Fig 1.
Schematic representation of the CDT loci from representative strains.
a, The full length CdtLoc from the ribotype 027 strains (M7404 and R20291). b, The CdtLoc from the ribotype 078 strain, JGS6133. The cdtR pseudogene is shown in black and is grey after the premature stop codon. c, The CdtLoc from the ribotype 012 strain 630 carrying cdtAB pseudogenes (shown in black). d, The CdtLoc in the CDT negative strain CD37 is replaced with a 68 bp sequence. The boundaries of the CdtLoc are indicated with vertical lines and the flanking genes are blue.
Fig 2.
a–c, Western immunoblot using CDTa-specific and cross-reactive Ib-specific antibodies and precipitated supernatants from the strains indicated. CD37 (non-toxigenic), V = vector control, R+ = cdtR complemented. The arrows indicate the 48 kDa CDTa and 99 kDa CDTb proteins. d–f, CDT activity assessed by ADP-ribosyltransferase assay. Samples were separated by SDS-PAGE and biotinylated (ADP-ribosylated) actin detected by HRP-streptavidin. Relative CDT activity was assessed by densitometry compared to the non-toxigenic control strain CD37. A = actin, A-A = ADP-ribosylated actin. Data represent the mean ± SEM (n = 3). *, p ≤ 0.05.
Fig 3.
Analysis of TcdA and TcdB production.
a, c, e, Western immunoblot using TcdA-specific and TcdB-specific antibodies with precipitated supernatant from the strains indicated. CD37 (non-toxigenic), V = vector control, R+ = cdtR complemented. Arrows indicate the 308 kDa TcdA and 270 kDa TcdB proteins. Supernatants were collected at 12, 24, 48 and 72 hours post inoculation and assayed by doubling dilution cytotoxicity assays. b, The panel of M7404 strains were assayed using HT29 cells and Vero cells. d, R20291 panel of strains assayed using HT29 cells and Vero cells. Data represent the mean ± SEM (n = 3–5). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Fig 4.
Transcriptional analysis of M7404 cdtR mutant and complemented strains compared to wild-type.
RNA was isolated from strains for analysis of (a) tcdA, (b) tcdB, (c) cdtA, (d) tcdR, (e) tcdC and (f) sigD expression. Levels of gene expression were normalised to rpoA. Data represent the mean normalised gene expression ± SEM from five independent biological replicates. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
Fig 5.
Virulence of M7404 wild-type, cdtR mutant and complemented strains in mice.
a, Kaplan-Meier survival curve showing time from infection to euthanasia of mice infected with different strains of C. difficile in hours. (n = 15). b, Time from inoculation of mice to death in hours. Data represents mean ± S.E.M. (n = 15). Data represent the mean ± SEM ****, p ≤ 0.0001.
Fig 6.
Histopathology of C. difficile infected tissues.
Representative images of sections of colon and caeca collected from uninfected mice or mice infected with different strains of C. difficile, fixed and strained with PAS-Alcian blue. Red brackets ([) indicate crypt hyperplasia, arrow heads (▲) represent surface epithelial damage and asterisks (*) represent oedema and inflammation. Scale bars (200 μm) are shown in yellow. Histopathology damage scores from uninfected or infected colons (b) and caeca (c). Data represent the mean ± SEM ****, p ≤ 0.0001.
Table 1.
Bacterial strains and plasmids.
Table 2.
Oligonucleotide primers.