| dc.description.abstract | The recent increase in availability of next generation sequencing methodologies
has led to extensive analysis of the genome of Clostridium difficile. In contrast,
protein expression analysis, crucial to the elucidation of mechanisms of disease,
has severely lagged behind. In this study, in-depth proteomic analysis of three
strains of varying virulence, demonstrated previously in an animal model, has
been undertaken against a background of the sequenced genomes. Strain B-1 is
a historic, virulent, ribotype 005 clone, strain A represents the emerging
hypervirulent 027 ribotype, while strain Tra5/5, ribotype 001, is of low
virulence.
To undertake a comprehensive overview of the expressed proteome, both 1D
and 2D gel electrophoresis were used to separate and display the protein
content of each isolate. This was coupled to MALDI-TOF and LC-MS/MS mass
spectrometry for protein identification. A total of 888 different proteins were
characterised by comparative analysis of isolates grown in parallel for 64 hours
on blood agar. Of these, only 38% were shared between all isolates. An
additional 350, 243 and 398 proteins were detected from broth cultures, and the
use of a hexapeptide bead library, designed to capture low abundance proteins,
led to the detection of a further 148, 127, and 171 proteins in strains A, B-1 and
Tra5/5 respectively.
Relative differential expression was investigated using Differential In Gel
Electrophoresis (DIGE), and five proteins were shown to have a statistically
higher concentration in strain A, twelve in strain B-1 and eight in strain Tra5/5.
A number of these were surface proteins, with selected S-layer proteins found to be up-regulated in each strain, and the flagellar protein, FliC, up-regulated in
both A and B-1. Furthermore, differential post-translation modification events
were seen in flagellar and S-layer proteins.
In-vivo expression of these proteins was mapped using Western blotting.
Immunodetection of the majority of these, including FliC and the high
molecular weight S-layer protein, were conserved between the three strains, but
a notable series of immunoreactive protein spots were present in strains A and
Tra5/5 but not B-1, most likely corresponding to an additional S-layer protein
present in the genomes these strains, but not that of B-1.
Protein expression differences for a number of previously proposed virulence
proteins were evident between strains, including toxin B, sporulation, flagella
and the S-layer proteins, metabolic enzymes, stress response proteins and ABC
transporters. This study strongly supports the view that the virulence of
Clostridium difficile is multifactorial, and that a number of related factors,
although not directly required for pathogenicity, may serve to modulate the
virulence of individual strains. | en |
