The genus Clostridium represents a heterogeneous group of anaerobic spore-forming bacteria, comprising prominent toxin-producing species, such as C. difficile, C. botulinum, C. tetani and C. perfringens, in addition to well-known non-pathogens like solventogenic C. acetobutylicum. In the last decade several clostridial genomes have been deciphered and post-genomic studies are currently underway. The advent of newly developed, genetic manipulation tools have permitted functional-based and systems biology analyses of several clostridial strains. Research in this area is at a very exciting stage.
In this book internationally recognised Clostridium experts critically review the most important aspects of clostridial research, providing the first coherent picture of the organism's molecular and cellular biology in this post-genomic era. The first major focus of the book is the genetics and molecular biology of the major clostridial toxins including: botulinum and tetanus neurotoxins, C. difficile large exotoxins, C. perfringens enterotoxin, pore-forming and binary bacterial toxins. Other topics include: molecular epidemiology of C. botulinum and C. difficile, metabolic networks in C. acetobutylicum, development of genetic knock-out systems for clostridia, surface structures, anti tumor potential of clostridia, and antibiotic resistance determinants in C. difficile.
Essential reading for every clostridia researcher, from the PhD student to the experienced scientist, as it provides a timely review of current research.
Chapter 1 Botulinum and Tetanus Neurotoxins, Molecular Biology and Toxin Gene Regulation, Mode of Action
S. Raffestin, A. Couesnon, Y. Pereira, C. Mazuet and M. R. Popoff
Botulinum neurotoxins (BoNT) and tetanus toxin (TeNT) are potent toxins which are responsible for severe diseases, botulism and tetanus, in men and animals. BoNTs induce a flaccid paralysis, whereas TeNT causes a spastic paralysis. Both toxins are zinc-dependent metalloproteases, which specifically cleave one of the three proteins (VAMP, SNAP25, and syntaxin) forming the SNARE complex within target neuronal cells which have a critical function in the release of neurotransmitter. BoNTs inhibit the release of acetylcholine at peripheral cholinergic nerve terminals, whereas TeNT blocks neurotransmitter release at central inhibitory interneurons. Only a single form of TeNT is known, but BoNTs are divided in 7 toxinotypes and various subtypes, which differ in amino acid sequences and immunological properties. In contrast to TeNT, BoNTs are associated to non-toxic proteins (ANTPs) to form highly stable botulinum complexes. TeNT is produced by Clostridium tetani, and BoNTs by Clostridium botulinum and atypical strains of Clostridium baratii and Clostridium butyricum. The genes encoding the neurotoxin and ANTPs are clustered in a DNA segment, called botulinum locus, which is located on chromosome, plasmid or phage. Neurotoxin synthesis is a highly regulated process, which occurs in late exponential growth phase and beginning of stationary phase, and which is dependent of alternative sigma factors (BotR or TetR). BotR and TetR are related to other clostridial sigma factors, TcdR and UviA, which are involved in the control of Clostridium difficile toxins A and B, and Clostridium perfringens bacteriocin, respectively. BotR, TetR, TcdR and UviA form a new subgroup of RNA polymerase sigma factors.
Chapter 2 Improved Understanding of the Action and Genetics of Clostridium perfringens Enterotoxin Suggests Potential Applications for Cancer Therapy and Drug Delivery
Sameera Sayeed, Susan L. Robertson, Justin A. Caserta and Bruce A. McClane
Clostridium perfringens enterotoxin (CPE) causes the intestinal symptoms of a common food-borne illness and ~5-15% of all antibiotic-associated diarrhea cases. In food poisoning isolates, the enterotoxin gene (cpe) is usually present on the chromosome, while cpe is carried by conjugative plasmids in antibiotic-associated diarrhea isolates. CPE action involves its binding to claudin receptors, oligomerization/prepore formation, and prepore insertion to form a functional pore that kills cells by apoptosis or oncosis. The C-terminal half of CPE mediates receptor binding, while its N-terminal half is required for oligomerization. CPE/CPE derivatives are being explored for cancer therapy/diagnosis and improved drug delivery.
Chapter 3 The Cholesterol-dependent Cytolysins and Clostridium septicum a-Toxin; Pore Forming Toxins of the Clostridia
Eileen M. Hotze and Rodney K. Tweten
Two classes of pore-forming toxins of the clostridia are represented by the cholesterol-dependent cytolysins (CDCs) and the Clostridium septicum a-toxin. The CDCs are found in a wide variety of clostridial species, but are also found in many species from other Gram-positive genera. As a result, various CDCs have evolved specific traits that appear to enhance their ability to complement the pathogenic mechanism of a specific bacterial species. In contrast, closely related toxins to C. septicum a-toxin (AT) have not been found in other species of the clostridia, although C. perfringens epsilon toxin appears to be distantly related. Remarkably, distant relatives of AT have been found in species of Gram-negative bacteria as well as certain species of mushrooms and the enterolobin tree seed. Although the CDCs appear to be restricted to Gram-positive bacterial pathogens it has recently been shown that the unusual protein fold of their membrane-penetrating domain is present in proteins of the eukaryotic complement membrane attack complex. Both toxins penetrate the membrane by the use of a ß-barrel pore but differ significantly in their pore-forming mechanisms. The contribution of both classes of toxins to disease is not yet well understood for the clostridia. It is clear that they play important, but likely different roles in clostridial disease.
Chapter 4 Binary Bacterial Toxins: Evolution of a Common, Intoxicating Theme
Bradley G. Stiles and Michel R. Popoff
Several proteins from Gram-positive, spore-forming bacilli use a synergistic binary mechanism for intoxicating eukaryotic cells. These toxins include Clostridium botulinum C2 toxin, Clostridium difficile toxin (CDT), Clostridium perfringens iota () toxin, and Clostridium spiroforme toxin (CST). Furthermore, closely related Bacillus species such as Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis produce strikingly similar binary toxins. As per existing literature, these latter proteins have provided a "model" for the clostridial binary toxins. Each of these clostridial and bacillus binary toxins consists of distinct enzymatic "A" and binding "B" proteins that work in concert. Conservation of a basic intoxication theme between different genera clearly suggests retention of an evolutionarily successful mechanism promoting bacterial survival and dissemination throughout Nature. In particular, this chapter highlights the existing literature linked to Clostridium binary toxins and their role in pathogenesis, structure, function, as well as current / future use as protein shuttles.
Chapter 5 Molecular Epidemiology of Group I and II Clostridium botulinum
Miia Lindström, Maria Fredriksson-Ahomaa, and Hannu Korkeala
Clostridium botulinum, producing highly potent botulinum neurotoxin, is a diverse species consisting of four genetically and physiologically distinct groups (Groups I-IV) of organisms. Groups I and II C. botulinum produce A, B, E, and/or F toxins which cause human botulism. In addition, some strains of Clostridium butyricum and Clostridium barati produce type E and F toxins, respectively, and have thus been related to human illness. Human botulism appears in five different forms, such as the classical food botulism, infant botulism, wound botulism, adult infectious botulism, and iatrogenic botulism. Typical of all forms of human botulism is descending flaccid paralysis which may lead to death upon respiratory muscle failure. While the research and diagnostics of botulinum neurotoxigenic clostridia and botulism were based on toxin detection by the mouse bioassay until mid 1990¹s, the subsequent development of molecular detection and typing assays enabled rapid, sensitive, specific, and ethically acceptable molecular epidemiological detection, identification and strain characterization of these organisms, increasing our understanding of the epidemiology of botulinum neurotoxigenic clostridia and botulism.
Chapter 6 Molecular variability in C. difficile large clostridial toxins
Maja Rupnik
Clostridium difficile, as all clostridia, is a toxin producing microorganism and the toxins are the main virulence factors. In early eighties it was clear that two large toxins are produced by bacterium and epidemiological studies have indicated that strains either produce both toxins (toxin A, TcdA, and toxin B, TcdB) or none of them. Toxigenic strains were usualy associated with the disease, while nontoxigenic were not. Few years later, however, this simple situation changed as strains producing only TcdB or strains producing an additional toxin (binary toxin CDT) were described. Such strains with unusual toxin production pattern were subsequently found to have changes in the genomic PaLoc region encoding the toxins TcdA and TcdB. These changes are the basis for a method that distinguish C. difficile strains into toxinotypes. The variability of genes coding for large clostridial toxins (LCTs) had consequences in changes of laboratory diagnosis, changes in understanding of the role of both toxins in the pathogenesis, in structure function relationships and partialy in understanding of the evolutions of LCTs.
Chapter 7 Comparative Genomics of Clostridium difficile
Lisa F. Dawson, Richard A. Stabler and Brendan W. Wren
The recent emergence of hypervirulent strains of Clostridium difficile and their ability to spread across continents, has caused alarm in both hospitals and the community. This has drawn attention away from other important pathogenic C. difficile strains, which are responsible for significant morbidity and mortality. Little is known about the genetic diversity of these strains and their less pathogenic counterparts. The recent publication of the genome sequence of strain 630 and advances in both microarray and mutagenesis technologies promises to revolutionise our understanding of the pathogenesis and population dynamics of C. difficile. This chapter summarises the salient findings of the 630 genome sequence and includes phylogenetic analysis of C. difficile strains from diverse origins.
Chapter 8 Surface Structures of C. difficile and Other Clostridia: Implications for Pathogenesis and Immunity
Jenny Emerson and Neil Fairweather
The cell wall of Clostridium difficile has an architecture typical of other Gram-positive bacteria. A thick peptidoglycan layer lies external to the cell membrane with many associated cell wall proteins. In C. difficile two major cell wall proteins constitute the S-layer, a paracrystalline two-dimensional array surrounding the entire cell. The sequences of these S layer proteins (SLPs) are variable between strains, perhaps reflecting immunological pressures on the cell. The genome sequence reveals a family of proteins with homology to the high molecular weight SLP; each of these proteins have a second unique domain but their functions remain largely uncharacterised. This family of cell wall proteins is also found in some other species, for example C. botulinum and C. tetani, but not in others such as C. perfringens. Some cell wall proteins of C. difficile, including the SLPs, have properties that imply an involvement in pathogenesis, particularly in binding to host cell tissues. The cell wall proteins of C. difficile may also act as immunogens to induce a partially protective immune response to infection, and may be considered as components of future vaccines against C. difficile associated disease.
Chapter 9 Antibiotic resistance determinants in Clostridium difficile
Paola Mastrantonio and Patrizia Spigaglia
Clostridium difficile, the well known nosocomial pathogen responsible for the majority of antibiotic associated diseases, is increasingly recognised also as the cause of community-associated disease and of enteric disease in animals. The organism is resistant to several antibiotics and can survive disruption of the normal intestinal flora after antibiotic treatment exploiting this advantage to colonize and cause disease. The study of the mechanisms responsible for resistance have highlighted the presence of mobile genetic elements in the C. difficile genome, potentially acquired from other microorganisms. C. difficile might be able to disseminate resistance determinants to other species, thus collaborating to the evolution of the antibiotic resistant patterns that characterise the bacteria circulating worldwide.
Chapter 10 Development of Genetic Knock-out Systems for Clostridia
John T. Heap, Stephen T. Cartman, Oliver J. Pennington, Clare M. Cooksley, Jamie C. Scott, Ben Blount, David Burns and Nigel P. Minton
Despite the medical and industrial importance of the genus Clostridium our understanding of their basic biology lags behind that of their more illustrious counterpart, Bacillus. The advent of the genomics era has provided new insights, but full exploitation of the data becoming available is being hindered by a lack of mutational tools for functional genomic studies. Thus, in the preceding decades the number of clostridial mutants generated has been disappointingly low. On the one hand, the absence of effective transposon elements has stymied random mutant generation. On the other hand, the construction of directed mutants using classical methods of recombination-based, allelic exchange has met with only limited success. Indeed, in the majority of clostridial species mutants are largely based on integration of plasmids by a Campbell-like mechanism. Such single crossover mutants are unstable. As an alternative, recombination-independent strategies have been developed that are reliant on retargeted group II intron. One element in particular, the ClosTron, has been devised which provides the facility for the positive selection of mutants. ClosTron-mediate mutant generation is extremely rapid, highly efficient and reproducible. Moreover the mutants made are extremely stable. Its deployment considerably expands current options for functional genomic studies in clostridia.
Chapter 11 Clostridia in Anti-tumor Therapy
Asferd Mengesha, Ludwig Dubois, Kim Paesmans, Brad Wouters, Philippe Lambin and Jan Theys
Although traditional anticancer therapies are effective in the management of many patients, there are a variety of factors that limit their effectiveness in controlling some tumors. These observations have led to interest in alternative strategies to selectively target and destroy cancer cells. In that context, Clostridium-based tumor targeted therapy holds promise for the treatment of solid tumors. Upon systemic administration, various strains of non-pathogenic clostridia have been shown to infiltrate and selectively replicate within solid tumors. This specificity is based upon the unique physiology of solid tumors, which is often characterized by regions of hypoxia and necrosis. Clostridial vectors can be safely administered and their potential to deliver therapeutic proteins has been demonstrated in a variety of preclinical models. However, there are several issues that are still unknown and remain major challenges. In this chapter, we will review the potential use of Clostridium in cancer treatment and discuss the major advantages, challenges and shortcomings of bacterial systems for tumor-specific therapy. In addition, we will highlight the requirements needed to advance the approach into clinical trials.
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