过氧化物酶(horseradish peroxidase, HRP)标记的金黄色葡萄球菌蛋白A(Staphylococal Protein A, SPA)作为二抗,结合实验设计,建立检测血清中抗BoNT-A中和抗体的间接ELISA,验证该方法的特异性、灵敏度和重复性,并与经典动物中和试验法检测抗BoNT-A中和抗体的结果进行比较。结果①间接ELISA条件的建立与优化,使用2.50μg/mL纯化的BoNT-A抗原包被,4℃过夜;1%明胶作为封闭剂,37℃封闭2 h;确定一抗优化条件:反应条件为1∶5 000稀释,37℃孵育90 min;二抗优化条件:反应条件为1∶10 000稀释,37℃孵育90 min;加入底物37℃显色10 min,加终止液终止后,用酶标仪测定A_(450 nm)值;②经过验证该方法特异性好,所包被抗原与抗伤寒杆菌兔血清、抗破伤风毒素兔血清、抗B型肉毒毒素兔血清、抗E型肉毒毒素兔血清、抗F型肉毒毒素兔血清均无交叉反应;灵敏度高,当抗BoNT-A兔血清按照1∶320 000稀释时,检测结果依然呈阳性;重复性良好,批内重复性验证CV分别为2.62%、1.40%、2.14%,变异系数均小于3%,批间差异无统计学意义(P=0.103,P>0.05);该方法检测抗BoNT-A中和抗体检出限为抗-0.03 LD_(50)/mL,大约是经典动物中和试验法的1/30,显著地提高了检测灵敏度。结论建立了一种可用于检测血清中抗BoNT-A中和抗体的间接ELISA。     

Nature of intracellular type A botulinum neurotoxin.

The neurotoxin in cells of young Clostridium botulinum type A culture was extracted with lysozyme. Highly purified neurotoxin preparation, obtained by processing the extract in two chromatographic steps had only unnicked (single-chain) molecules of molecular weight comparable to that of the dichains isolated from type A crystals. Trypsinization converted the unnicked molecules into dichains whose component subunits were of sizes indistinguishable from those of the neurotoxin from crystals. The enzymatic treatment increased toxicity of crude extract 30-fold but did not activate the purified intracellular neurotoxin preparation. The results indicated that intracellular type A botulinum neurotoxin is unnicked, is not fully activated, and is activated in the time between its extraction and purification. Since trypsinization nicked all of the single chains without increasing toxicity, nicking was not causally related to toxicity activation.     

Characterization of the subunits of botulinum neurotoxin type A

A comparative amino acid analysis of botulinum neurotoxin type A and its subunits has been carried out. The heavy and light chains of neurotoxin have the same ratios of polar and non-polar amino acids (1.3:1), the amount of tryptophan residues in the heavy chain is 4 times as much as that in the light chain, and the number of SH-groups exceeds that in the light chains 2-fold. In neurotoxin, two N-terminal amino acid residues--alanine and leucine--were identified. Alanine was found to be the N-terminus of the heavy chain. The fluorescence spectra of neurotoxin subunits indicate differences in the conformational state of the polypeptide chains. The antigenic non-identity of botulinum neurotoxin A subunits suggests the presence in the neurotoxin molecule of at least two antigenic determinants, corresponding to the heavy and light chains.     

Proteolysis of synthetic peptides by type A botulinum neurotoxin

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17-to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187–191 were required for efficient hydrolysis, but residues 167–186 and 203–206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.     

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

AIMS: An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). METHODS AND RESULTS: Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. CONCLUSION: Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.     

A型肉毒神经毒素理化及生物学特性研究

目的探究A型肉毒神经毒素的理化及生物学特性,为A型肉毒神经毒素制品的研制提供依据。方法采用SDS-PAGE、HPLC测定A型肉毒神经毒素的纯度;采用毛细管凝胶电泳分析其在非还原条件及还原条件下各组分的相对分子质量;采用等电点聚焦电泳测定其等电点;对其N-末端氨基酸序列进行测序,并将测序结果与NCBI blast数据库中A型肉毒梭菌Hall株的氨基酸序列作比对,确证其各组分的成分;对A型肉毒毒素复合物及A型肉毒神经毒素进行口服毒性分析,并对3批A型肉毒神经毒素原液和其半成品进行生物学稳定性研究。结果 A型肉毒神经毒素纯度99.00%;非还原条件下其相对分子质量约为152 000,N-末端氨基酸序列ALNDLQINVN,为完整的A型肉毒神经毒素;还原条件下,由相对分子质量约为101 000、N-末端氨基酸序列为ALNDLQINVN的重链和相对分子质量约为101 000、N-末端氨基酸序列为PFVNKQFNYK的轻链组成;等电点为4.91;口服A型肉毒神经毒素的小鼠LD50为1.50×107U/kg,是其复合物的7.9倍;3批A型肉毒神经毒素原液在2~8℃放置28 d生物学活性无下降,平均比活性为2.13×108U/mg,是其复合物的7.1倍;3批半成品在18~26℃放置28 d生物学活性无下降。结论 A型肉毒神经毒素纯度较高,非还原条件下为单链,还原条件下裂解为轻链和重链,其原液及其半成品的生物学稳定性较好。     

Elimination of botulinum neurotoxin (BoNT) type B from drinking water by small-scale (personal-use) water purification devices and detection of BoNT in water samples

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 microm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of < 0.1 log10 units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of > 2.3 log10 units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.     

Characterization of LC-HCC fusion protein of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.     

A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity

The peptide N-acetyl-CRATKML-amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61-64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl-containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin-converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2-mercapto-3-phenylpropionyl yielded a peptide with K(i) of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1' arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.     

Mucosal vaccine targeting improves onset of mucosal and systemic immunity to botulinum neurotoxin A

Absence of suitable mucosal adjuvants for humans prompted us to consider alternative vaccine designs for mucosal immunization. Because adenovirus is adept in binding to the respiratory epithelium, we tested the adenovirus 2 fiber protein (Ad2F) as a potential vaccine-targeting molecule to mediate vaccine uptake. The vaccine component (the host cell-binding domain to botulinum toxin (BoNT) serotype A) was genetically fused to Ad2F to enable epithelial binding. The binding domain for BoNT was selected because it lies within the immunodominant H chain as a beta-trefoil (Hcbetatre) structure; we hypothesize that induced neutralizing Abs should be protective. Mice were nasally immunized with the Hcbetatre or Hcbetatre-Ad2F, with or without cholera toxin (CT). Without CT, mice immunized with Hcbetatre produced weak secretory IgA (sIgA) and plasma IgG Ab response. Hcbetatre-Ad2F-immunized mice produced a sIgA response equivalent to mice coimmunized with CT. With CT, Hcbetatre-Ad2F-immunized mice showed a more rapid onset of sIgA and plasma IgG Ab responses that were supported by a mixed Th1/Th2 cells, as opposed to mostly Th2 cells by Hcbetatre-dosed mice. Mice immunized with adjuvanted Hcbetatre-Ad2F or Hcbetatre were protected against lethal BoNT serotype A challenge. Using a mouse neutralization assay, fecal Abs from Hcbetatre-Ad2F or Hcbetatre plus CT-dosed mice could confer protection. Parenteral immunization showed that the inclusion of Ad2F enhances anti-Hcbetatre Ab titers even in the absence of adjuvant. This study shows that the Hcbetatre structure can confer protective immunity and that use of Hcbetatre-Ad2F gives more rapid and sustained mucosal and plasma Ab responses.     

Spectroscopic analysis of pH-induced changes in the molecular features of type A botulinum neurotoxin light chain

Clostridial botulinum neurotoxins (BoNTs) cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. While the toxin's heavy chain (HC) is involved in binding and internalization, the light chain (LC) acts as a unique Zn(2+)-endopeptidase against a target protein in the exocytotic docking/fusion machinery. During the translocation of the LC to the cytosol, it is exposed to the endosomal low pH. Low pH showed a dramatic change in the BoNT/A LC polypeptide folding as indicated by differential heat denaturation. Furthermore, binding of 1-anilinonaphthalenesulfonate (ANS) revealed exposure of hydrophobic domains of BoNT/A LC at low pH. Low-pH-induced structural (and by implication the endopeptidase activity) changes were completely reversible. Exposure of BoNT/A LC to low pH (4.7) did not, however, evoke the loss of Zn(2+) bound to its active site. Implications of these observations to the delivery of active BoNT/A LC to the nerve cell are discussed. We further analyzed the nature of low-pH-induced change in the polypeptide folding of BoNT/A LC by Trp fluorescence measurements. The Trp fluorescence peak was observed at 322 nm, and the two fluorescence lifetime components estimated at 2.1 ns (88%) and 0.6 ns (12%) did not change much at low pH. These observations suggested that the two Trp residues are buried and constrained in a hydrophobic environment, and it is likely that the core of the BoNT/A LC protein matrix does not participate in the low-pH-induced structural alteration. This conclusion was further supported by the near-UV circular dichroism spectra under two pH conditions.     

Synthesis of substrates and inhibitors of botulinum neurotoxin type A metalloprotease.

Botulinum neurotoxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the active sites of the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes for enzyme activation, we synthesized a series of transition-state analog inhibitors in which the dipeptide cleavage site is replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. In this paper, we report our efforts to design inhibitors of BoNT/A metalloprotease. We confirm that an effective substrate sequence for BoNT/A metalloprotease is a 17-mer peptide corresponding to residues 187-203 of SNAP-25. A more stable substrate, Nle202SNAP-25 [187-203] was synthesized in order to develop an assay for proteolytic activity of BoNT/A metalloprotease that can be used to monitor time-dependent inhibition. Alpha-thiol amide analogs of Gln-197 were incorporated via solid-phase peptide synthesis into both 17-mer minimal peptide substrate sequences. The synthesis, characterization and inhibition kinetics for the alpha-thiol amide analogs of holotoxin A substrate are described. These substrate-derived inhibitors were shown to be submicromolar inhibitors of BoNT/A catalytic activity.     

Improved detection of botulinum neurotoxin type A in stool by mass spectrometry

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind. Rapid and sensitive detection of BoNTs is necessary for timely clinical confirmation of the disease state in botulism. BoNTs cleave proteins and peptide mimics at specific sites. A mass spectrometry (MS)-based method, Endopep–MS, can detect these cleavages and has detection limits of 0.05–0.5 mouse LD₅₀ (U) in serum, depending on the BoNT serotypes. In this method, the products generated from cleavage of peptide substrates using antibody affinity-purified toxins are detected by MS. Nonspecific bound endogenous proteases or peptidases in stool can coextract with the toxin, cleaving the peptide substrates and reducing the sensitivity of the method. Here we report a method to reduce nonspecific substrate cleavage by reducing stool protease coextraction in the Endopep–MS assay.     

Improved detection of botulinum neurotoxin type A in stool by mass spectrometry

A novel electrochemical method, termed flash chronopotentiometry (FCP), is used to develop a rapid and sensitive method for detecting protease activities. In this method, an appropriate current pulse is applied across a polycation-selective polymer membrane to induce a strong flux of the polycationic peptides from the sample phase into the organic membrane of the electrode. During this current pulse, the cell potential (EMF) is monitored continuously, and is a function of the polypeptide concentration. The imposed current causes a local depletion of the polypeptide at the sample/membrane interface, which yields a drastic potential change in the observed chronopotentiogram at a characteristic time, called the transition time (τ). For a given magnitude of current, the square root of τ is directly proportional to the concentration of the polypeptide. Proteases cleave polypeptides into smaller fragments that are not favorably extracted into the membrane of the sensor. Therefore, a decrease in the transition time is observed during the proteolysis process. The degree of change in the transition time can be correlated to protease activity. To demonstrate this approach, the activities of trypsin and α-chymotrypsin are detected using protamine and synthetic polycationic oligopeptides that possess specific cleavage sites that are recognized by these proteases.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism

The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.     

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.     

Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.