Elsevier

Toxicon

Volume 39, Issue 11, November 2001, Pages 1703-1722
Toxicon

Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective

https://doi.org/10.1016/S0041-0101(01)00157-XGet rights and content

Abstract

Clostridium botulinum comprises a diverse assemblage of clostridia that have the common property of producing a distinctive protein neurotoxin (BoNT) of similar pharmacological activity and extraordinary potency. BoNTs are produced in culture as molecular complexes consisting of BoNT, hemagglutinin (HA) and associated subcomponent proteins, nontoxic nonhemagglutinin (NTNH), and RNA. The genes encoding the protein components reside as a cluster on the chromosome, on bacteriophages, or on plasmids depending on the C. botulinum serotype. A gene BotR coding for a regulatory protein has been detected in toxin gene clusters from certain strains, as well as ORFs coding for uncharacterized components. The gene encoding TeNT is located on a large plasmid, and expression of the structural gene is controlled by the regulatory gene, TetR, located immediately upstream of the TeNT structural gene. TeNT is not known to be assembled into a protein/nucleic acid complex in culture. Cellular synthesis of BoNT and TeNT have been demonstrated to be positively regulated by the homologous proteins, BotR/A and TetR. Evidence suggests that negative regulatory factors and general control cascades such as those involved in nitrogen regulation and carbon catabolite repression also regulate synthesis of BoNTs. Neurotoxigenic clostridia have attracted considerable attention from scientists and clinicians during the past decade, and many excellent reviews are available on various aspects of these organisms and their neurotoxins. However, certain areas have not been well-studied, including metabolic regulation of toxin formation and genetic tools to study neurotoxigenic clostridia. These topics are the focus of this review.

Introduction

Clostridium botulinum and Clostridium tetani have attracted much interest in recent years due largely to extensive research on the biochemistry, structure, pharmacology, and cell biology of their neurotoxins (BoNTs and TeNT) (Bigalke and Shoer, 2000, Kreydon et al., 2000, Rosetto et al., 2001, Schiavo et al., 2000), as well as the application of botulinum toxin complexes as therapeutic agents for the treatment of human diseases (Brin, 1997, Dressler, 2000, Jankovic and Hallett, 1994). Several hundred research papers have been published in these areas, while relatively few studies have addressed the physiology and genetics of neurotoxigenic clostridia and the regulation of neurotoxin formation.

Early interest in the molecular biology of clostridia mainly involved studies of metabolism and physiology of nonpathogenic clostridia such as solvent-producing species (e.g. C. acetobutylicum), clostridia producing extracellular hydrolytic enzymes or other products of industrial interest, nitrogen-fixing species, those forming lethal toxins, and species with perplexing degradative pathways for amino acids and other nitrogenous compounds (Andreesen et al., 1989, Barker, 1978, Johnson, 1999, Minton and Clarke, 1989, Van Heyningen, 1950). Kato (1970) addressed the relation of metabolism and cell structure to toxin production, but this subject has not been reviewed recently for the neurotoxigenic clostridia. The necessity to produce vaccines for prevention of clostridial human and animal diseases necessitated study of environmental and nutritional factors to increase toxin production, and methods to isolate the toxic fractions from culture broths (Brown and Williamson, 1997, Johnson and Goodnough, 1995, Schantz and Johnson, 1992). Early studies on toxin production and isolation were later complemented by characterization of plasmids, bacteriophages, bacteriocins, and the isolation of mutants in clostridia (Eklund et al., 1989, Young et al., 1989). Beginning in the 1980’s, various genes were cloned from clostridia, and the structure of genetic elements and mechanisms involved in the regulation of transcription and translation began to be studied (Young et al., 1989, Minton, 1995, Rabinowitz, 1993). The cloning, expression and sequencing of genes encoding toxins led to the field of molecular toxinology (Alouf, 1988, Niemann, 1991). The detection of toxin genes on plasmids and bacteriophages in neurotoxigenic clostridia (Inoue and Iida, 1970, Eklund et al., 1971) was followed by the demonstration of lateral transfer of toxin genes in C. botulinum serotypes C and D and C. novyi (Eklund et al., 1989, Eklund, 1993). The findings that certain C. botulinum isolates produce more than one serotype of toxin or contain silent (unexpressed) gene clusters supported the lateral transfer of genes encoding neurotoxigenicity (Gimenez and Ciccarelli, 1970, Franciosa et al., 1994, Hatheway and McCroskey, 1987, Hutson et al., 1996). Toxin gene transfer also was suspected from investigations of intestinal botulism cases, which showed that type E and F BoNT genes were present in rare strains of normally nontoxigenic clostridial species (Aureli et al., 1986, Hall et al., 1985, McCroskey et al., 1986). Despite these observations and their importance to toxinology and medicine, the molecular mechanisms governing toxin synthesis and interspecies transfer of the toxin genes remain largely unknown. Genetic tools for study of these mechanisms are in early stages of development. Genetic manipulation of neurotoxigenic clostridia has developed slowly compared to other clostridia such as C. perfringens and C. acetobutylicum (Lyras and Rood, 2000).

Section snippets

Clostridium botulinum—the organism

As presently defined, the genus Clostridium consists of a phenotypically and phylogenetically diverse assemblage of Eubacteria that have four common phenotypic properties: (a) the formation of heat- and chemical-resistant endospores; (b) the presence of a Gram-positive cell wall structure in vegetative cells; (c) an anaerobic, fermentative metabolism and; (d) a low guanine+cytosine (G+C) content of 38–56 mol% (26–32% for toxigenic species) (Cato et al., 1986, Hippe et al., 1999). Members of the

Neurotoxin and antibody detection

Irrespective of their taxonomic affiliations, the defining character of neurotoxigenic clostridia is the synthesis of a large neurotoxic protein of ~150 kDa with remarkable neuronal specificity (Schiavo et al., 2000) and extraordinary potency (Gill, 1982, Sugiyama, 1980). The toxicity of BoNT/A has been estimated to be ~0.2 ng/kg body weight, and as little as 0.1–1 μg may be lethal for humans (Schantz and Johnson, 1992, Scott and Suzuki, 1988). Consequently, considerable care and safety

Disease states of neurotoxigenic clostridia and isolation of novel strains

The classic diseases of neurotoxigenic clostridia are tetanus wound infections followed by CNS intoxication and foodborne botulism with characteristic flaccid paralysis of motor and respiratory functions. Wound botulism, infant botulism, and intestinal botulism in non-infants were discovered in the 1940’s, 1970’s and 1980’s respectively (Hatheway and Johnson, 1998). The first descriptions of the symptoms of tetanus were attributed to Hippocrates (Major, 1965, Montecucco, 1995) and those of

The genome of C. botulinum

Initial studies of the genomes of toxigenic clostridia and generation of genetic maps were performed by pulsed-field gel electrophoresis (PFGE) and detection of markers on restriction fragments (Canard and Cole, 1989, Lin and Johnson, 1995). Our laboratory was the first to apply PFGE for genome analysis of C. botulinum type A (Lin, 1992. Thesis, University of Wisconsin–Madison; Lin and Johnson, 1995). The genomes of four group I (type A) strains examined had unique restriction digestion

Nutritional regulation of BoNT and TeNT synthesis

Very little is known of the nutritional and environmental factors that influence BoNT and TeNT formation in media, foods, wounds, and in the human gastric and intestinal tracts. Understanding the control of toxin synthesis and post-translational modification events such as nicking will be of considerable value in enhancing the quality and potency of toxin preparations. Early studies showed that supplementation of complex media with nutrients, such as meat digests, casein hydrolysates, corn

Structure and organization of BoNT complexes

Although the neurotoxigenic clostridia are physiologically and phylogenetically diverse, there is uniformity in the organization of the genes encoding the toxin complexes (also termed progenitor toxins). The association of BoNT with other proteins was initially shown by ultracentrifugation in alkaline conditions (Wagman and Bateman, 1951) and by the demonstration that the neurotoxic organisms and hemaglutinin activity could be dissociated (Lamanna and Lowenthal, 1951). Sakaguchi and colleagues

Positive and negative regulation of BoNT expression

The positive regulatory proteins, BotR and TetR, have characteristics of transcriptional regulators including basic pIs (10.4 and 9.3, respectively) and helix-turn-helix motif structures. BotR and TetR possess considerable homology (~50% identity) have 20–29% identity to some other regulatory proteins including UviA, a putative activator of bacteriocin synthesis in C. perfringens, and TxeR from C. difficile, a positive activator of toxins A and B (Marvaud et al., 1998a,b). Studies have been

Overview

Although gene manipulation methods have been established in C. perfringens (Lyras and Rood, 2000; Rood, 1998) and in C. acetobutylicum (Mitchell, 1998), genetic methods have developed comparatively slowly in C. botulinum. Genetic analysis of toxinogenesis and other properties in C. botulinum has been hindered because of the lack of certain needed tools, including directed mutation systems, efficient gene transfer methods, lack of cloning and expression vectors, and absence of gene replacement

Evidence for lateral gene transfer of BoNT and other clostridial genes and evolution of toxigenesis

An intriguing and important genetic property of certain toxin genes in clostridia is that they are often associated with genetic elements that can be laterally transferred across phylogenetic barriers, occasionally to nontoxigenic clostridia. The possibility for transfer of BoNT genes was first suspected in the investigation of infant botulism cases in Italy and in the USA, when strains of C. butyricum or C. baratii were recovered from infants with botulism (Aureli et al., 1986, Hall et al.,

Acknowledgements

Research in EAJ's laboratory has been supported by the National Institutes of Health, the United States Department of Agriculture, the US Department of Defense, Food Industry sponsors of the Food Research Institute, University of Wisconsin, Madison.

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