Meeting with Prof Selkirk
This will be at 1pm at Base Camp (3.03 RSM) and anyone can come!
- The specificity of the protease is fundamental to the detection system.
- The sensitivity is also really important; it will affect the volume of water you need to test. The sensitivity of the cercariae for the lipids is not an issue (they're extremely sensitive) but the actual concentration of the proteases produced by them may not be high enough.
- We wondered if we could engineer our bacterium to produce the induction lipids on it's surface, but this may prove difficult. However, as certain lipids are chemoattractant, it may help to draw the cercariae close to the bacterium and so increase the protease concentration close to the bacterium's surface.
- If we can use a single lipid to induce the invasive behaviour of the cercariae, this may help us when we are quantifying the results.
- Elastase is the major protease secreted by cercariae, but there are others, depending on the stage of the infection and the glands they are stored in. But as we want a fast response, we will probably focus on the elastase (which is the first protease to be secreted).
- Assaying the protease activity would be very important. Prof Selkirk said he may be able to get hold of the actual schistosoma elastase, which would be fantastic.
- The specific cleavage site of elastase is after a lysine residue.
- Our membrane-anchored protein could have, for example, many FMLP sequences (a common autoinducer) separated by a linker, which would contain a cleavage site for the cercariae protease.
- Other water-borne parasites that might use proteases would be Fasciola and hookworm.
- We discussed cryptosporidium, amoeba and other parasites that are ingested, and whether or not we could induce invasive behaviour in them. However, this is likely to take a long time (possibly hours) and as we want to focus on a fast response detection, we will not concentrate on these parasites.
- We wondered if there would be other similar proteases present, and whether we would get false read-outs. However, this seems to be unlikely.
- On the other hand, other species of schistosoma that infect birds and cattle might also secrete proteases and give a false read-out.
- Some species of schistosoma secrete different proteases. However, they are usually located in different parts of the world and so this shouldn't be too much of an issue.
- The elastase is around 37kD and is synthesised as a proenzyme. It may be calcium dependent. This should be considered when we assay it.
- If we could amplify the signal using a positive feedback loop, we would get a much stronger response. Wolf suggested that our bacterium could signal to each other after detecting the peptide and so induce a stronger response.
- As a final step, we could use the cercariae from the Natural History Museum to measure the sensitivity of the system.
- Before that, we could use a recombinant elastase to test it.
We are very grateful to Prof Selkirk for his time and guidance.