Trends in Microbiology
ReviewThe role of toxin A and toxin B in the virulence of Clostridium difficile
Section snippets
Clostridium difficile infections and virulence factors
C. difficile is a Gram-positive anaerobic bacterium that is one of the most important causes of antibiotic-associated diarrhoea in the developed world, leading to significant morbidity and mortality and placing a considerable economic burden on healthcare systems 1, 2. The organism causes a range of intestinal diseases collectively referred to as C. difficile infections (CDI) or C. difficile associated disease (CDAD). This disease can range from mild self-limiting diarrhoea, through to
The pathogenicity locus
Toxin A and toxin B are encoded by the genes tcdA and tcdB, respectively, which are both located within a 19.6 kb region of the chromosome known as the pathogenicity locus or PaLoc (Figure 1) [24]. In addition to the major toxin genes, the PaLoc region encodes three accessory genes: tcdR and tcdC, which encode proteins involved in regulating the expression of TcdA and TcdB, and tcdE, whose product is postulated to facilitate the secretion of the toxins from the cell. There is now substantial
Hypervirulent or epidemic strains of C. difficile
Recently, morbidity and mortality resulting from CDI have increased significantly, most probably as a result of changes in the virulence of the causative strains coupled with changes in antibiotic usage patterns [29]. Hypervirulent strains, belonging predominantly to the BI/NAP1/027 group, appear to cause epidemics more readily than other C. difficile strains, leading to an increased incidence of CDI and greater disease severity 5, 30, 31. Infections by these strains are conservatively
The structure and function of toxin A and toxin B
Toxins A and B are both members of the large clostridial toxin (LCT) family, which includes the lethal and haemorrhagic toxins (TcsL and TcsH, respectively) of Clostridium sordellii [41], alpha toxin (TcnA) from Clostridium novyi [42] and TpeL from Clostridium perfringens isolates from domestic livestock [43] (Box 1). The LCTs are an important family of bacterial toxins; they are monoglycosyltransferases that inactivate Rho family GTPases, including Rho, Rac, Ras, Ral and Cdc42, through the
Potential impact of variant toxins in CDI
C. difficile is a genetically heterogeneous species with substantial chromosomal variation between strains 7, 40, 51. This genetic variation extends to the PaLoc region, including the toxin structural genes tcdA and tcdB, facilitating the development of a C. difficile toxinotyping scheme. This typing scheme classifies strains, based on defined PCR analysis and subsequent restriction endonuclease analysis of DNA fragments, into ‘toxinotypes’, reflecting specific nucleotide changes that are
Toxin A as the major mediator of disease
Although the majority of C. difficile strains produce both toxin A and toxin B, toxin A was thought to be the major virulence factor for many years on the basis of early studies using purified toxins. In these experiments, hamsters were challenged intragastrically with purified toxin and then monitored for signs of disease. Hamsters succumbed to disease and displayed symptoms typical of C. difficile infection when challenged with purified toxin A, including fluid accumulation, inflammation and
A paradigm shift: the role of toxin B in the virulence of C. difficile
The identification of clinical strains of C. difficile that do not produce toxin A but still cause symptomatic disease [55] was perplexing. Experiments using purified toxins clearly indicated that toxin A was the major mediator of CDI, yet the occurrence of toxin A-negative, toxin B-positive isolates suggested otherwise. Additionally, isolation of these strains from diseased patients indicated that toxin A was not required for toxin B functional activity in vivo. At a clinical level, the
Determining the roles of TcdA and TcdB using a genetic approach
Despite the detailed studies performed using purified toxins, the individual contribution of toxin A and toxin B to disease remained ambiguous primarily because the research findings did not correlate with the variant clinical isolates that were becoming increasingly common. This situation was exacerbated by an inability to generate isogenic toxin gene mutants in C. difficile. This problem was recently overcome in research that resulted in the successful construction of multiple independently
Concluding remarks and future directions
An in-depth understanding of the role of each toxin in the development of CDI is crucially important for the rational development of the next generation of treatments to effectively combat CDI. The recent finding that toxin B is the major virulence factor of C. difficile [67] could, for example, account for the unexpectedly poor performance of the toxin-binding polymer telovamer in phase III clinical trials [74] because this drug preferentially binds to toxin A (note: the authors have no
Acknowledgements
This work was supported by Project Grants from the Australian National Health and Medical Research Council and a Discovery Grant from the Australian Research Council.
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