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The role of toxin A and toxin B in Clostridium difficile infection

Abstract

Clostridium difficile infection is the leading cause of healthcare-associated diarrhoea in Europe and North America1,2. During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have indicated that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A or there is prior damage to the gut mucosa3. However, a recent study indicated that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent4. This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knockout system5,6 to inactivate the toxin genes permanently, we found that C. difficile producing either one or both toxins showed cytotoxic activity in vitro that translated directly into virulence in vivo. Furthermore, by constructing the first ever double-mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue to consider both toxins in the development of diagnostic tests and effective countermeasures against C. difficile.

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Figure 1: Characterization of C. difficile toxin mutants.
Figure 2: In vitro cytotoxicity.
Figure 3: Virulence of C. difficile strains in hamsters.

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Acknowledgements

S.A.K., S.T.C., A.C. and N.P.M. acknowledge the financial support of the UK Medical Research Council (G0601176). Support for M.L.K. was provided by the European Union (HEALTH-F3-2008-223585) and for J.T.H. by the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/F003390/1). We thank C. von-Eichel Streiber for supplying the neutralizing antibody serums and Y. Mahida for the Vero cells. We are also grateful to A. Olling and R. Gerhard for methodological advice on western blots.

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Contributions

The study was conceived by N.P.M. and designed by S.A.K., S.T.C. and J.T.H. Construction of mutants and in vitro characterization was carried out by S.A.K. In vivo work was carried out by S.T.C., M.L.K. and A.C. Analysis of data was carried out by S.A.K. and M.L.K. with assistance from S.T.C. and J.T.H. The manuscript was written by S.A.K. and S.T.C. with critical input from all other authors. Funding for the study was sourced by N.P.M. and A.C.

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Correspondence to Nigel P. Minton.

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The authors declare no competing financial interests.

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Kuehne, S., Cartman, S., Heap, J. et al. The role of toxin A and toxin B in Clostridium difficile infection. Nature 467, 711–713 (2010). https://doi.org/10.1038/nature09397

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