Team:Dundee

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Clostridium difficile (C. diff) - associated disease of the gut is a major health problem, and current treatment methods are both ineffective and unpalatable.  Previous research identified a C. diff-specific endolysin from the phage ΦCD27, which could be used to kill C. diff cells. Type VI secretions systems, found in a variety of organisms including Salmonella typhimurium, are characterised by a needle structure, the primary component of which is encoded by the gene Hcp. The tip of the needle is encoded by VgrG. The aim of this project was to create a new type of synthetic Escherichia coli expressing a simplified version of the Type VI Secretion System, with the C. diff-specific endolysin fused to VgrG, and which could be delivered to the gut to combat serious C. diff infections. Mathematical modelling was used to assist in the biological planning and a variety of relevant software applications were made.
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<b>Six, Lyse and Obliterate: a synthetic silver bullet against healthcare acquired infection.</b><br>
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Hospital acquired infections are a global problem. One example is <I>Clostridium difficile</I>, a bacterial pathogen that infects patients undergoing prolonged antibiotic treatment and results in pseudomembranous colitis, a potentially fatal gut infection. This project aimed to design a synthetic bacterium that would respond to <I>C. difficile</I> infection and kill the pathogen <i>in situ</i>. <I>Escherichia coli</I> was engineered to secrete an endolysin from a bacteriophage that would specifically attack the <I>C. difficile</I> cell wall. The endolysin was fused to the extracellular components of an engineered Type VI Secretion System from <i>Salmonella</i>, which itself comprised 13 different proteins. In addition, a synthetic ‘inflammation biosensor’ was developed, based on a two-component system from <i>Salmonella</i>, with the aim of restricting endolysin secretion to the diseased colon only. Mathematical modelling was used to assist in the development of the laboratory work and to investigate potential therapeutic strategies beyond the scope of the experimental programme.
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  <h3 class="white">Clostridium Difficile</h3>
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    <em>Clostridium difficile (C. diff)</em> is a gram-positive bacterium that lives
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    naturally in the gut. In healthy individuals, the levels of <em>C. diff</em> &nbsp;are kept
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    constant due to competition with other naturally occurring bacterial species in the gut flora. However, when patients
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    receive large doses of antibiotics, competing gut flora can be wiped out. This can
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    allow the population of <em>C. diff</em> &nbsp;to increase to a level where infection can be caused and in some cases
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    resulting in severe colitis. <em>C. diff</em> &nbsp;has therefore become a major cause of
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    hospital acquired infections, with, for example, some 2645 patients in hospitals in
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    England and Wales found suffering from <em>C. diff</em> &nbsp;induced colitis in
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    March-May 2010.
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    Infection rates have also been high at Dundee's Ninewells Hospital,
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    which is affiliated with the University of Dundee. As a result, in 2010 a ward at the hospital was closed following the deaths of 5 elderly patients due to <em>C. diff</em> &nbsp; infections. So for us, this is also a very
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    local health problem. Up until now, there have been two ways of treating this problem:
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    prescribe more antibiotics, with the added difficulty of possibly causing more
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    resistance to build-up, or by means of faecal transplant. A faecal transplant
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    involves the faeces of a closely related person being transplanted directly into the patient's
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    colon or through a drip into the stomach. This has been proven to be effective in
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    test cases, but is obviously an unsavoury form of treatment for many patients and
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    so the idea of creating an alternative gave rise to this project.
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    <h3 class="black">Clostridium Difficile</h3>
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        <img src="https://static.igem.org/mediawiki/2012/5/50/Dundee12cdiff.jpg" alt="C.Diff microscopy image" width="415px" />
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    Micrograph depicting Gram-positive <em>C. diff</em> bacteria using a .1µm filter.
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    Public Domain : Obtained from CDC image library (http://phil.cdc.gov)
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    <h3 class="black">Type VI Secretion System</h3>
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alt="Type VI Secretion System image, courtesy of Eric Cascales" width="380px" />
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    Type VI Secretion System image, courtesy of Eric Cascales.
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                    &copy; Eric Cascales, reproduced with kind permission.
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             <h3 class="white">Type VI Secretion System</h3>
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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 <em>Serratia</em> species, <em>Vibrio cholerae</em> and <em>Pseudomonas aeruginosa</em>. A type VI secretion system has also been found in <em>Salmonella typhimurium</em>, which is closely related to <em>Escherichia coli (E. coli)</em>. The proteins for <em>Salmonella</em> type VI secretion systems are encoded by more than 13 genes, including <em>Hcp</em>, 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 <em>VgrG</em>. 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.
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            <h3 class="white">Project Summary</h3>
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    The aim of our project is to create a new type of synthetic <em>E. coli</em>, expressing a type VI secretion system that will incorporate a <em>C. diff</em> specific endolysin fused with the needle. It is hoped that the needle will either be able to puncture the <em>C. diff</em> cells and secrete the endolysin directly into the organism. We hope to prove that this engineered strain of <em>E. coli</em> will be able to produce enough needles on the cell surface to interact with and kill <em>C. diff</em> cells and that enough endolysin is secreted to optimise this killing. We hope to carry out in vitro experiments to test this hypothesis. In addition to this main experiment, we hope to do some proof of theory experiments which will involve fusing endolysin with Hcp and VgrG in <em>Salmonella typhimurium</em>.
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            <h3 class="white">Mathematical Modelling</h3>
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    The depth and range of the mathematical modelling will evolve throughout this project, from models of growth for <em>Salmonella</em> and the strain of <em>E. coli</em> that we will engineer, to systems ordinary and partial differential equations that will show us not only how two populations interact, but how the shape and natural processes of the colon affect this interaction.
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Other aspects that we will look at modelling are the number of "needles" that we can engineer the <em>E. coli</em> to create and the amount of endolysin that must be secreted to lyse all of the <em>C. diff</em> bacteria. Using software such as MATLAB® and COMSOL® we are able to create visual representations of population interactions such as graphs and animations of the colon as we introduce the <em>E. coli</em> to lyse the excessive levels of <em>C. diff</em>.
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    <h3 class="white">Software Development</h3>
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    Beyond the team's Wiki and blog site, ongoing work is taking place to develop an Android based application framework. It shall encompass any tools that the team finds useful, and will be released at the end of the project so that it may be utilised as a starting point for other similar projects.
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The team is also working on a desktop modelling tool that utilises the principles of cellular automata
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<a href="https://2012.igem.org/Team:Dundee/References" target="_blank">[4]</a> to graphically illustrate the cell interactions that are taking place between the modified <em>E. coli</em> and <em>C. diff</em> cells. The application currently models cell mitosis, movement, lysis and flow. Work on the application is continuing to encompass further aspects such as nutrients, colony sizes and initial placement of cells within the model. The completed application, including source code, will be released for Windows and Linux development environments to be used by others in the future for practical and educational purposes.
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Latest revision as of 21:03, 26 September 2012


Six, Lyse and Obliterate: a synthetic silver bullet against healthcare acquired infection.
Hospital acquired infections are a global problem. One example is Clostridium difficile, a bacterial pathogen that infects patients undergoing prolonged antibiotic treatment and results in pseudomembranous colitis, a potentially fatal gut infection. This project aimed to design a synthetic bacterium that would respond to C. difficile infection and kill the pathogen in situ. Escherichia coli was engineered to secrete an endolysin from a bacteriophage that would specifically attack the C. difficile cell wall. The endolysin was fused to the extracellular components of an engineered Type VI Secretion System from Salmonella, which itself comprised 13 different proteins. In addition, a synthetic ‘inflammation biosensor’ was developed, based on a two-component system from Salmonella, with the aim of restricting endolysin secretion to the diseased colon only. Mathematical modelling was used to assist in the development of the laboratory work and to investigate potential therapeutic strategies beyond the scope of the experimental programme.




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