Team:Dundee/Solution
From 2012.igem.org
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© Eric Cascales, reproduced with kind permission
Secretion systems: Why type VI?
Type I secretion is a simple 3 protein system and was ruled out because it does not allow targeted secretion of a substrate.
Type II and type V secretion systems were also ruled out on the premise that they are unable to specifically secrete substrates to a target organism.
Type III secretion is a key system used by bacteria to deliver effector proteins to eukaryotic cells during pathogenesis. This was ruled out as we want to target C. difficile cells exclusively.
Type IV secretion is thought to be mainly associated with exchange of genetic material between cells. In addition this system can target eukaryotic cells so was ruled out.
Type VI secretion systems were discovered in 2007 in Vibrio cholera and Pseudomonas aeruginosa. In these organisms, T6S is essential for their disease-causing ability. To date, T6SS genes have been shown to be present in the genomes of most Proteobacteria. Most of the original research on T6SS focused on its role in pathogenesis of higher organisms. However, more recent work tends to focus on the broad role T6SS plays in inter-bacterial interactions. In Salmonella typhimurium, the T6SS is encoded by 13 genes. Two of the proteins in this system, Hcp and VgrG, are known to universally secreted. As a result, these proteins will be fused with the endolysin. The other gene products are membrane proteins and thought to be involved in the assembly and function of the system.
The Endolysin
The endolysin we have chosen to use is from ΦCD27. This has been shown by Mayer et. al. to specifically kill 30 strains of C. difficile. We will aim to isolate the gene and fuse it to Hcp and VgrG genes which have been cloned to not possess a stop codon. This endolysin interferes with the peptidoglycan layer of bacteria and causes the cell to lyse. By fusing this protein to proteins known to be exported during T6SS expression it should be able to target and kill C. difficile.Inflammation Biosensor
Although E. coli is a natural member of the gut flora, the presence of a T6SS may lead to increased competition with other organisms in the colon. In addition, there is a possibility that it could damage the cells of the gut epithelium. As a result, we concluded that the system must only be active in the event of infection. The biosensor we intend to use comes from the bacteria Salmonella enteritica Typhimurium.
Hydrogen sulphide is made in large quantities by colonic bacteria. This compound is highly toxic so it is converted to thiosulfate by the gut mucosa. During inflammation, thiosulfate is thought to be oxidised to tetrathionate during the migration of neutrophils and production of oxygen radicals. Thiosulfate cannot be used by Salmonella as an electron acceptor, but tetrathionate can. As a result, when the gut becomes inflamed Salmonella can alter their metabolism to use tetrathionate. The genes responsible for this phenomenon are named TtrS and TtrR. These genes code for the sensory and response regulators of this system which acts as a biosensor to ensure this metabolic pathway is only used in the presence of tetrathionate. We plan to clone this biosensor and use it to control our system in E. coli so that the secretion system and endolysin are only produced in times of gut inflammation.