Team:Dundee/Solution

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Continuing with the idea of reintroducing the natural gut bacteria to the colon by a faecal transplant in order to combat Clostridium difficile (C. difficile), our project aims to engineer a harmless Escherichia coli (E. coli) strain to target and kill C. difficile during infection of the large intestine. Our idea is to take a type VI secretion system (T6SS) from Salmonella typhimurium LT2 and express it in E. coli. In addition, we want to isolate a gene from a bacteriophage that encodes a C. difficile specific endolysin and fuse this gene with genes from the T6SS. In order to control the system, we will incorporate a synthetic inflammation biosensor which will ensure that the system is only activated during times of infection.

Type VI Secretion System image, courtesy of Eric Cascales.
© 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 products of these genes, Hcp and VgrG, are known to universally secreted when the system is present. As a result, these proteins will be fused with the endolysin. These proteins form the needle and the tip of the system respectively. The other gene products are membrane proteins and thought to be involved in the assembly and function of the system. 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.

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-                 Many bacterial species have evolved various types of secretion system. Type VI secretion systems are naturally found in gram negative organisms, including Serratia species, Vibrio cholerae and Pseudomonas aeruginosa. A type VI secretion system has also been found in Salmonella typhimurium, which is closely related to Escherichia coli (E. coli). The proteins for Salmonella type VI secretion systems are encoded by more than 13 genes, including Hcp, which encodes for the main structural component of the needle. This projects through the periplasm and outer membrane and can inject directly into competing cells, via the tip protein which is encoded by the gene VgrG. In this way, the type VI secretion system punctures other cells. Hcp and VgrG are largely conserved across all species expressing these systems. TypeVi secretion systems can also be associated with secreted effector molecules, these are thought to play a role in the pathogenesis of higher organisms and could help facilitate interactions with other bacteria.
+                 Continuing with the idea of reintroducing the natural gut bacteria to the colon by a faecal transplant in order to combat <I>Clostridium difficile</I> (<i>C. difficile</I>), our project aims to engineer a harmless Escherichia coli (E. coli) strain to target and kill <i>C. difficile</I> during infection of the large intestine. Our idea is to take a type VI secretion system (T6SS) from <I>Salmonella typhimurium</I> LT2 and express it in E. coli. In addition, we want to isolate a gene from a bacteriophage that encodes a <i>C. difficile</I> specific endolysin and fuse this gene with genes from the T6SS. In order to control the system, we will incorporate a synthetic inflammation biosensor which will ensure that the system is only activated during times of infection.
 <br><br><center><img src="https://static.igem.org/mediawiki/2012/5/55/Solutionpic3.jpg"><br>
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 © Eric Cascales, reproduced with kind permission</div>
 </center><br>
+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 <i>C. difficile</I> 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 <I>Vibrio cholera</I> and <I>Pseudomonas aeruginosa</I>. 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 products of these genes, Hcp and VgrG, are known to universally secreted when the system is present. As a result, these proteins will be fused with the endolysin. These proteins form the needle and the tip of the system respectively. The other gene products are membrane proteins and thought to be involved in the assembly and function of the system.
+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 <i>C. difficile</I>. 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 <i>C. difficile</I>.
+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.