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Team:Newcastle University/Nina's Lab Journal
From 2008.igem.org
Contents |
17/05
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.
19/05
Today I will be working backwards to identify gram-positive bacteria that have well characterized quorum sensing systems (QSS). This should be a more efficient way of finding a suitable peptide.
List of Bacteria with well characterized QSS
Streptococcus pneumoniae - currently being looked at by Megan
Streptoccoccus mutans and gordonii - cause dental plaque and sub-acute endocarditis. Possibly not severe enough for us to use?
Bacillus subtillis - not pathogenic
Lactococcus lactis - not pathogenic. Used in dairy products.
Staphylococcus aureus - currently being looked at by Mark
Pseudomonas aeruginosa - Gram-negative
Carnobacterium piscicola - not pathogenic but releases a bacteriocin inhibiting QSS of Listeria monocytogene (approved and already looked at by Mark).
Yersinia enterocoliticia - Gram negative!!
Lactobacillus sake - not pathogenic (used in fermented sausages)
Note to self... a lot of gram-negative well characterized QSS..annoying. What if I focus search to only G+ve disease causing bacteria? Another note to self... I cannot consider any of the bacteria which solely adopt the LuxS QSS (such as Clostridium difficile) since autoinducer emitted is the same for many bacteria and would not serve as a suitable protein to target. (Lerat et al., 2004).
Streptococcus pyogenes- myositis, toxic shock syndrome, puerperal fever, pharyngitis, cellulitis, rhematic fever etc..
Streptococcus agalactiae - important human pathogen..meningistis, pneumonia, sepsis and major cause of infections in new borns.
Bacillus cereus - Food pathogen particularly in rice. Has a Plc-PapR quorum sensing system.
Conclusion
Bacillus cereus, Streptococcus agalactiae and Streptococcus pyogenes all serve as extra pathogenic bacteria which we can detect in addition to Staph. aureus, Streptococcus pneumoniae and Listeria monocytogene. They all have very well characterized quorum sensing systems and peptides which we can detect.
21/05
The matrix.
To populate the compatibility matrix I used three methods.
Literature searching (google the parts and look through journals for conformation that they can interact/control gene expression of the connecting part)
Look at homologous structures of parts that have been confirmed to interact. If the structure is the same, they may also be compatible.
Use blast of the DNA sequence two parts together to see the sequence exists at all. This would confirm compatibility.
22/05
Find out for sure whether B. subtilis has the PapR/PlcR system like B. cereus
The expression of various chromosomal genes encoding extracellular factors (i.e., phospholipases C, proteases, cell wall proteins, enterotoxins and hemolysins) is activated by a pleiotropic regulator, PlcR, that is specific to the B. cereus group. The activity of PlcR depends on the presence of PapR, a small signaling peptide that acts as a quorum-sensing effector. PapR is exported by the bacterial cell, processed, presumably as a pentapeptide, and then reimported into the cell, where it interacts with PlcR to facilitate its binding to its DNA targets. This activating mechanism is strain specific, and this specificity is determined by the first residue of the pentapeptide.
(Slamti et al., 2005)
