Newcastle University Drylab/17 May 2008

Nina
Yesterday I researched unique peptides in the Gram-positive anaerobic bacterium Clostridium difficile. Background

This pathogen is associated with antibiotic-related diarrhoea and life threatening pseudomembranous colitis so can be regarded as an important species to research. The current gold standard for diagnosis is toxigenic culture in which organisms are cultured on selected medium and tested for specific toxin production (A and B). This procedure is sensitive and specific but is slow and labour intensive.

Potential Peptides

I started by looking at quorum sensing peptides in C. difficile. Studies (Carter et al., 2005) have shown that at least one signalling molecule exists which can induce bioluminescence in a Vibrio harveyi luxS reporter strain. Carter then concluded that a luxS homologue (luxScd) quorum sensing system existed in C. difficile. The signalling molecule corresponded to autoinducer-2 (AI-2). However this diffusable molecule, regulated by the rolA/B two component system in C. difficile, is ubiquitous amongst many species of bacteria (Kleerebezem et al., 1997), (Barth et al., 2004), (Carter et al., 2005), (Sebaihia et al., 2006). It would therefore be foolish to consider a receptor of AI-2 as a potential diagnostic tool of C. difficile related diseases alone. In addition there is no direct evidence that C. difficile actually employs a quorum sensing system!! But I didn't want to give up here...'' I decided to then explore the (unique to C. difficile) exotoxins A and B released. These toxins bind to receptors of target cells and are then endocytosed (Aktories et al., 2007). I had some difficulty identifying these receptors initially.

I then found out why. The nature of toxin A (TcdA) and B (TcdB) receptors are not well defined, although carbohydrate structures may be involved (Aktories et al., 2007). I tried to dig deeper and read through more literature for clues about these receptors. I wanted to know if they were specific to the exotoxins of C. difficile and exactly which cells of the host they were present on. I then found out that the receptor for TcdA was indeed only on the surface of intestinal epithelial cells and was indeed made of carbohydrates (Ho et al., 2005). These cell surface carbohydrates included Gal-a1,3-Gal-b1,4-GlcNAc. However it also seems that they are not specific to the C. difficile toxins. I came to this conclusion since Ho et al mentioned that the binding site on TcdA for the receptor, consisting of a run of up to 50 amino acids, was similar to other extracellular bacterial carbohydrate-binding proteins.

Conclusion

Further inspection of the relationship between the unique exotoxins of C. difficile and their relative receptors revealed a low binding affinity and attempts to crysallize the complex have thus so far failed (Ho et al., 2005). In addition, the fact that the receptor is firstly carbohydrate and secondly, mammalian, contributes to my view that exploiting the exotoxins would not appropriate for our application.

Since a quorum sensing system has not been formally established in C. difficile I would also conclude that this species is not viable for our use. I would recommend concentrating on Staphylococcus aureus, Bacillus anthracis and Streptococcus pneumoniae. Having read quite a bit, I can see these have much better characterised QS systems.