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Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

Clostridioides difficile infection (CDI) is a toxin-mediated infection in the gut and a major burden on healthcare facilities worldwide. We rationalized that it would be beneficial to design an antibody therapy that is delivered to, and is active at the site of toxin production, rather than neutralizing the circulating and luminal toxins after significant damage of the layers of the intestines has occurred. Here we describe a highly potent therapeutic, OraCAb, with high antibody titers and a formulation that protects the antibodies from digestion/inactivation in the gastrointestinal tract. The potential of OraCAb to prevent CDI in an in vivo hamster model and an in vitro human colon model was assessed. In the hamster model we optimized the ratio of the antibodies against each of the toxins produced by C. difficile (Toxins A and B). The concentration of immunoglobulins that is effective in a hamster model of CDI was determined. A highly significant difference in animal survival for those given an optimized OraCAb formulation versus an untreated control group was observed. This is the first study testing the effect of oral antibodies for treatment of CDI in an in vitro gut model seeded with a human fecal inoculum. Treatment with OraCAb successfully neutralized toxin production and did not interfere with the colonic microbiota in this model. Also, treatment with a combination of vancomycin and OraCAb prevented simulated CDI recurrence, unlike vancomycin therapy alone. These data demonstrate the efficacy of OraCAb formulation for the treatment of CDI in pre-clinical models.

OBJECTIVES: Escherichia coli is the leading cause of bacteraemia. In an era of emerging multi-drug-resistant strains, development of effective preventative strategies will be informed by knowledge of strain diversity associated with specific infective syndromes/patient groups. We hypothesised that the number of virulence factor (VF) genes amongst bacteraemia isolates from neutropaenic patients would be lower than isolates from immunocompetent patients.METHODS: Immunocompetent and neutropaenic adults with E. coli bacteraemia were recruited prospectively and the source of bacteraemia determined. VF gene profiles were established in silico following whole genome sequencing.RESULTS: Isolates from individual patients were monoclonal. Strains from immunocompetent patients with urinary tract infective foci (UTIF) harboured more VF genes (median number of VF genes 16, range 8-24) than isolates from both immunocompetent patients with non-UTIF (10, 2-22, p = 0.0058) and neutropaenic patients with unknown focus of infection (NPUFI) (8, 3-13, p < 0.0001). Number of VF genes (OR 1.21, 95% CIs 1.01-1.46, p = 0.039) and urinary catheter/recurrent urinary tract infection (OR 12.82, 95% CIs 1.24-132.65, p = 0.032) were independent predictors of bacteraemia secondary to UTIF vs. non-UTIF in immunocompetent patients. papA, papC, papE/F, papG, agn43, tia, iut, fyuA, kpsM and sat were significantly more prevalent amongst UTIF- vs non-UTIF-originating isolates amongst immunocompetent patients, while papC, papE/F, papG, agn43, tia, fyuA, hlyA, usp and clb were significantly more prevalent amongst UTIF- vs NPUFI-associated isolates.CONCLUSIONS: Bacteraemia-associated E. coli strains originating from UTIF have distinct VF gene profiles from strains associated with non-UTIF- and NPUFI. This diversity must be addressed in the design of future vaccines to ensure adequate coverage of strains responsible for site-specific disease.

The nosocomially acquired pathogen Clostridium difficile is the primary causative agent of antibiotic associated diarrhoea and causes tens of thousands of deaths globally each year. C. difficile presents a paracrystalline protein array on the surface of the cell known as an S-layer. S-layers have been demonstrated to possess a wide range of important functions, which, combined with their inherent accessibility, makes them a promising drug target. The unusually complex S-layer of C. difficile is primarily comprised of the high- and low- molecular weight S-layer proteins, HMW SLP and LMW SLP, formed from the cleavage of the S-layer precursor protein, SlpA, but may also contain up to 28 SlpA paralogues. A model of how the S-layer functions as a whole is required if it is to be exploited in fighting the bacterium. Here, we provide a summary of what is known about the S-layer of C. difficile and each of the paralogues and, considering some of the domains present, suggest potential roles for them.

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera-monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

Two structures of Cwp84, a cysteine protease from the S-layer of C. difficile, are presented after propeptide cleavage. They reveal the movement of three loops, two in the active-site groove and one on the surface of the lectin-like domain, exposing a hydrophobic pocket., In recent decades, the global healthcare problems caused by Clostridium difficile have increased at an alarming rate. A greater understanding of this antibiotic-resistant bacterium, particularly with respect to how it interacts with the host, is required for the development of novel strategies for fighting C. difficile infections. The surface layer (S-layer) of C. difficile is likely to be of significant importance to host–pathogen interactions. The mature S-layer is formed by a proteinaceous array consisting of multiple copies of a high-molecular-weight and a low-molecular-weight S-layer protein. These components result from the cleavage of SlpA by Cwp84, a cysteine protease. The structure of a truncated Cwp84 active-site mutant has recently been reported and the key features have been identified, providing the first structural insights into the role of Cwp84 in the formation of the S-layer. Here, two structures of Cwp84 after propeptide cleavage are presented and the three conformational changes that are observed are discussed. These changes result in a reconfiguration of the active site and exposure of the hydrophobic pocket.

Clostridium difficile is a burden to healthcare systems around the world, causing tens of thousands of deaths annually. The S‐layer of the bacterium, a layer of protein found of the surface of cells, has received a significant amount of attention over the past two decades as a potential target to combat the growing threat presented by C. difficile infections. The S‐layer contains a wide range of proteins, each of which possesses three cell wall‐binding domains, while many also possess a “functional” region. Here, we present the high resolution structure of the functional region of one such protein, Cwp19 along with preliminary functional characterisation of the predicted glycoside hydrolase. Cwp19 has a TIM barrel fold and appears to possess a high degree of substrate selectivity. The protein also exhibits peptidoglycan hydrolase activity, an order of magnitude slower than that of lysozyme and is the first member of glycoside hydrolase‐like family 10 to be characterised. This research goes some way to understanding the role of Cwp19 in the S‐layer of C. difficile. Database Structural data are available in the PDB under the accession numbers 5OQ2 and 5OQ3.

Objectives Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

Summary 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.

Summary Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E. Specific inhibitors of thioredoxin reductase or thioredoxin prevent intoxication of cultured neurons in a dose-dependent manner and are also very effective inhibitors of the paralysis of the neuromuscular junction. We found that this group of inhibitors of botulinum neurotoxins is very effective in vivo. Most of them are nontoxic and are good candidates as preventive and therapeutic drugs for human botulism.

Highlights • Definition of Clostridium difficile toxin-derived antigens for soluble expression in E. coli. • Demonstration of their potent neutralising immune response against key epidemic strain toxins. • TcdA and TcdB were different with respect to the domains that evoke a neutralising immune response. • TcdB central domains dominate the generation of a toxin-neutralising response. • Generated antibodies prevent C. difficile infection in passive immunisation studies., Infection with the bacterium Clostridium difficile causes symptoms ranging from mild to severe diarrhoea with life-threatening complications and remains a significant burden to healthcare systems throughout the developed world. Two potent cytotoxins, TcdA and TcdB are the prime mediators of the syndrome and rapid neutralisation of these would afford significant benefits in disease management. In the present study, a broad range of non-toxic, recombinant fragments derived from TcdA and TcdB were designed for soluble expression in E. coli and assessed for their capacity to generate a potent toxin-neutralising immune response as assessed by cell-based assays. Significant differences between the efficacies of isolated TcdA and TcdB regions with respect to inducing a neutralising immune response were observed. While the C-terminal repeat regions played the principal role in generating neutralising antibodies to TcdA, in the case of TcdB, the central region domains dominated the neutralising immune response. For both TcdA and TcdB, fragments which comprised domains from both the central and C-terminal repeat region of the toxins were found to induce the most potent neutralising immune responses. Generated antibodies neutralised toxins produced by a range of C. difficile isolates including ribotype 027 and 078 strains. Passive immunisation of hamsters with a combination of antibodies to TcdA and TcdB fragments afforded complete protection from severe CDI induced by a challenge of bacterial spores. The results of the study are discussed with respect to the development of a cost effective immunotherapeutic approach for the management of C. difficile infection.

Tetanus and botulinum neurotoxins act inside nerve terminals and, therefore, they have to translocate across a membrane to reach their targets. This translocation is driven by a pH gradient, acidic on the cis side and neutral on the cytosol. Recently, a protocol to induce translocation from the plasma membrane was established. Here, we have used this approach to study the temperature dependence and time course of the entry of the L chain of tetanus neurotoxin and of botulinum neurotoxins type C and D across the plasma membrane of cerebellar granular neurons. The time course of translocation of the L chain varies for the three neurotoxins, but it remains in the range of minutes at 37 °C, whilst it takes much longer at 20 °C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates.

Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity., Highlights • ADP-ribosyltransferase activity determines cytotoxicity of CDTa from Clostridium difficile. • CDT ADP-ribosylation follows SN1 mechanism. • A single amino acid mutation is sufficient to impair CDTa cytotoxicity.

Purpose The focus of this study was to characterize the effect of clinically relevant stress-inducing conditions on the production and composition of proinflammatory outer membrane vesicles (OMVs) produced from ST131 extraintestinal pathogenic Escherichia coli (ExPEC) clinical isolates. Experimental design A label-free method (relative normalized spectral index quantification, SINQ) was used to identify changes in the respective OMV proteomes following exposure of the ExPEC strains to antibiotics and low iron. Nanoparticle tracking analysis was used to quantify changes in abundance and size of OMVs produced by the gentamicin-resistant (GenR) and gentamicin-sensitive (GenS) ExPEC strains. Results Up to a 13.1-fold increase in abundance of particles were detected when the gentamicin-sensitive strain was exposed to a range of gentamicin concentrations. In contrast, no increase was observed for the gentamicin-resistant strain. Iron-limiting conditions had minimal effect on OMV production for either strain. Marked changes in the OMV proteome were observed for both strains including increases in Hsp100/Clp proteins, ATP-dependent ClpP protease, and regulatory proteins. Conclusion These data provide information on changes in the composition of OMV particles derived from ExPEC strains generated in response to clinically relevant conditions. We show that the levels of the proinflammatory OMVs increase for gentamicin-sensitive ExPEC exposed to the antibiotic

Tetanus and botulinum neurotoxins cause paralysis by cleaving SNARE proteins within the cytosol of nerve terminals. They are endocytosed inside acidic vesicles and the pH gradient across the membrane drives the translocation of their metalloprotease L domain in the cytosol. This domain is linked to the rest of the molecule by a single interchain disulfide bridge that has to be reduced on the cytosolic side of the membrane to free its enzymatic activity. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-thioredoxin reductase-thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins.

Summary Tetanus and botulinum neurotoxins are di-chain proteins that cause paralysis by inhibiting neuroexocytosis. These neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, whose acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage was made accessible to investigation by making it to occur at the surface of neurons. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxin B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Based on these and previous findings, we propose a stepwise sequence of molecular events that lead from toxin binding to membrane insertion.

Clostridium difficile, a highly infectious bacterium, is the leading cause of antibiotic-associated pseudomembranous colitis. In 2009, the number of death certificates mentioning C. difficile infection in the U.K. was estimated at 3933 with 44% of certificates recording infection as the underlying cause of death. A number of virulence factors facilitate its pathogenicity, among which are two potent exotoxins; Toxins A and B. Both are large monoglucosyltransferases that catalyse the glucosylation, and hence inactivation, of Rho-GTPases (small regulatory proteins of the eukaryote actin cell cytoskeleton), leading to disorganization of the cytoskeleton and cell death. The roles of Toxins A and B in the context of C. difficile infection is unknown. In addition to these exotoxins, some strains of C. difficile produce an unrelated ADP-ribosylating binary toxin. This toxin consists of two independently produced components: an enzymatic component (CDTa) and the other, the transport component (CDTb) which facilitates translocation of CDTa into target cells. CDTa irreversibly ADP-ribosylates G-actin in target cells, which disrupts the F-actin:G-actin equilibrium leading to cell rounding and cell death. In the present review we provide a summary of the current structural understanding of these toxins and discuss how it may be used to identify potential targets for specific drug design.

Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulence factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.

Clostridium difficile is a major and growing problem as a hospital-associated infection that can cause severe, recurrent diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood but undoubtedly involves protein components within the surface layer (S-layer), which play a role in adhesion. In C. difficile, the S-layer is composed of two principal components, the high and low molecular weight S-layer proteins, which are formed from the post-translational cleavage of a single precursor, SlpA. In the present study, we demonstrate that a recently characterized cysteine protease, Cwp84 plays a role in maturation of SlpA. Using a gene knock-out approach, we show that inactivation of the Cwp84 gene in C. difficile 630DeltaErm results in a bacterial phenotype in which only immature, single chain SlpA comprises the S-layer. The Cwp84 knock-out mutants (CDDeltaCwp84) displayed significantly different colony morphology compared with the wild-type strain and grew more slowly in liquid medium. SlpA extracted from CDDeltaCwp84 was readily cleaved into its mature subunits by trypsin treatment. Addition of trypsin to the growth medium also cleaved SlpA on CDDeltaCwp84 and increased the growth rate of the bacterium in a dose-dependent manner. Using the hamster model for C. difficile infection, CDDeltaCwp84 was found to be competent at causing disease with a similar pathology to the wild-type strain. The data show that whereas Cwp84 plays a role in the cleavage of SlpA, it is not an essential virulence factor and that bacteria expressing immature SlpA are able to cause disease.

The clostridial neurotoxins include tetanus toxin and the seven antigenically different botulinum neurotoxins (BoNTs), all of which exert their action by blocking the calcium-mediated release of neurotransmitters (24). The BoNTs act principally on the peripheral nervous system, where they inhibit the release of acetylcholine at the neuromuscular junction, an action that results in a widespread descending flaccid paralysis and ultimately the syndrome botulism. Because of the high potencies of the BoNTs, they are considered potential reagents for bioterrorist use and are currently designated by the Centers for Disease Control and Prevention as category A biothreat agents (1). In their most active forms, the BoNTs consist of two subunits: a light chain (∼50 kDa) linked by a disulfide bond to a heavy chain (∼100 kDa). Structurally, these subunits are arranged into three distinct domains (17, 30): a 50-kDa HC domain that consists of two subdomains (of which the C-terminal subdomain is involved in neuronal acceptor binding), a translocation domain represented by the N-terminal half of the heavy chain (HN domain), and a light-chain, effector domain (LC). Collectively, these domains enable the BoNTs to bind and translocate to within the presynaptic nerve terminal (6), where they act, via highly specific, zinc-dependent protease actions within the LC domain, to disable the process of calcium-mediated transmitter release (24). While architecturally and mechanistically similar, the various serotypes of the BoNTs differ significantly in their primary structures (19) with the result that antibodies raised against one BoNT serotype offer no, or very little, protection against the biological action of another. Separate antigens are therefore required for each serotype to provide complete protection against the full spectrum of BoNTs. Vaccine development is further complicated by the occurrence of subtypes within most of the BoNT serotypes (13). For BoNT serotype A, for example, four subtypes have thus far been identified (designated BoNT/A1 to BoNT/A4) which display between 7 and 16% heterology in their primary nucleic acid sequences (2). These sequence variations occur primarily within surface-exposed regions on the molecule, thus maximizing their impact on antibody binding and neutralization and hence vaccine efficacy. Providing adequate cross-protection against the principal subtypes of each BoNT serotype must therefore be an important consideration in design of both vaccines and antibody-based therapeutics for the BoNTs. Current vaccines for the BoNTs consist of formaldehyde-inactivated toxin complexes which were first developed in the 1950s. Although these vaccines are effective, they require specialized high containment manufacturing facilities and are difficult and expensive to manufacture in large quantities (9). The initial design of recombinant vaccines was undertaken with the rationale of inhibiting a key facet of the biological activity of the BoNTs, such as receptor binding. Thus, first-generation recombinant vaccines under development are based on the receptor-binding domains (HC fragments) of each BoNT. These fragments, produced in Pichia pastoris, have been shown to provide a protective immune response in mice and have recently entered clinical trials (3, 4, 27). The HC fragments derived from the various BoNTs, however, differ markedly in their isoelectric points (pIs 5.7 to 9.1), which make formulation of a multivalent vaccine difficult. More recent studies indicate that antibodies directed against the light chain and the HN region of the BoNT molecule can also provide a neutralizing immune response (5, 6). The LHN fragment of the BoNTs is a polypeptide of ∼100 kDa consisting of the light-chain domain in close association with the translocation domain (Fig. ​(Fig.1).1). A polypeptide belt from the latter surrounds the light chain under nonreducing conditions. In initial studies, the LHN fragment of BoNT/A was produced by prolonged trypsin digestion of the neurotoxin and shown to be a soluble, immunoreactive fragment (26). Subsequently, LHN fragments from several BoNT serotypes have been produced by recombinant DNA technology and demonstrated to be useful as the core of a range of potential novel therapeutics (10, 29). In the present study, LHN fragment-based vaccines for BoNT/A and BoNT/B are described. A derivative of the LHN/A vaccine is shown to have exceptional efficacy in animal studies providing single-dose protection against BoNT/A subtypes A1, A2, and A3. The LHN/B vaccine is shown to provide protection against BoNT/B subtypes B1 and B4 (nonproteolytic). FIG. 1. Structure and function of the BoNTs. A diagram of the structure of BoNT/A shows the organization of the domains and the composition of the LHN fragment. The HC (binding) domain binds to neuronal receptors, after which the HN (translocation) domain mediates ...

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.

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins.

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile -specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans ). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

Sortase enzymes are responsible for covalent anchoring of specific proteins to the peptidoglycan of the cell wall of gram-positive bacteria. In some gram-positive bacteria (e.g. Staphylococcus aureus), sortases have been found to be essential for pathogenesis and their inhibitors are under development as potential novel therapeutics. Here we provide the first report on the structural characterisation of the C. difficile sortase. An active site mutant was crystallised and its structure determined to 2.55 Å by X-ray diffraction to provide structural insight into its catalytic mechanism. In order to elucidate the role of the sortase in the cell wall biogenesis, a C. difficile sortase knockout strain was constructed by intron mutagenesis. Characterisation of this mutant led to the discovery that the putative adhesin CD0386 is anchored to the peptidoglycan of C. difficile by the sortase SrtB and that an SPKTG peptide motif is involved in the transpeptidation reaction with the C. difficile peptidoglycan. In an animal model for C. difficile infection, the SrtB mutant caused disease at a similar rate of onset as the wild type strain. In conclusion, our detailed study shows that the SrtB enzyme from C. difficile does not play an essential role in pathogenesis.

The development of diagnostic tests for the botulinum neurotoxins is complicated by their extremely high potencies and the considerable diversity observed within the neurotoxin family. Current approaches for the detection of the toxins and the organism include amplified immunoassays and PCR techniques. Assays which exploit the biological activities within the botulinum toxins are also in development. These are based on both antibody and mass spectrometric techniques which measure the endopeptidase activities of the neurotoxins. This overview of theAssays and Detection Workshop of the 5th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Neurotoxins discusses recent progress in the development of these assay systems and the issues that need to be overcome prior to their implementation.

Botulinum neurotoxins (BoNTs) form a growing family of metalloproteases with a unique specificity either for VAMP, SNAP25 or syntaxin. The BoNTs are grouped in seven different serotypes indicated by letters from A to G. These neurotoxins enter the cytosol of nerve terminals via a 100 kDa chain which binds to the presynaptic membrane and assists the translocation of a 50 kDa metalloprotease chain. These two chains are linked by a single disulfide bridge which plays an essential role during the entry of the metalloprotease chain in the cytosol, but thereafter it has to be reduced to free the proteolytic activity. Its reduction is mediated by thioredoxin which is continuously regenerated by its reductase. Here we show that inhibitors of thioredoxin reductase or of thioredoxin prevent the specific proteolysis of VAMP by the four VAMP-specific BoNTs: type B, D, F and G. These compounds are effective not only in primary cultures of neurons, but also in preventing the in vivo mouse limb neuroparalysis. In addition, one of these inhibitors, Ebselen, largely protects mice from the death caused by a systemic injection. Together with recent results obtained with BoNTs specific for SNAP25 and syntaxin, the present data demonstrate the essential role of the thioredoxin-thioredoxin reductase system in reducing the interchain disulfide during the nerve intoxication mechanism of all serotypes. Therefore its inhibitors should be considered for a possible use to prevent botulism and for treating infant botulism.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH(N)/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH(N)/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram-positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram-positive bacteria, it is likely that other Gram-positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools.

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.

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.

mAbs were produced in mice against highly purified, renatured light chain (LC) of botulinum neurotoxin A (BoNT A) that was immobilised on nitrocellulose to avoid the undesirable use of toxoids. Subcutaneous implants of relatively high amounts (up to 10 micrograms each) of LC allowed its slow release into the systemic circulation and, thus, yielded much higher antibody titres against the underivatized antigen than had hitherto been obtained by conventional immunization. Seven stable hybridoma cell lines were established which secrete mAb of IgG1 and IgG2b subclasses reactive specifically with BoNT A and LC, in native and denatured states, without showing any cross-reactivity with types B, E, F or tetanus toxin. The pronounced reactivities of three mAbs towards refolded LC or intact toxin, observed in immunobinding and precipitation assays, relative to that seen in Western blots imply a preference for conformational epitopes. Though mAbs 4, 5 and 7 failed to neutralize the lethality of BoNT in vivo, administration intraneurally of mAb7 prevented the inhibition of transmitter release normally induced by subsequent extracellular administration of BoNT A. Notably, the latter mAb reacted with a synthetic peptide corresponding to amino acids 28-53 in the N-terminus of the LC, a highly conserved region in Clostridial neurotoxins reported to be essential for maintaining the tertiary structure of the chain. Most importantly, when mAbs 4 or 7 were microinjected inside ganglionic neurons of Aplysia, each reversed, though transiently, the blockade of acetylcholine release by the toxin; this novel finding is discussed in relation to the nature of the zinc-dependent protease activity of the toxin.

Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.

Tetanus and botulinum neurotoxins are di-chain proteins that cause paralysis by inhibiting neuroexocytosis. These neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, whose acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage was made accessible to investigation by making it to occur at the surface of neurons. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxin B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Based on these and previous findings, we propose a stepwise sequence of molecular events that lead from toxin binding to membrane insertion.

Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution., Cwp19 is a putatively surface-located protein from Clostridium difficile. A recombinant N-terminal protein (residues 27–401) lacking the signal peptide and the C-terminal cell-wall-binding repeats (PFam04122) was crystallized using the sitting-drop vapour-diffusion method and diffracted to 2 Å resolution. The crystal appeared to belong to the primitive monoclinic space group P21, with unit-cell parameters a = 109.1, b = 61.2, c = 109.2 Å, β = 111.85°, and is estimated to contain two molecules of Cwp19 per asymmetric unit.

ADP-ribosylation is one of the favored modes of cell intoxication employed by several bacteria. Clostridium difficile is recognized to be an important nosocomial pathogen associated with considerable morbidity and attributable mortality. Along with its two well known toxins, Toxin A and Toxin B, it produces an ADP-ribosylating toxin that targets monomeric actin of the target cell. Like other Clostridial actin ADP-ribosylating toxins, this binary toxin, known as C. difficile toxin (CDT), is composed of two subunits, CDTa and CDTb. In this study, we present high resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5, and 9.0) and in complex with ADP-ribose donors, NAD and NADPH (at pH 9.0). The crystal structures of the native protein show "pronounced conformational flexibility" confined to the active site region of the protein and "enhanced" disorder at low pH, whereas the complex structures highlight significant differences in "ligand specificity" compared with the enzymatic subunit of a close homologue, Clostridium perfringens iota toxin. Specifically in CDTa, two of the suggested catalytically important residues (Glu-385 and Glu-387) seem to play no role or a less important role in ligand binding. These structural data provide the first detailed information on protein-donor substrate complex stabilization in CDTa, which may have implications in understanding CDT recognition.

Introduction The main objective of this study was to develop, validate and implement a novel microarray platform to enable the simultaneous quantification of systemic IgG immune responses to a 7-plex panel of highly purified C. difficile -specific virulence factors, including whole toxins A and B (toxinotype 0, strain VPI10463, ribotype 087), recombinant fragments of toxin A and B, PCR ribotype-specific surface layer proteins (001, 002, 027) and control proteins (tetanus and candida). We evaluated the performance of the microarray technique against tradional ELISA. Method Microbial antigens were diluted to 200 μg/ml in printing buffer in a 384-well plate and spotted in quadruplicate onto poly-L-Lysine-coated slides using a Biorobotics MicroGridII arrayer. The slides were blocked with 5% BSA diluted in PBS-Tween-20 wash buffer. After washing, all slides were incubated with sera diluted 1:500 in antibody diluent. Following washing, slides were incubated with biotinylated anti-human IgG diluted 1:20,000, washed and then incubated with Streptavidin Cy5 diluted 1:2,000 in 5% BSA. After final washes, slides were dried by centrifugation and scanned using a GenePix 4200AL scanner. The resultant TIFF images were processed with Axon GenePix Pro-6 microarray image analysis software. Protein signals were finally determined with background subtraction using RPPanalyzer, a module within the R statistical language on CRAN (http://vran.r-project.org). Kruskall Wallis was used with Dunn’s post test. Correlation was evaluated using Spearman rank correlation coefficient. P values of Results A total of 327 serum samples from patients with C. difficile infection (CDI), cystic fibrosis (CF) and healthy controls (HC) were tested by microarray for the presence of specific IgG antibody. The microarray assay fell within acceptable limits of precision for both intra- and inter-assay variability for all antigens tested ( p .0001) between antibody detection levels using the microarray system and the ELISA format. Conclusion This is the first reported use of protein microarray to assess serum immunoreactivities to C. difficile -specific antigens. Our results suggest that the microarray platform allows accurate, precise and reproducible specific antibody quantifcation. Additional modifications of this approach could be used to simultaneously study multiple isotype specificities to several C. difficile strain-specific antigens for large collections of patient sera. Disclosure of interest O. Negm: None Declared, M. Hamed: None Declared, E. Abbott: None Declared, P. Tighe: None Declared, C. Shone: None Declared, C. Loscher: None Declared, L. Edwards: None Declared, M. Wilcox Grant/ Research Support from: Multiple therapeutic and diagnostic companies, Consultant for: Multiple therapeutic and diagnostic companies, T. Monaghan: None Declared.

INTRODUCTION AND HISTORICAL PERSPECTIVE, K.A. Foster, P. Hambleton, and C.C. Shone Introduction Neurotoxigenic Clostridia Diseases of Botulinum and Tetanus Neurotoxins Clinical Use of Botulinum Neurotoxins THREE-DIMENSIONAL PROTEIN STRUCTURES OF LIGHT CHAINS OF BOTULINUM NEUROTOXIN SEROTYPES A, B, AND E AND TETANUS NEUROTOXIN, S. Swaminathan and R.C. Stevens Structure of Botulinum Neurotoxin 150kDa Holotoxin Proteolysis of Snare Proteins by Botulinum Neurotoxin Light Chains Structure of Botulinum Neurotoxin Serotype A Light Chain Strucuture of Botulinum Neurotoxin Serotype B Light Chain A Possible Proteolytic Mechanism of Botulinum Neurotoxins Strudture of Botulinum Neurotoxin Light Chain E Structure of Tetanus Neurotoxin Light Chain Conclusion References MECHANISMS OF ACTIONS OF NEUROTOXINS, H. Bigalke and T. Binz Introduction Toxin Uptake, Toxicity, and Clinical Disease Manifestation Binding to Nerve Cell Receptors Internalization into the Neuronal Cytosol References VACCINES TO PROTECT AGAINST NEUROTOXINS, L.A. Smith and I. Henderson Introduction Toxoid Vaccines Recombinant Vaccines The Vaccine Candidate RBV A/B Conclusion References INHIBITORS OF CLOSTRIDIAL NEUROTOXIN PROTEINASE ACTIVITIES, J.J. Schmidt and K.A. Foster Inhibitors if Clostridial Neurotoxin Proteinase Activities Neutralizing Antibodies Conclusion References INTRODUCTION TO CLINICAL USE OF BOTULINUM NEUROTOXINS, M.P. Barnes Introduction Theory of Clinical Use Practical Aspects of Injection Technique Longer Term Side Effects and Problems Logistics and Cost Dystonia Spasticity Conclusions References EXPANDING CLINICAL USES OF BOTULINUM NEUROTOXINS, A.P. Moore Introduction New Indications The Future: Other Possible Indications and Speculation Conclusions DEVELOPMENT OF THERAPEUTICS BASED UPON CLOSTRIDIAL ENDOPEPTIDASE ACTIVITY, K.A. Foster and J.A. Chaddock Introduction Clostridial Neurotoxin Structure and Function: The 'Endopeptidase' Domain Recombinant Expression of the LHN/A Rationale for Use of the LHN Endopeptidase Pain The Potential Use of Neurotoxins Endopeptidase Fragments for Pain Releif Alternative Therapeutic Possibilities Conclusions References USE OF NEUROTOXINS AS DRUG DELIVERY SYSTEMS, P. Foran Overview Generation of Non-Toxic Derivatives Retaining Neuronal Targeting and Delivery Capabilities Using Classical Biochemical Mehtods or Recombinant Means The Diverse Neuronal Targeting and Cell Trafficking Pathways Exerted by Clostridial Neurotoxins In Vitro or In Vivo Neuronal Targeting Achievements Made Using Innocuous Derivatives: But Not Demonstrating Cytosolic Transfer Achievements of Cell or Neuronal Targeting, as well as, Cytoplasmic Delivery of Foreign Cargos Using Innocuous Derivatives Important Structural Features Proven Essential For Efficient In Vivo Neuronal Targeting and Cytoplasmic Delivery of Cargo Strategies for the Efficient Linkage of Therapeutic Adducts to Transporters: Approaches that Ensure Optimal Linkage Efficiency and Orientation Potential Diseases that May be Targeted Using Innocuous BoNT- or TeNT-based Neuron Delivery Vehicles How Much Cargo can BoNT-Based Transporters Deliver? The Varied Intracellular Trafficking and Site-Specific Delivery Addresses Anticipated for Cargos Linked to BoNT-or TeNT-based Vehicles Conclusion References CONCLUSIONS AND FUTURE PERSPECTIVES: BOTULINUM NEUROTOXINS AS PARADIGMS FOR DEVELOPMENT OF MICROBIAL EFFECTOR-DERIVED DRUGS, J.M. Sutton Introduction To Kill or Not to Kill: Strategies for Toxin Therapeutics General Themes for the Future Development of Bacterial Effector-Based Therapeutics References Index