Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

Confirmation of botulism diagnosis in Australian bird samples by ELISA and RT rtPCR.

We developed a sandwich ELISA that detects Clostridium botulinum C and D toxins and reverse-transcription real-time PCRs (RT-rtPCRs) that detect botulinum C and D toxin genes, respectively, to replace the mouse bioassay. The toxin genes were closely associated with the toxin molecules and used as surrogates for the presence of toxin. Samples (638) from 103 clinical cases of birds (302) with suspected botulinum toxicity came from wild birds and poultry (9 cases). Samples tested included blood serum, other body fluids, various tissues, gut contents, maggots, water, and sediment. Botulism was diagnosed in 34 cases (all of which had positive samples in the ELISA, the C toxin gene RT-rtPCR, or both assays). Botulism was suspected in 16 cases (each of which had 1 positive sample either in the ELISA or the C toxin gene RT-rtPCR). In the remaining 53 cases, no samples were positive, but botulism could not be excluded in 32 of these cases, whereas there was no indication of botulism or another diagnosis in 21 cases. The D toxin gene was not detected in any of the clinical samples. No C or D toxin genes were detected in 71 pooled cloacal swabs from 213 healthy migratory birds. The use of an ELISA that detects botulinum C and D toxins in combination with a RT-rtPCR for the botulinum C toxin gene can help confirm the diagnosis of botulism in birds.

Emergency response to a cluster of suspected food-borne botulism in Abuja, Nigeria: challenges with diagnosis and treatment in a resource-poor setting.

Food-borne botulism is a rare, acute and potentially fatal neurologic disorder that results from ingestion of food contaminated by botulinum toxin released from the anaerobic, spore-forming, gram-positive bacterium Clostridium botulinum. We reported an unusual cluster of botulism outbreak with high case fatality affecting a family following ingestion of home-made fish. A suspected outbreak of botulism affecting three patients in a family of six was reported to the Nigeria Centre for Disease Control. A rapid response team investigated by line-listing all the family members, interviewed extended family members, caregivers, clinicians, and nurses to collect socio-demographic and clinico epidemiological information using a semi-structured questionnaires. We collected blood from patients and food samples and locally made drink from the family home for laboratory testing. All family members ingested the same home-made food within the 48hrs before onset of symptoms in the index case. The clinical presentation of the three affected cases (AR=50.0%) was consistent with botulinum poisoning. Two of the affected cases died (CFR=66.7%) within 48hrs of admission, before antitoxin was made available. The third case had a milder presentation and survived, after administration of appropriate antitoxin. The remaining three children developed no symptoms. None of the samples cultured Clostridium botulinum. The blood samples were negative for mouse lethality test. Our report describes the challenges of diagnosis and management of rare emerging infectious disease outbreaks in resource-constrained settings.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score (DAS) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all BoNT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well-tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species-specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Real-Time Monitoring of a Botulinum Neurotoxin Using All-Carbon Nanotube-Based Field-Effect Transistor Devices.

The possibility of exposure to botulinum neurotoxin (BoNT), a powerful and potential bioterrorism agent, is considered to be ever increasing. The current gold-standard assay, live-mouse lethality, exhibits high sensitivity but has limitations including long assay times, whereas other assays evince rapidity but lack factors such as real-time monitoring or portability. In this study, we aimed to devise a novel detection system that could detect BoNT at below-nanomolar concentrations in the form of a stretchable biosensor. We used a field-effect transistor with a p-type channel and electrodes, along with a channel comprising aligned carbon nanotube layers to detect the type E light chain of BoNT (BoNT/E-Lc). The detection of BoNT/E-Lc entailed observing the cleavage of a unique peptide and the specific bonding between BoNT/E-Lc and antibody BoNT/E-Lc (Anti-BoNT/E-Lc). The unique peptide was cleaved by 60 pM BoNT/E-Lc; notably, 52 fM BoNT/E-Lc was detected within 1 min in the device with the antibody in the bent state. These results demonstrated that an all-carbon nanotube-based device (all-CNT-based device) could be produced without a complicated fabrication process and could be used as a biosensor with high sensitivity, suggesting its potential development as a wearable BoNT biosensor.

Purification and Characterization of Recombinant Botulinum Neurotoxin Serotype FA, Also Known as Serotype H.

We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum. BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3. BoNT/FA cleaves the same site in VAMP-2 as BoNT/F5, which is different from the cleavage site of other F serotype BoNTs. BoNT/FA has slower enzyme kinetics than BoNT/F1 in a cell-free protease assay and is less potent at inhibiting ex vivo nerve-stimulated skeletal muscle contraction. In contrast, BoNT/FA is more potent at inhibiting neurotransmitter release from cultured neurons.

Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium.

Botulinum neurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocytosis in neurons, at sites distinct from known BoNT cleavage sites on these two proteins. Comparative genomic analysis determines that the E. faecium strain carrying BoNT/En is a commensal type and that the BoNT/En gene is located within a typical BoNT gene cluster on a 206 kb putatively conjugative plasmid. Although the host species targeted by BoNT/En remains to be determined, these findings establish an extended member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multi-drug-resistant (MDR) infections, to horizontally acquire, and possibly disseminate, a unique BoNT gene cluster.

[Infant botulism ­ why honey should be avoided for children up to one year]. / Spädbarnsbotulism ­ skäl att inte ge honung till barn under ett år.

Infant botulism - why honey should be avoided for children up to one year Infant botulism means that Clostridium botulinum colonize and produce toxin in the infant gut, usually during the first year of life. Illness severity varies widely and the incidence may be under-estimated. Infant botulism should be considered in cases of acute muscle weakness or floppiness in infants, especially when accompanied by constipation or feeding difficulties. Respiratory failure and need for mechanical ventilation is common, but full recovery is gradually obtained. Diagnosis is based on stool culture and toxin detection in stool. Botulinum spores are frequently present in honey, which should consequently be avoided for infants.

Molecular and Epidemiological Characterization of Infant Botulism in Beijing, China.

Laboratory-based pathogen isolation, identification, and toxicity determination were performed on samples from a suspected case of infant botulism. Mice injected with cultures generated from the enema sample and ingested Powered infant formula (PIF) presented typical signs of botulism. Antitoxins to polyvalent botulinum neurotoxins (BoNTs) and monovalent BoNT type B antitoxin had protective effects. Clostridium botulinum isolated from the enema and residual PIF samples were positive for type B toxin. Pulsed-field gel electrophoresis (PFGE) revealed that the two strains of C. botulinum isolated from the two samples produced indistinguishable pulsotypes. These findings confirmed this case of type B infant botulism associated with the ingestion of PIF contaminated by type B C. botulinum spores.

Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria.

Evaluation of the VIDAS glutamate dehydrogenase assay for the detection of Clostridium difficile.

We evaluated the performance of the VIDAS GDH assay for the detection of Clostridium difficile. In total, 350 fecal specimens collected from patients clinically suspected of having CDI were analyzed by C. difficile culture and enzyme-linked fluorescent immunoassay (VIDAS GDH); the results were compared with those of toxigenic C. difficile culture (TC), PCR (Xpert C. difficile assay), and toxin AB EIA (VIDAS CDAB). The numbers of culture-positive and culture-negative samples were 108 and 242, respectively. The concordance between the GDH assay and C. difficile culture was 90.3%. With PCR, 12 more samples were found to be positive in GDH-positive/C. difficile culture-negative specimens. Thus, the concordance between GDH assay and C. difficile culture/PCR was 93.7%. The sensitivity, specificity, positive predictive value, and negative predictive value of the VIDAS GDH assay were 97.2%, 87.2%, 77.2%, and 98.6%, respectively, based on the C. difficile culture, and 97.5%, 91.7%, 86.0%, and 98.6%, respectively, based on C. difficile culture/PCR. Positivity rates of the GDH assay were partially associated with those of semi-quantitative C. difficile cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). We evaluated the two-step or three-step algorithm using GDH assay as a first step. No toxin EIA-positive case was found among GDH-negative samples, and 60.8% (48/79) were TC- and/or PCR-positive among the GDH-positive/toxin EIA-negative samples. Thus, approximately 25% of the 350 samples required a confirmatory test (TC or PCR) in the GDH-toxin EIA algorithm, whereas only 2.3% of the total samples in GDH-PCR algorithm was discrepant and required another confirmatory test like TC.

Structural and functional analysis of botulinum neurotoxin subunits for pH-dependent membrane channel formation and translocation.

The structure-function relationship of Botulinum Neurotoxin (BoNT) proteins is greatly influenced by pH. While the low pH of endosome favors membrane interaction of the heavy chain (HC) for the formation of a membrane channel and translocation of the light chain (LC), the catalytic activity of the LC requires a neutral pH for cleavage of the soluble NSF attachment protein receptor (SNARE) complex in the cytosol. In this study, we monitored secondary structural characteristics of LC, HC and holotoxin at individual pHs 4.5 and 7.2 and at the transition pH4.5 to 7.2 to identify the structural signatures underlying their function. The HC showed higher thermal stability at pH4.5 with a melting temperature (Tm) of 60.4°C. The structural analysis of HC in the presence of liposomes showed no difference in ellipticity with that of HC at pH7.2 at 208 and 222 nm but a 25.2% decrease in ellipticity at 208 nm at acidic pH, indicating low pH-induced structural changes that might facilitate interaction with the membrane. Further, HC showed 18% release of K+ ions from liposomes at pH4.5 as against 6% at neutral pH, reinforcing its role in membrane channel formation. LC on the other hand, showed maximum ellipticity at pH7.2, a condition that is relevant to its endopeptidase activity in the cytosol of the neurons. Also, the similarity in the structures at pH7.2 and transition pH4.5 to 7.2 suggested that the flexibility acquired by the protein at low pH was reversible upon exposure to neutral pH for cleavage of SNARE proteins.

Expression and biochemical characterization of light chains of Botulinum neurotoxin subtypes F5 and F7.

Botulinum neurotoxins are the most potent protein toxins known to human. To date, seven subtypes of the BoNT/F serotype (BoNT/F1 to BoNT/F7) have been identified, among which BoNT/F5 and BoNT/F7 are the most divergent. However, little structural and functional information is available for these two subtypes due to a lack of suitable recombinant proteins for biochemical characterization, except that they appear to possess unique substrate recognition mechanisms, thereby impeding development of vaccine or inhibitors against these proteins. In the present study, we utilized a combinatorial approach which involved examining the effects of different affinity tags, mapping C-terminal truncation mutants and optimization of expression and purification conditions, that allowed us to successfully express and purify soluble and highly active recombinant LC/F5 and LC/F7 proteins. GST-LC/F5(1-450) and 6× His-LC/F5(1-405) were the formats which exhibit the highest level of solubility and activity, whereas GST-LC/F7(1-405) was the most active form of LC/F7. In comparison, GST-LC/F5(1-450) was more active than GST-LC/F7(1-405), which was in turn more active than the LC/F1 control. Our data suggest that solubility of these proteins strongly correlated with their catalytic activity. Successful expression and purification of LC/F5 and LC/F7 in this work will, for the first time, provide materials for further characterization of these two subtypes of BoNT/F, which is essential for future development of protective vaccine or other therapeutic strategies, as well as BoNT/F protein engineering.

Clostridium difficile ribotype 027: relationship to age, detectability of toxins A or B in stool with rapid testing, severe infection, and mortality.

BACKGROUND: Clostridium difficile infection (CDI) can cause severe disease and death, especially in older adults. A better understanding of risk factors for adverse outcomes is needed. This study tests the hypotheses that infection with specific ribotypes and presence of stool toxins independently associate with severity and constructs predictive models of adverse outcomes. METHODS: Cases of non-recurrent CDI were prospectively included after positive stool tests for toxins A and/or B by enzyme immunoassay (EIA) or tcdB by polymerase chain reaction. Outcomes included severe CDI (intensive care unit admission, colectomy, or death attributable to CDI within 30 days of diagnosis) and 30-day all-cause mortality. Adjusted models were developed to test hypotheses and predict outcomes. RESULTS: In total, 1144 cases were included. The toxin EIA was positive in 37.2% and 35.6% of patients were of age >65 years. One of the 137 unique ribotypes was ribotype 027 (16.2%). Detectable stool toxin did not associate with outcomes. Adjusting for covariates, including age, Ribotype 027 was a significant predictor of severe CDI (90 cases; odds ratio [OR], 1.73; 95% confidence interval [CI], 1.03-2.89; P = .037) and mortality (89 cases; OR, 2.02; 95% CI, 1.19-3.43; P = .009). Concurrent antibiotic use associated with both outcomes. Both multivariable predictive models had excellent performance (area under the curve >0.8). CONCLUSIONS: Detection of stool toxin A and/or B by EIA does not predict severe CDI or mortality. Infection with ribotype 027 independently predicts severe CDI and mortality. Use of concurrent antibiotics is a potentially modifiable risk factor for severe CDI.

Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows.

Botulism caused by neurotoxins of Clostridium (C.) botulinum is a rare, but serious life-threatening disease in humans and animals. Botulism in livestock is usually caused by the oral uptake of C. botulinum neurotoxins (BoNT) via contaminated feed and is characterized by flaccid paralysis. In the recent past a new syndrome caused by BoNT in dairy cattle was postulated. It was supposed that C. botulinum is able to colonize the lower intestine and may subsequently produce neurotoxin. The continuous resorption of small amounts of these BoNT may then provoke the so called syndrome of "chronic" or "visceral" botulism involving unspecific clinical symptoms, reduced performance of dairy cows and massive animal losses in the affected herd. To test this hypothesis a case-control study was conducted involving 92 affected farms and 47 control farms located in Northern Germany. Fecal samples of 1388 animals were investigated for the presence of BoNT to verify the key requirement of the hypothesis of chronic botulism. BoNT was not detected in any of the fecal samples using the most sensitive standard method for BoNT detection, the mouse bioassay. Therefore, the existence of "chronic" or "visceral" botulism could not be proven.