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Clostridium botulinum and its toxins: Pathogenic germ and potential biological weapon? Current aspects of the scientific knowledge
Abstract
The focus on the bacterium Clostridium botulinum and its toxins covers at least four interesting aspects: First there is the historical and taxonomical aspect of the empirical discovering of bacteria as disease causes, which could exemplarily be shown at this organism. Secondly the so called botulism is a for long times known disease which could also in recent days present dramatic until deadly courses if not identified and treated properly. The botulinum toxin, existing in several distinct serotypes, is furthermore the most poisonous substance known to man and because of its ubiquitous occurrence it could be bred without difficulties and ranks therefore among the first category of potential bioterroristic weapons. The Janus-faced capability now is the aspect that this deadly toxin is taken in a purified version mostly of the serotype A (also known as Botox ®) as an effective therapeutical agent in the medical field since almost three decades and its application areas expand. Concerning the occupational medicine special emphases lie in prevention and ken of transmission of botulism on the one hand and in the awareness of its clinical symptoms as well as a realistic evaluation of the bioterroristic threat on the other hand.
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A frequently forgotten old malady called botulism has been recognized for more than a century. This ailment occurs worldwide, afflicts human of all age groups from infants to elderly and affects Oriental people more often in several regions of China. Occurrence in Taiwan is uncommon, and therefore, it is often overlooked. The outbreaks of human botulism in various regions of the world, the clinical types, the molecular mechanisms, and the electrophysiologic findings will be highlighted.
For most of its time, the history of botulinum toxin (BT) has been the history of botulism, i. e. of an intoxication with BT. By the end of the 1960's a paradigm shift took place which in this radicalness had never occurred before in the history of mankind. At that time BT was first used therapeutically to treat strabismus. From ophthalmology BT rapidly spread into numerous medical specialties. For most of its indications BT is the therapy of choice, for some it has revolutionized their treatment altogether. The widespread therapeutic use of BT allowed detailed clinical and technical investigations of BT's action upon the human body. Applying this knowledge we diagnosed for the first time chronic botulism in adults living on a farm with chronic bovine botulism. This constitutes another radical paradigm shift. The history of BT is the history of a dual paradigm shift each time induced by a complete reversal of the viewing perspective. Knowledge gain can be a linear process. It can, however, also be a circular one. Changes of the viewing perspective are crucial. Changing the viewing perspective may facilitate knowledge gain. This might be used to develop an instrument to facilitate knowledge gain.
(c) Georg Thieme Verlag KG Stuttgart-New York.
This review presents a brief account of the most significant biological effects and clinical applications of botulinum neurotoxins, in a way comprehensive even for casual readers who are not familiar with the subject. The most toxic known substances in botulinum neurotoxins are polypeptides naturally synthesized by bacteria of the genus Clostridium. These polypeptides inhibit acetylcholine release at neuromuscular junctions, thus causing muscle paralysis involving both somatic and autonomic innervation. There is substantial evidence that this muscle-paralyzing feature of botulinum neurotoxins is useful for their beneficial influence on more than 50 pathological conditions such as spastic paralysis, cerebral palsy, focal dystonia, essential tremor, headache, incontinence and a variety of cosmetic interventions. Injection of adequate quantities of botulinum toxins in spastic muscles is considered as a highly hopeful procedure for the treatment of people who suffer from dystonia, cerebral palsy or have experienced a stroke. So far, numerous and reliable studies have established the safety and efficacy of botulinum neurotoxins and advocate wider clinical therapeutic and cosmetic applications.
In an attempt to determine which elements of the axon reaction are essential for early axonal outgrowth, axonal sprouting was induced with botulinum toxin (BoTx) and the nerve cell body changes compared with those accompanying axonal growth after nerve trauma. Anterior horn cells of mice were examined histologically at times ranging from 3 days to 3 weeks after either BoTx hindlimb injection or sciatic nerve crush. After sciatic nerve crush there was dispersion of Nissl substance, increase in cell body size, and an increase in neurofilament protein staining. None of these changes were found after BoTx-induced terminal axonal sprouting, suggesting that these morphological features of the axon reaction are not essential for early axonal outgrowth.
The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)
The experimentally produced syndrome of botulism (Type A toxin) in the monkey, when the toxin is administered intravenously, mimics the human counterpart, which usually results from ingestion. All the animals developed the same signs, which appeared in a definite sequence. Onset and duration of signs were dose-related, and signs were reversible in survivors. The only consistent findings on autopsy were atelectasis and bronchopneumonia. Animals that died showed no morphological changes.In the rhesus monkey, the intravenous LD50 of Type A partially purified toxin is 40 MU/kg; the intragastric LD50 is 30,000 MU/kg. In the squirrel monkey, the intravenous LD50 is 66 MU/kg.
Paralysis of the mouse levator auris longus muscle by in vivo injection of Clostridium botulinum type-D neurotoxin (BoNT/D) triggered a marked outgrowth of the motor nerve from the original terminal arborization. The increase in total nerve terminal length was due to both increase in the number of terminal branches and in average branch length. Asynchronous quantal transmitter release in response to nerve impulses was a prominent feature in paralysed junctions that started 24 h after poisoning and lasted for about 15 days. The functional recovery of poisoned junctions occurred 25-30 days after poisoning and was characterized by the synchronous quantal transmitter release upon nerve stimulation that triggered synaptically evoked action potentials and muscle fibre contraction.
Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an ∼150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun.
183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the ∼50–kDa light chain, Lys 422–Lys 1251 constitute the ∼100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The ∼150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.
Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a ∼150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1992), Appl. Environ. Microbiol.
58, 2345–2354] The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulflde bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a ∼50-kDa light chain (Pro 1 through Lys 440) and a ∼100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440–Ala 441 bond appear to be due to the adjacent Ala 441–Pro 442 imide bond's probable cis configuration in a mixed population of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT. A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysis; thus 29.7% of the protein's entire amino acid sequence predicted from the nucleotide sequence was confirmed. The 6 amino acids, residues 945–950, did not match with the sequence predicted in 1992, but did match with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp–Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without 14C label), separation and isolation of the fragments by SDS–PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.
To the Editor: A historical incident illustrates a number of features of botulinum toxin not discussed in the review of bioweaponry by Dr Arnon and colleagues.1During World War II, the US Office of Strategic Services (OSS) developed a plan for Chinese prostitutes to assassinate high-ranking Japanese officers with whom they sometimes consorted in occupied Chinese cities. Concealing traditional weapons on the women at the appropriate time would obviously be difficult. Therefore, under the direction of Stanley Lovell, the OSS prepared gelatin capsules "less than the size of the head of a common pin"2 containing a lethal dose of botulinum toxin. Wetted, a capsule could be stuck behind the ear or in scalp hair, later to be detached and slipped into the officer's food or drink. The OSS recognized that the normal background of botulism cases would deflect suspicion from the women.2
Botulinum toxin is the etiologic agent responsible for the disease botulism, which is characterized by peripheral neuromuscular blockade. Botulism is ordinarily encountered as a form of oral poisoning. The toxin is absorbed from the lumen of the gut to reach the general circulation and is then distributed to peripheral cholinergic nerve endings. However, there is a widespread presumption that botulinum toxin can also act as an inhalation poison, which would require that it be absorbed from the airway. Experiments have been done to show that both pure toxin and progenitor toxin (a complex with auxiliary proteins) are inhalation poisons. Interestingly, the data indicate that auxiliary proteins are not necessary to protect the toxin or to facilitate its absorption. When studied on rat primary alveolar epithelial cells or on immortalized human pulmonary adenocarcinoma (Calu-3) cells, botulinum toxin displayed both specific binding and transcytosis. The rate of transport was greater in the apical-to-basolateral direction than in the basolateral-to-apical direction. Transcytosis was energy dependent, and it was blocked by serotype-specific antibody. The results demonstrated that the holotoxin was not essential for the process of binding and transcytosis. Both in vivo and in vitro experiments showed that the heavy-chain component of the toxin was transported across epithelial monolayers, which indicates that the structural determinants governing binding and transcytosis are found in this fragment. The heavy chain was not toxic, and therefore it was tested for utility as an inhalation vaccine against the parent molecule. This fragment was shown to evoke complete protection against toxin doses of at least 10(4) times the 50% lethal dose.
Justinius Kerner collected data on 230 cases of botulism in the 1820s, suggested the therapeutic use of toxin, and gave a remarkably complete and accurate description of clinical botulism: its symptoms, time course, and the physical findings that the tear fluid disappears, the skin is dry, the eye, gut, and somatic muscles are paralyzed, and mucus and saliva secretion is suppressed. These effects are the clinical targets of botulinum therapy today. Inspired by Drachman's use of toxin to safely paralyze the hind limb in chicks, we worked out the procedures for its safe medical application and licensure from 1972 to 1989, applying it first to correct strabismus, blepharospasm, leg muscle spasm, and torticollis. This list is now extended by others to well over 100 uses. For many years, blepharospasm patients returning for injection around the eyes and upper face would mention as a joke that they were "back to get the wrinkles out." Working in aesthetic dermatology and ophthalmology, Alistair and Jean Carruthers could envision the intentional cosmetic application of botulinum toxin, probably its greatest single use today.
In studies from this laboratory, we localized the regions on the H chain of botulinum neurotoxin A (BoNT/A) that are recognized by anti-BoNT/A antibodies (Abs) and block the activity of the toxin in vivo. These Abs were obtained from cervical dystonia patients who had been treated with BoNT/A and had become unresponsive to the treatment, as well as blocking Abs raised in mouse, horse, and chicken. We also localized the regions involved in BoNT/A binding to mouse brain synaptosomes (snp). Comparison of spatial proximities in the three-dimensional structure of the Ab-binding regions and the snp binding showed that except for one, the Ab-binding regions either coincide or overlap with the snp regions. It should be folly expected that protective Abs when bound to the toxin at sites that coincide or overlap with snp binding would prevent the toxin from binding to nerve synapse and therefore block toxin entry into the neuron. Thus, analysis of the locations of the Ab-binding and the snp-binding regions provides a molecular rationale for the ability of protecting Abs to block BoNT/A action in vivo.