Lay Summary
Proposal No. IBD-0073
Principal Investigator: Gerald Tannock, Ph.D.
Applicant Organization: University of Otago (Dunedin, New Zealand)
Project Title: Cloning the gut metagenome: a strategy to detect pro-inflammatory substances produced by intestinal bacteria
Period of Award: September 1, 2003 - December 31, 2006
Inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) result from an abnormal reaction by the immune system to the presence of bacteria that are normally residents of the bowel. Usually, the presence of these bacteria is tolerated by the immune system, but in IBD loss of tolerance results in continuous inflammation of the bowel wall. The particular bacteria or bacterial products against which the immune system reacts is not known. The response of the immune system has been studied extensively, however, and is characterized by the production of a pro-inflammatory substance called gamma interferon (IFN-γ).
Two kinds of experimental animals have been used in IBD research to understand the interactions of gut bacteria and the immune system. Rats that carry the HLA-B27 gene of humans (transgenic rats) develop inflammation of the colon and other body sites. Similarly, mice whose gene encoding a component of the immune system (interleukin-10) has been deleted suffer from inflammation of the large bowel.
The bowel of these animals is home to hundreds of different types of bacteria, but only a few can be grown and studied in the laboratory. We have shown previously that the bacterial collection (gut microflora) can be described using analytical methods that are DNA-based (molecular methods). The gut microfloras of HLA-B27 rats and interleukin-10-deficient mice are very different, yet the immune system of these animals produces a similar inflammation of the bowel wall.
We propose that, although the compositions of the gut microfloras of the two kinds of animal are different, similar bacterial products are produced and that the immune system reacts against these.
We want to detect the bacterial products that the immune system reacts against. This will be done by extracting bacterial DNA from the gut contents of HLA-B27 transgenic rats and interleukin-10-deficient mice. Large pieces of DNA will be inserted (cloned) into carrier DNA molecules (plasmids) that will be introduced into the cells of a bacterium called E. coli. The large pieces of DNA will have come from the chromosomes of the members of the gut microflora, so we will build gene libraries representing all of these gut bacteria in E. coli. The E. coli will make products coded for by the cloned genes, and we will test these products in the laboratory to see which ones stimulate immune cells to produce IFN-γ. We will also make gene libraries in other kinds of bacteria to ensure that we provide appropriate conditions for a wide range of bacterial products to be made.
These experiments will identify the bacterial substances that the immune system reacts against in these animal models of IBD, and will help us develop better research tools to study these diseases.