Protective antibodies against Clostridium difficile are present in intravenous immunoglobulin and are retained in humans following its administration.

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

An assay for botulinum toxin types A, B and F that requires both functional binding and catalytic activities within the neurotoxin.

AIM: To develop a novel assay technique for the botulinum neurotoxin family (BoNTs) which is dependent on both the endopeptidase and receptor-binding activities of the BoNTs and which is insensitive to antigenic variation with the toxin family. METHODS AND RESULTS: An endopeptidase activity, receptor-binding assay (EARB assay) has been developed which captures biologically active toxin from media using brain synaptosomes. After capture, the bound toxin can be incubated with its substrate, and cleavage detected using serotype-specific antibodies raised against the cleaved product of each toxin serotype. The EARB assay was assessed using a range of BoNT serotypes and subtypes. For BoNT/A, detection limits for subtypes A(1), A(2) and A(3) were 0.5, 3 and 10 MLD(50) ml(-1), respectively. The limit of detection for BoNT/B(1) was 5 MLD(50) ml(-1) and a novel antibody-based endopeptidase assay for BoNT/F detected toxin at 0.5 MLD(50) ml(-1). All these BoNTs can be captured from media containing up to 10% serum without loss of sensitivity. BoNT/A(1) could also be detected in dilutions of a lactose- containing formulation similar to that used for clinical preparations of the toxin. Different serotypes were found to possess different optimal cleavage pHs (pH 6.5 for A(1), pH 7.4 for B(1)). CONCLUSIONS: The EARB assay has been shown to be able to detect a broad range of BoNT serotypes and subtypes from various media. SIGNIFICANCE AND IMPACT OF THE STUDY: The EARB assay system described is the first convenient in vitro assay system described which is requires multiple functional biological activities with the BoNTs. The assay will have applications in instances where it is essential or desirable to distinguish biologically active from inactive neurotoxin.

Re-engineering the target specificity of Clostridial neurotoxins - a route to novel therapeutics.

The ability to chemically couple proteins to LH(N)-fragments of clostridial neurotoxins and create novel molecules with selectivity for cells other than the natural target cell of the native neurotoxin is well established. Such molecules are able to inhibit exocytosis in the target cell and have the potential to be therapeutically beneficial where secretion from a particular cell plays a causative role in a disease or medical condition. To date, these molecules have been produced by chemical coupling of the LH(N)-fragment and the targeting ligand. This is, however, not a suitable basis for producing pharmaceutical agents as the products are ill defined, difficult to control and heterogeneous. Also, the molecules described to date have targeted neuroendocrine cells that are susceptible to native neurotoxins, and therefore the benefit of creating a molecule with a novel targeting domain has been limited. In this paper, the production of a fully recombinant fusion protein from a recombinant gene encoding both the LH(N)-domain of a clostridial neurotoxin and a specific targeting domain is described, together with the ability of such recombinant fusion proteins to inhibit secretion from non-neuronal target cells. Specifically, a novel protein consisting of the LH(N)-domains of botulinum neurotoxin type C and epidermal growth factor (EGF) that is able to inhibit secretion of mucus from epithelial cells is reported. Such a molecule has the potential to prevent mucus hypersecretion in asthma and chronic obstructive pulmonary disease.

Resonance Raman study of reconstituted carotenoproteins incorporating astaxanthin and 15,15'-didehydroastaxanthin.

Two reconstituted carotenoproteins have been studied by resonance Raman spectroscopy. They were prepared from the apoprotein of the Asterias rubens carotenoprotein, asteriarubin and either astaxanthin or 15,15'-didehydroastaxanthin. Spectral properties of dehydrocarotenoids are first discussed. The spectral properties of the complexes are compared to those of the free carotenoids and of other carotenoproteins containing astaxanthin, and possible protein-carotenoid interactions are discussed. Greater delocalisation of the pi-electron system in the central part of the polyene chain, and the role of lateral methyl groups in binding is emphasised.

Analysis of the substrate recognition domain determinants of botulinum type B toxin using phage display.

The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.

The interaction of synaptic vesicle-associated membrane protein/synaptobrevin with botulinum neurotoxins D and F.

Botulinum neurotoxins type D and F are zinc-endopeptidases with a unique specificity for VAMP/synaptobrevin, an essential component of the exocytosis apparatus. VAMP contains two copies of a nine residue motif, termed V1 and V2, which are determinants of the interaction with tetanus and botulinum B and G neurotoxins. Here, we show that V1 plays a major role in VAMP recognition by botulinum neurotoxins D and F and that V2 is also involved in F binding. Site-directed mutagenesis of V1 and V2 indicates that different residues are the determinants of the VAMP interaction with the two endopeptidases. The study of the VAMP-neurotoxins interaction suggest a pairing of the V1 and V2 segments.

Substrate residues N-terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity.

Clostridium botulinum type B neurotoxin is a highly specific zinc-endopeptidase which cleaves vesicle-associated membrane protein (VAMP/synaptobrevin), a critical component of the vesicle docking/fusion mechanism. In this study, substrate residues flanking the N-terminal side of the cleavage site are shown to play a key role in enzyme substrate recognition. Two aspartate residues in this region are identified as critical determinants of the neurotoxin's specificity. These findings are discussed in relation to the mechanism by which botulinum type B neurotoxin cleaves its substrate.

Tyrosine-1290 of tetanus neurotoxin plays a key role in its binding to gangliosides and functional binding to neurones.

Tetanus toxin acts by blocking the release of glycine from inhibitory neurones within the spinal cord. An initial stage in the toxin's action is binding to acceptors on the nerve surface and polysialogangliosides are a component of these acceptor moieties. Using site-directed mutagenesis, we identify tyrosine-1290 of tetanus toxin as a key residue that is involved in ganglioside binding. This residue, which is located at the centre of a shallow pocket on the beta-trefoil domain of the tetanus H(c) fragment, is also shown to play a key role in the functional binding of tetanus toxin to spinal cord neurones leading to the inhibition of neurotransmitter release.

Development of in vitro assays for the detection of botulinum toxins in foods.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bioassay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bioassay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin of the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bioassay. It is envisaged that such assays will prove realistic alternatives to animal-based tests.

Modification of surface histidine residues abolishes the cytotoxic activity of Clostridium difficile toxin A.

Clostridium difficile toxin A displays both cytotoxic and enterotoxic activities. It has recently been demonstrated that toxin A exerts its cytotoxic effect by the glucosylation of the small GTP-binding proteins of the Rho family. Diethyl pyrocarbonate, at pH 7.0, was used to chemically modify exposed histidine residues on toxin A. Modification of toxin A with diethyl pyrocarbonate abolished both its cytotoxic activity and the ability of the toxin to bind Zn-Sepharose gel. Treatment of toxin A with [(14)C]-diethyl pyrocarbonate revealed concentration dependent labelling of histidine residues on the toxin molecules. The effects of diethyl pyrocarbonate could be reversed by hydroxylamine treatment. These data suggest the modified histidine residues on toxin A are critical to its cytotoxic activity. Histidine modification had no effect on the glucosyl transferase enzyme activity of toxin A. However, modification abolished the 'cold' binding of toxin to bovine thyroglobulin in an ELISA and reduced ligand binding activity in a rabbit erythrocyte haemagglutination assay. The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A. Exposed histidines on toxin A are available for zinc chelation, and these have been exploited in the development of a novel purification protocol for toxin A using zinc-chelating chromatography.

Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin.

Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of human/rat VAMP-2 [VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (Km = 3.3 x 10(-4) M) and by its purified light subunit (Km = 3.5 x 10(-4) M). The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Zn2+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94). The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P'1 position. The properties of the P1 amino acid were less critical; the neurotoxin cleaving the C-terminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P1 position corresponding to the sequence of rat VAMP-1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.

Inhibition of calcium-dependent release of noradrenaline from PC12 cells by botulinum type-A neurotoxin. Long-term effects of the neurotoxin on intact cells.

(a) Clostridium botulinum type-A neurotoxin (BoNTA) inhibited the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37 degrees C, a neurotoxin concentration of approximately 0.12 +/- 0.03 microM was required to inhibit 50% of the calcium-dependent noradrenaline release. (b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose-dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I-labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high-affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non-specific binding to the cell membrane or via a low-concentration low-affinity acceptor molecule. (c) A study of the long-term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0.3 microM) for 24 h was reduced to less than 20% of control values over a subsequent 4-day period. After 8 days, release levels were significantly lower (60-65%) than control values, despite a more than 10-fold increase in the cell mass. (d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94-98%) to be associated with the cell membrane fraction. Of this, 50-80% of the BoNTA was associated with the nuclear and cell debris fraction and 11-25% was recovered in the large-granule-vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar. (e) Examination of the form of the cell-associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non-reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Steady-state and pre-steady-state kinetics of coenzyme A linked aldehyde dehydrogenase from Escherichia coli.

Coenzyme A linked aldehyde dehydrogenase from Escherichia coli strain B has been purified to a specific activity of 14 units/mg of protein, and initial rate and substrate analogue inhibition experiments have been performed. On the basis of these steady-state measurements, a bi-uni-uni-uni ping-pong mechanism is proposed in which NAD+ binds to the free enzyme followed by acetaldehyde. The product NADH is then released before coenzyme A (CoA) can bind, and acetyl-CoA is the final product released. A pre-steady-state time-dependent activation of the enzyme was observed when assays were initiated with NAD+. This lag phase of the reaction was studied as a function of the NAD+ concentration and found to be first order. Furthermore, the presence of NAD+ was demonstrated to be necessary to maintain the enzyme in the active conformation. Evidence that the enzyme contains two distinct NAD+ binding sites, an activator site and a catalytic site, has been obtained from pre-steady-state experiments with the NAD+ analogues AMP and 3-pyridine-carboxaldehyde adenine dinucleotide. AMP, a potent competitive inhibitor with respect to NAD+ under steady-state conditions, did not affect the rate of enzyme activation during pre-steady-state measurements. The analogue 3-pyridine-carboxaldehyde adenine dinucleotide, a potent activator of the aldehyde dehydrogenase, was a poor substrate compared with NAD+. At concentrations of this analogue that fully activated the enzyme, no alternate substrate inhibition was observed with respect to NAD+. A model incorporating two binding sites for NAD+ has been put forward to explain these observations.

Clostridium botulinum toxins: nature and preparation for clinical use.

C. botulinum neurotoxins are acutely toxic materials and act by inhibiting release of the neurotransmitter acetylcholine. The specific nature of this inhibition is discussed and the preparation and purification of Type A toxin specifically for clinical use is described.

Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity.

Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit. A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit. Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin. The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action. It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site.

Differences in the protease activities of tetanus and botulinum B toxins revealed by the cleavage of vesicle-associated membrane protein and various sized fragments.

Botulinum neurotoxin serotype B (BoNT/B) and tetanus toxin (TeTx) block neuroexocytosis through selective endoproteolysis of vesicle-associated membrane protein (VAMP). The enzymological properties of both toxins were compared for the first time in their cleavage of VAMP and various sized fragments using a sensitive chromatographic assay. The optimal substrate sizes for the zinc-dependent protease activities of the light chains of TeTx and BoNT/B were established using synthetic peptides corresponding to the hydrophilic core of VAMP (30-62 amino acids in length). TeTx was found to selectively cleave the largest peptide at a single site, Gln76-Phe77. It exhibited the most demanding specificity, requiring the entire hydrophilic domain (a 62-mer) for notable hydrolysis, whereas BoNT/B efficiently cleaved the much smaller 40-mer. Thus, an unusually long N-terminal sequence of 44 amino acids upstream of the scissile bond is required for the selective hydrolysis of VAMP by TeTx. Using the largest peptide, BoNT/B and TeTx exhibited approximately 50% and 35%, respectively, of the activities shown toward intact VAMP, detergent solubilized from synaptic vesicles. Given the large size of the smallest substrates, it is possible that these neurotoxins recognize and require a three-dimensional structure. Although both toxins were inactivated by divalent metal chelators, neither was antagonized by phosphoramidon or ASQFETS (a substrate-related peptide that spans the cleavage site), and TeTx was only feebly inhibited by captopril; also, they were distinguishable in their relative activities at different pHs, temperatures, and ionic strengths. These collective findings are important in the design of effective inhibitors for both toxins, as well as in raising the possibility that TeTx and BoNT/B interact somewhat differently with VAMP.

A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles.

1. A 50-kDa fragment representing the NH2-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH2-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2-terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665). 3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin-binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.

Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities.

A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.

Purification of bacterial exotoxins. The case of botulinum, tetanus, anthrax, pertussis and cholera toxins.

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.