质谱技术在肉毒毒素检测中的应用

∙1Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA. skalb@.∙2Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA. aboyer@.∙3Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA. jbarr@.AbstractMass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.KEYWORDS:anthrax; anthrax lethal factor; botulinum neurotoxin; botulism; mass spectrometryPMID:26404376PMCID:PMC4591662DOI:10.3390/toxins7093497[PubMed - indexed for MEDLINE]Free PMC ArticleFormat∙Summary∙Summary (text)∙Abstract∙Abstract (text)∙MEDLINE∙XML∙PMID ListApplyChoose Destination∙File∙Clipboard∙Collections∙E-mail∙Order∙My Bibliography ∙Citation managerCreate File1Fox Chase Chemical Diversity Center, Inc., 3805 Old Easton Rd., Doylestown, PA 18902, USA. areitz@.AbstractWe describe here the state of the art of certain aspects concerning potential small molecule therapy directed toward botulism, by inhibition of the zinc-protease containing light chain (LC) of botulinum neurotoxin BoNT/A from the anaerobic bacillus Clostridium botulinum. Botulinum neurotoxins (BoNTs) are comprised of eight serologically-distinct proteins (A - H), several of which are further divided, such as BoNT/A which has five subtypes. The BoNTs are the most toxic substances known to mankind, causing a form of flaccid paralysis that can be rapid and is often lethal. BoNT/A is comprised of a ~100 kDa heavy chain (HC) attached via a single disulfide Cys-Cys bond to a ~50 kDa LC. The HC mediates transport to and uptake by presynaptic glutamatergic neurons, where the LC cleaves the protein SNAP-25 and thus prevents vesicular trafficking and release of acetylcholine. The Zn-endoprotease activity of the LC of BoNT/A is a target for the development of small molecule inhibitors of BoNT/A-mediated toxicity. A variety of BoNT/A LC inhibitors have been described to date and we focus here primarily on the Zn-binding 8-hydroxyquinoline structural type as well as some of the previously-described hydroxamic acids.PMID:25335884[PubMed - indexed for MEDLINE]1Department of Neurology, Medical University of Warsaw, Banacha 1a Street, 02-097 Warsaw, Poland. apotulska@.pl.AbstractBACKGROUND:Botulism is an acute form of poisoning caused by one of four types (A, B, E, F) toxins produced by Clostridium botulinum, ananaerobic, spore forming bacillus. Usually diagnosis of botulism is considered in patients with predominant motor symptoms: muscle weakness with intact sensation and preserved mental function.CASE PRESENTATION:We report a case of 56-year-old Caucasian female with a history of arterial hypertension, who presented with acute respiratory failure and bilateral ptosis misdiagnosed as brainstem ischemia. She had severe external and internal ophtalmoplegia, and autonomic dysfunction with neither motor nor sensory symptoms from upper and lower limbs. Diagnosis of botulinum toxin poisoning was made and confirmed by serum antibody testing in the mouse inoculation test.CONCLUSIONS:Ophtalmoplegia, autonomic dysfunction and respiratory failure can be caused by botulism. Early treatment and intensive care is essential for survival and recovery. The electrophysiological tests are crucial to correct and rapid diagnosis. Botulism (especially type B) should be considered in any case of acute or predominant isolated autonomic dysfunction.PMID:24172031PMCID:PMC3816205DOI:10.1186/1756-0500-6-438[PubMed - indexed for MEDLINE]Free PMC ArticleJ Sci Food Agric. 2014 Mar 15;94(4):707-12. doi: 10.1002/jsfa.6310. Epub 2013 Aug 12.An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.Sachdeva A1, Singh AK, Sharma SK.Author information1Division of Microbiology, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, 5100 Paint Branch Parkway,College Park, MD, 20740, USA.AbstractBACKGROUND:Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products.RESULTS:1Biotechnology Division, Defence Research & Development Establishment, Gwalior, India. ramkumardhaked@AbstractBotulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholinerelease into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility . It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin.PMID:21149997PMCID:PMC3028942[PubMed - indexed for MEDLINE]Free PMC ArticleJ Food Prot. 2005 May;68(5):1005-11.Growth and toxin production by Clostridium botulinum in steamed rice aseptically packed under modified atmosphere. Kasai Y1, Kimura B, Kawasaki S, Fukaya T, Sakuma K, Fujii T.Author information1Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan.AbstractSales and consumption of ready-to-eat aseptic steamed rice products have increased manyfold in Japan over the past 10 years. To determine the safety of steamed rice (water content 60%, pH 6.5) aseptically packaged under modified atmosphere, challenge studies were performed using a mixture of Clostridium botulinum proteolytic strains (five strains of type A and five strains of type B). Atmospheric conditions of 0 and 15% oxygen (with 5% CO2 and 5% N2 as the balance) were used. No neurotoxins were detected, and organoleptically acceptable conditions persisted for 24 weeks at 15% oxygen conditions. However, botulinum neurotoxin was found in one of three samples at 12 weeks and in one of two samples at 24 weeks at 0% oxygen and 30 degrees C. When samples were inoculated with C. botulinum with amylase (0% oxygen), neurotoxin and sample spoilage was detected after only 1 week of storage. Challenge studies using proteolytic strains of C. botulinum mixed with Bacillus subtilis (amylase formers) also were performed with atmosphere conditions of oxygen at 0, 5, 10, and 15% (with 5% CO2 and 5% N2 as the balance). Under 10 and 15% oxygen conditions, neurotoxin was not detected after 1 week of storage, but sample spoilage was detected afterthe same period. Under 0% oxygen conditions, neurotoxin was detected at 1 week, but the sample remained organoleptically acceptable even after 2 weeks of storage. Both neurotoxin and sample spoilage were detected at 1 week of storage under 5% oxygen conditions. Based on these results, cocontamination of amylase-producing Bacillus with C. botulinum would increase the risk of foodborne botulism when aseptic rice samples are packed underlow-oxygen conditions (<5%). Therefore, to ensure the safety of these products, packing under atmospheric containing more than 10% oxygen is recommended.PMID:15895734[PubMed - indexed for MEDLINE]Crit Rev Microbiol. 2005;31(1):11-8.Clostridium botulinum: a bug with beauty and weapon.Shukla HD1, Sharma SK.Author information1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, USA. shukla@AbstractClostridium botulinum, a Gram-positive, anaerobic spore-forming bacteria, is distinguished by its significant clinical applications as well as its potential to be used as bioterror agent. Growing cells secrete botulinum neurotoxin (BoNT), the most poisonous of all known poisons. While BoNT is the causative agent of deadly neuroparalytic botulism, it also serves as a remarkably effective treatment for involuntary muscle disorders such as blepharospasm, strabismus, hemifacial spasm, certain types of spasticity in children, and other ailments. BoNT is also used in cosmetology for the treatment of glabellar lines, and is well-known as the active component of the anti-aging medications Botox and Dysport. In addition, recent reports show that botulinum neurotoxin can be used as a tool for pharmaceutical drug delivery. However, BoNT remains the deadliest of all toxins, and is viewed by biodefense researchers as a possible agent of bioterrorism (BT). Among seven serotypes, C. botulinum type A is responsible for the highest mortality rate in botulism, and thus has the greatest potential to act as biological weapon. Genome sequencing of C. botulinum type A Hall strain (ATCC 3502) is now complete, and has shown the genome size to be 3.89 Mb with a G+C content of approximately 28.2%. The bacterium harbors a 16.3 kb plasmid with a 26.8% G+C content--slightly lower than that of the chromosome. Most of the virulence factors in C. botulinum are chromosomally encoded; bioinformatic analysis of the genome sequence has shown that the plasmid does not harbor toxin genes or genes for related virulence factors. Interestingly, the plasmid does harbor genes essential to replication, including dnaE, which encodes the alpha subunit of DNA polymerase III which has close similarity with its counterpart in C. perfringens strain 13. The plasmid also contains similar genes to those that encode the ABC-type multidrug transport ATPase, andpermease. The presence of ABC-type multidrug transport ATPase, and permease suggests putative involvement of efflux pumps in bacteriocin production, modification, and export in C. botulinum. The C. botulinum plasmid additionally harbors genes for LambdaBa04 prophage and site-specific recombinase that are similar to those found in the Ames strain of Bacillus anthracis; these genes and their products may play a role in genomic rearrangement. Completion of genome sequencing for C. botulinum will provide an opportunity to design genomic and proteomic-based systems for detecting different serotypes of C. botulinum strains in the environment. The completed sequence may also facilitate identification of potential virulence factors and drug targets, as well as help characterizeneurotoxin-complexing proteins, their polycistronic expression, and phylogenetic relationships between different serotypes.PMID:15839401DOI:10.1080/10408410590912952[PubMed - indexed for MEDLINE]Bioorg Med Chem. 2004 Jun 1;12(11):3055-62.Small tripeptide surrogates with low nanomolar affinity as potent inhibitors of the botulinum neurotoxin Bmetallo-proteolytic activity.Blommaert A1, Turcaud S, Anne C, Roques BP.Author information1Département de Pharmacochimie Moléculaire et Structurale, INSERM U266, UFR des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270Paris cedex 06, France.AbstractBotulinum neurotoxin type B is a high-weight (150 kDa) protein produced by the anaerobic bacillus Clostridium botulinum. This metallo-protease neurotoxin cleaves synaptobrevin, a protein, which is crucial to neurotransmission, resulting in the muscle paralysis, which characterizes botulism. Inhibition of the metallo-peptidase activity is a possible approach to obtain specific therapeutics to treat botulism. We have previously reported a successful attempt to block the proteolytic activity of this neurotoxin with new, selective amino-thiol inhibitors endowed with Ki values in the 15-20 nanomolar range. With the aim of increasing the affinity and bioavailability of this first series of inhibitors we have optimized the residue that fits the P(1) subsite of the enzyme by comparing a series of ligands that contain subtle but significant variants of the parent structure. In addition, this strategy provided a simplification ofthe synthesis of BoNT/B inhibitors by reducing the possible number of stereoisomers. As such we were able to enhance the inhibitory potency whilst reducing the size as compared to the initial privileged structure yielding the first pseudo-tripeptide inhibitors with Ki values in the low nanomolar range.PMID:15142564DOI:10.1016/j.bmc.2004.03.006[PubMed - indexed for MEDLINE]Clin Lab Sci. 2004 Winter;17(1):30-4.Botulin toxin: a weapon in terrorism.Josko D1.Author information1School of Health Related Professions, Department of Clinical Laboratory Science, University of Medicine and Dentistry of New Jersey, Newark, NJ 07107, USA.joskotda@AbstractClostridium botulinum, the causative agent of botulism is an anaerobic, spore forminggram-positive bacillus. C. botulinum causes three types of botulism; foodborne botulism, wound botulism, and infant botulism. Most strains of the bacterium produce a potent,muscle-paralyzing neurotoxin. Respiratory failure secondary to paralysis of the respiratory muscles can lead to death unless appropriate therapy is promptly initiated. Due to the severity and potency of this neurotoxin, its importance as a biological weapon is of major concern to public health officials.PMID:15011978[PubMed - indexed for MEDLINE]JuNihon Rinsho. 2003 Feb;61 Suppl 2:81-91.[Germs and toxins in bioterrorism].[Article in Japanese]Inoue N1.∙1Department of Hygiene, Faculty of Medicine, Kyushu University.∙1Department of Biomedical Sciences, University of Padua, Via G. Colombo 3, 35121, Padua, Italy.AbstractThe recent determination of their primary sequence has lead to the discovery of themetallo-proteolytic activity of the bacterial toxins responsible for tetanus, botulism and anthrax. The protease domain of these toxins enters into the cytosol where it displays a zinc-dependent endopeptidase activity of remarkable specificity. Tetanus neurotoxin and botulinum neurotoxins type B, D, F and G cleave VAMP, an integral protein of the neurotransmitter containing synaptic vesicles. Botulinum neurotoxins type A and E cleave SNAP-25, while the type C neurotoxin cleaves both SNAP-25 and syntaxin, two proteins located on the cytosolic face of the presynaptic membrane. Such specific proteolysis leads to an impaired function of the neuroexocytosis machinery with blockade of neurotransmitter release and consequent paralysis. The lethal factor of Bacillus anthracis is specific for the MAPkinase-kinases which are cleaved within their amino terminus. In this case, however, such specific biochemical lesion could not be correlated with the pathogenesis of anthrax. The recently determined sequence of the vacuolating cytotoxin of Helicobacter pylori contains within its amino terminal domain elements related to serine-proteases, but such an activity as well as its cytosolic target remains to be detected.PMID:10675723[PubMed - indexed for MEDLINE]∙Share on Facebook∙Share on Twitter∙Share on Google+1AFRC Institute of Food Research, Norwich Laboratory.PMID:1977014[PubMed - indexed for MEDLINE]Can Med Assoc J. 1966 Mar 19;94(12):582-90.Food-borne bacterial toxins.Thatcher FS.AbstractFood poisoning caused by the ingestion of preformed bacterial toxins is considered in relation to comparative symptoms, procedures for extraction and purification of the causal toxins, their chemistry, serology, assay procedures and pharmacology, in so far as these are known.The bacteria discussed in this context are Clostridium botulinum, C. perfringens, Staphylococcus aureus, Bacillus cereus, and Vibrio parahemolyticus. The possible roles of the enterococci, Proteus, E. coli and of unknown species, in relation to production of non-antigenic toxic substances, are discussed briefly.Requirements for prevention of the various forms of bacterial food poisoning are outlined.PMID:5905949PMCID:PMC1935354[PubMed - indexed for MEDLINE]Free PMC ArticleAnn Inst Pasteur (Paris). 1962 Jul;103:84-7.[Mechanism of the activation of botulism D toxin by B. licheniformis].[Article in French]PREVOT AR, SILLIOC R.PMID:14488696[PubMed - indexed for MEDLINE]。