Micrograph depicting Gram-positive C. diff bacteria using a .1µm filter.
Public Domain : Obtained from CDC image library (http://phil.cdc.gov)
Clostridium Difficile
Clostridium difficile (C. diff) is a gram- positive bacterium that lives naturally in the gut. In healthy individuals, the levels of C. diff are kept constant as it is outcompeted by other species in the gut flora. However, when patients receive large doses of antibiotics, competing gut flora can be wiped out. This can allow numbers of C. diff to increase to a level where infection can be caused, resulting in severe colitis. C. diff has therefore become a major cause of hospital acquired infections, with, for example, some 2645 patients in hospitals in England and Wales found suffering from C. diff induced colitis in March- May 2010. Infection rates have also been high at Dundee's Ninewells Hospital, which is affiliated with the University of Dundee, and so for us, this is also a very local health problem. Up until now, there have been two ways of treating this problem: prescribe more antibiotics, with the added difficulty of possibly causing more resistance to build-up, or by means of faecal transplant. A faecal transplant involves the faeces of another person being inserted directly into the patient's colon. This has been proven to be effective in test cases, but is obviously an unsavoury form of treatment for many patients and so the idea of creating an alternative gave rise to this project.
Type VI Secretion System
Many bacterial species have evolved secretion systems Type VI secretion systems are naturally found in gram negative organisms, including Serratia species, Vibrio cholerae and Pseudomonas aeruginosa, but a putative type VI 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 feature of the system, the needle. This projects through the periplasm and outer membrane, secreting effector molecules, via the tip protein which is encoded by the gene VgrG. Hcp and VgrG are largely conserved across all species expressing these systems. The secreted effector molecules are thought to play a role in the pathogenesis of higher organisms and could help facilitate interactions with other bacteria.
Project Summary
The aim of our project is to create a new type of synthetic E. coli, expressing a simplified type VI secretion system that will secrete a C. diff specific effector, endolysin. It is hoped that the needle will either be able to puncture the C. diff cells and secrete the endolysin directly into the organism or that the endolysin will come into contact with the C. diff cell surface after secretion. We hope to prove that the new strain of E. coli will be able to produce enough needles on the cell surface which can then come into contact with C. diff cells and that enough endolysin is secreted to kill them, so in vitro experiments will be carried out to test this hypothesis. Experiments will also be set up that show that our strain of E. coli is able to out-compete C. diff, resulting in reduced numbers of the bacterium and so less chance of infection.
Mathematical Modelling
The depth and range of the mathematical modelling will evolve throughout this project, from models of growth for Salmonella and the strain of E. coli 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.
Other aspects that we will look at modelling are the number of "needles" that we can engineer the E. coli to create and the amount of endolysin that must be secreted to lyse all of the C. diff 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 E. coli to lyse the excessive levels of C. diff.
Software Development
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.
The team is also working on a desktop modelling tool that utilises the principles of cellular automata to graphically illustrate the cell interactions that are taking place between the modified E. coli and C. diff 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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