Scientific Abstract

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

The etiologies of human inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) remain unknown. There is compelling evidence from animal models that a loss of immunologic tolerance to the gut microflora in genetically susceptible hosts leads to chronic IBD.

In most experimental animal models of chronic colitis, T-helper 1 (Th1) CD4+ lymphocytes secreting high levels of interferon (IFN-γ) mediate disease. However, the individual bacterial species critical in stimulating this response in Specific Pathogen Free (SPF) IBD animals are not known. We have demonstrated previously that the composition of the gut microflora generated by PCR/denaturing gradient gel electrophoresis (DGGE) changes in the large bowel of HLA-B27 rats and in that of interleukin-10-deficient mice as they age and develop colitis. Since in both of these experimental animal models bowel inflammation develops in the presence of gut microflora profiles that are radically different, we postulate that the important factor in the etiopathogenesis of disease is the nature of the bacterial products to which the immune system preferentially reacts.

While hundreds of bacterial species are believed to reside in the gut, most of them cannot be cultured in the laboratory. Recent developments in functional genomics provide a tool by which gut bacteria could be identified. The genomes of the collective microbes found in nature are termed the “metagenome.” Large fragments of DNA extracted from the gut contents of HLA-B27 transgenic rats and IL-10 gene knockout mice will be cloned using bacterial artificial chromosome (BAC) (single-copy vectors). These vectors can be used to maintain very large DNA inserts (>100,000 basepairs) in Escherichia coli. In addition, low copy number plasmids will be used to derive metagenomic clone libraries in Bacillus subtilis and/or Lactococcus lactis. Because even whole gene operons may be present in these large inserts, expression of these operons and subsequent processing of their gene products can occur in the cloning host. Therefore, BAC libraries of DNA, representing hundreds of genomes, will be screened for the production of substances that stimulate IFN-γ production in an in vitro assay system.

CD4⁺ T-cells will be enriched from mesenteric lymph node cell preparations from interleukin-10-deficient mice or HLA-B27 rats by negative selection to remove B cells and CD8⁺ T-cells using magnetic beads coated with appropriate antibodies. T-cell depleted antigen presenting cells will be prepared from spleens by complement-mediated lysis. After pulsing the antigen presenting cells with pools of 100 BAC library gene expression products overnight, the cells will be washed to remove non-processed antigen, then incubated for 3 days with purified CD4+ mesenteric lymph node cells. IFN-γ will be measured in supernatants by ELISA.

We aim to identify the bacterial products to which the immune systems of HLA-B27 transgenic rats and IL-10-deficient mice react preferentially. The information will permit the future development of sophisticated immunological models of IBD.