Novel high-molecular-weight, R-type bacteriocins of Clostridium difficile

Abstract

Clostridium difficile causes one of the leading nosocomial infections in developed countries, and therapeutic choices are limited. Some strains of C. difficile produce phage tail-like particles upon induction of the SOS response. These particles have bactericidal activity against other C. difficile strains and can therefore be classified as bacteriocins, similar to the R-type pyocins of Pseudomonas aeruginosa. These R-type bacteriocin particles, which have been purified from different strains, each have a different C. difficile-killing spectrum, with no one bacteriocin killing all C. difficile isolates tested. We have identified the genetic locus of these "diffocins" (open reading frames 1359 to 1376) and have found them to be common among the species. The entire diffocin genetic locus of more than 20 kb was cloned and expressed in Bacillus subtilis, and this resulted in production of bactericidal particles. One of the interesting features of these particles is a very large structural protein of ~200 kDa, the product of gene 1374. This large protein determines the killing spectrum of the particles and is likely the receptor-binding protein. Diffocins may provide an alternate bactericidal agent to prevent or treat infections and to decolonize individuals who are asymptomatic carriers.

Fig 1

Electron micrograph of negatively stained purified diffocin particles. (A) Diffocins isolated from CD4. (B) Diffocins isolated from strain ATCC 43593. Note the large flower-like appendages, which are likely the receptor-binding protein.

Fig 2

Bactericidal activity of diffocin 4 on strain 19137. (A) Lawn spot assay, showing zones of clearing due to the bactericidal activity of diffocin. (B) Liquid survival titration assay. A fixed number of target cells were incubated with dilutions of the diffocin preparation. The cells were then diluted and spotted onto an agar plate.

Fig 3

Silver stained SDS-PAGE gel of purified diffocin particles from CD4 (left lane). The arrows indicate bands that were excised for mass spectrometric analysis. The molecular mass markers (right lane) were pasted from a different lane of the same gel.

Fig 4

Map of the genetic locus of the diffocin cluster. ORFs encoding putative structural components are shown in blue, while putative regulatory proteins are shown in red. Those ORFs for which no function can be predicted based on sequence similarity are shown in gold. The intergenic sequence shown between ORFs 1360 and 1360a represents a potential operator region with two strong DinR consensus-binding sites (sequence portions indicated in red). Further annotations of the ORFs are in Table 2.

Fig 5

Spot plate assays of diffocin 4 and the hybrid diffocin 4-16 on target strains 19145, 19099, 19137, and 19135. Diffocin 4 was produced in B. subtilis BDG45, and the hybrid diffocin 4-16 was produced in BDG55. The diffocins were purified, and serial 5-fold dilutions of diffocins were spotted onto lawns of target bacteria, with diffocin 4 on the left and diffocin 4-16 on the right of each lawn. The plates were incubated overnight at 37°C before imaging.