IGEM:Imperial/2010/Detection module/Signal Peptide Bearing Protein

The signal peptide bearing protein (SPBP):

In order to set off our signalling cascade in the presence of the cercaria, we have to design a protein that carries our signal peptide out of the cell where the protease has access to it. The protease can then proceed to cleave the peptide (or multiple peptides) off the protein, allowing quorum sensing to take place. One big problem we have to overcome is the cell wall that will obstruct the protease’s access to the SPBP.

The Secretion System (Sec):

The system responsible for secretion of proteins out of the cell seems to be partially conserved across both bacteria and eukaryotes and is called Secretion system or short “Sec”. In B. subtilis the following six components form the sec-dependent secretion machinery:

1. Cytosolic Chaperones

The Ffh protein, a GTPase, forms a complex with a small cytosolic RNA forming the Signal Recognition Particle (homologous structures are found in E. coli and S. cerevisiae). It ensures that the protein to be exported remains a secretion competent i.e. linear form by binding to it as it emerges from the ribosome (co-translational export). Unlike in eukaryotes, translation is not arrested until the N-Terminus reaches the membrane but continues as normal. SRP is also thought to be involved in targeting the peptide to the secretion pore and motor. However the protein FtsY plays the most important part in targeting. There is evidence for a second, post-translational, route of protein secretion via CsaA which targets preproteins to SecA. CsaA shows affinity to preproteins and exhibits some chaperone-like activity.

1. Translocation motor

In B. subtilis the gene which was originally called div and was identified to be crucial for cell division and sporulation, turned out to be a homologue of E. coli’s SecA protein. SecA is an ATPase responsible for translocation of the preproteins through the translocation channel out of the cell.

1. Translocation channel

A heterotrimeric complex of the membrane proteins SecY, SecE, and SecG forms the main core of the translocation channel. SecE and signal peptides bind to the same or overlapping regions in SecY and SecE probably functions as a surrogate signal peptide when the SecY channel is in its closed form in the absence of a translocating protein. When a signal peptide is present, it displaces SecE and allows opening of the channel. SecG is not strictly required for preprotein translocation and cell viability but it is necessary for efficient translocation, possibly by facilitating the movement of preproteins through the translocation channel in concert with the insertion and deinsertion cycles of SecA. Deficiency caused secretion defects that resulted in cold-sensitive growth in B. subtilis as well as E. coli.

Unlike other organisms B. subtilis has a natural fusion protein of two components called SecDF. This protein as well is not essential for cell viability but it is thought the protein regulates SecA activity and might also be involved in translocase assembly and clearing of signal peptides from the channel.

1. SPases:

1. SPPases:

1. Folding factors (on trans site):

Transmembrane protein:

Initially we considered a fusion of some transmembrane protein with our signal peptide sequence. There are several problems to consider: The N-terminus cannot be modified as it specifies export via the Sec system, so only the C-Terminus would be available for modification. This implies that a protein would have to be chosen that has an exposed C-Terminus from which the proteases could remove the signal peptide easily. Additionally the cell wall problem would not be solved by this.

Flagellin:

This protein is the most abundant subunit of flagella and makes up most of the structure. The advantages this protein would have to offer is that it is abundantly synthesised and transported is through the cell wall where it would be accessible to the cercarias’ proteases. However structural studies have shown that both C and N-Temini of flagellin are inside the flagellar structure and would thus not be accessible. I believe that modifying the structure of flagellin so that one of the termini is exposed is not feasible and would probably not work as the structure of flagellin is highly conserved across bacterial species, suggesting that deviation from the natural structure is not functional.

Sortase:

Sortases are proteins used by Gram positive bacteria to attach specific proteins to their cell wall. B. subtilis has two of these – YhcS and YwpE – but they are not well characterized. S. Aureus however has one – SrtA – which has been tested and is well understood. Sortases recognize a sequence on the C-Terminus of a protein and attach it covalently to the cell wall. About 20 proteins are attached to the cell wall in this way naturally by S. aureus, for example the IG-binding protein A, but other proteins have successfully been attached to the cell wall by creating a signal peptide protein fusion. The sortases themselves are membrane bound via their N-Termini which suggests to me that they will not add the proteins on the outside of the cell wall, however the function of some of these proteins suggests otherwise. Unfortunately I have not been able to determine the localization of the proteins yet. SrtA has a well characterized specificity for LPXTG motifs which it recognizes with its catalytic TLXTC domain near the C-Terminus. Alternatively to SrtA of S. aureus, close relatives to B. subtilis, such as B. anthacis also use sortases of known specificity.

I think this approach is the most promising as it seems best suited to overcome the cell wall problem and also because we might be able to target a synthetic protein (that only contains the secretion sequence on its N-Terminus and the sorting signal at its C-Terminus with many signal peptide-protease cleavage site repeats in the middle) to the cell wall.