Nazari Shirvan, Ali
Molecular analysis of surface proteins of Clostridium difficile.
PhD thesis, University of Glasgow.
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Since the Gram positive anaerobe Clostridium difficile was first isolated and described, it has emerged as an increasingly important nosocomial pathogen in
Europe, North America and elsewhere, and the prime causative agent of antibiotic
associated dirrhoea and pseudomembranous colitis in humans.
The two large toxins, A and B, are the main virulence factors, proteins that are expressed in the gastrointestinal tract after colonisation by C. difficile. The pathological symptoms mediated by these toxins include disruption of the integrity
of the epithelium, fluid loss, intestinal inflammation and tissue destruction. Important as the toxins are to C. difficile pathogenesis, several other proteins are known to contribute to colonisation and other aspects of the disease process remain poorly understood. Immunological studies using antisera from the patients revealed a number of candidates and amongst these, proteins present, or thought to be present, at the bacterial surface contribute to adhesion, motility and other interactions with the human host.
The aims of this study were to produce a number of surface proteins from C. difficile as recombinant products and to isolate antibodies against these targets via phage display. The goal was to assess if these antibodies could inhibit the normal function of these targets and to confirm their location in C. difficile. Of 11 clostridial proteins, expression and purification of 3 proved impossible (Cwp84, FbpA and Acd) but 8 others (CspA, GroEL, FliC, FliD, a putative sortase, Cwp66, and its amino and carboxy terminal regions) were used for antibody isolation along with recombinant and native forms of SlpA. Phage display yielded a large panel of specific single chain variable fragments (scFv) antibodies that were expressed, purified and characterised.
Reaction between the scFvs and their targets took place in ELISA and Western blotting suggesting the recognition of linear rather than conformational epitopes. The binding of scFvs to SlpA and its components showed strain specificity 3 with good recognition of protein from C. difficile 630 but no reaction towards SlpA from R20291, and 027 ribotype. Binding of scFvs of a range of specificities to extracts from C. difficile M120 indicated that a component of the S layer from this strain might possess immunoglobulin binding activities in the manner of a superantigen.
The scFvs against flagellar proteins FliC and FliD were able to inhibit bacterial motility and therefore there would be potential in testing whether other scFvs generated in this study were able to inhibit the biological activity of their targets.
Some scFvs were tested in immunofluorescence microscopy. The positive results from these experiments showed that the reagents and the strategy pursued could be used to establish surface exposure of the targets and other components of the bacterial surface. Given the high specificity of the reagents, and in the case of Cwp66, the ability to isolate scFvs against defined regions of the protein, the strategy has the capacity to define the orientation of proteins in the bacterial surface. In contrast, the use of scFvs to locate their targets in electron microscopy using immunogold reagents was unsuccessful. As this approach has been successful in other studies, it deserves further investment of effort.
Overall, expression of proteins from C. difficile in an E.coli host was generally successful and phage display provided a rapid, highly efficient method for the isolation of specific immunological reagents. These have the potential to explore the location, orientation and activity of proteins from the pathogen.
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