Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers

Abstract

Clostridium difficile is a major nosocomial pathogen and the main causative agent of antibiotic-associated diarrhoea. The organism produces two potent toxins, A and B, which are its major virulence factors. These are chromosomally encoded on a region termed the pathogenicity locus (PaLoc), which also contains regulatory genes, and is absent in non-toxigenic strains. Here we show that the PaLoc can be transferred from the toxin-producing strain, 630Δerm, to three non-toxigenic strains of different ribotypes. One of the transconjugants is shown by cytotoxicity assay to produce toxin B at a similar level to the donor strain, demonstrating that a toxigenic C. difficile strain is capable of converting a non-toxigenic strain to a toxin producer by horizontal gene transfer. This has implications for the treatment of C. difficile infections, as non-toxigenic strains are being tested as treatments in clinical trials.

Figure 1. Cytotoxicity assay comparing toxin production.

Toxin production by the donor, transconjugant and recipient strains were compared using a cytotoxicity assay. (a) HFF-1 cells incubated with the culture supernatant of transconjugant PaLoc386 show rounding of the cells. (b) The cytotoxic effect of PaLoc386 can be neutralized using antibodies against TcdB. Scale bar, 1 mm in a and b. (c) The toxin end point titre was determined for culture supernatants of donor strain 630 Derm, transconjugants PaLoc386 and the recipient CD37. The mean±s.d. is shown for three independent experiments.

Figure 2. Schematic representation of the donor-specific DNA fragments present in the transconjugants.

The circular chromosome of C. difficile strain 630 is shown. The boxed region on the left is magnified on the right. The green fragments in the outer ring represent the 10 kb fragments used to differentiate transconjugants from donors; see text for more details. The dark blue fragments in the second ring indicate the mobile genetic elements present in the donor; the red fragment in this ring represents the PaLoc. The donor-specific fragments that are present in the transconjugants are shown in the seven inner-most rings as coloured fragments with the number of each transconjugant indicated. The red fragments in these represent the transferred PaLoc. The dark blue fragments in the track of PaLoc386 depict CTn1 and CTn5 that are both present in this transconjugant.