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Lipopolysaccharide (LPS) is known to play a variety of important roles in the virulence of Gram-negative bacteria. It has been implicated in processes as diverse as attachment to host tissues, evasion of phagocytosis, molecular mimicry, and (most importantly) immune stimulation. Periodontal disease is the result of a chronic host inflammatory response to oral bacteria. One of the most commonly isolated microbes from the gingiva is the Gram-negative anaerobe, Fusobacterium nucleatum. Given the predominance of F. nucleatum in oral infections, it seems logical to test the role of its LPS in promoting the inflammatory response observed in advance periodontal disease. From the literature, it is clear that LPS, proteinaceous virulence factors, metabolic end products, and host factors all contribute to the tissue damage seen in periodontal disease. Furthermore, in many cases these factors may perform the same or similar functions. The complexity of this clinical situation makes it very difficult to determine causal relationships between bacterial species, their products, and tissue damage. My research is focused upon establishing a experimental system to explore the structure, function, and diversity of LPS in F. nucleatum. A clear understanding of this important molecule will allow us to determine the role(s) that it plays in forming biofilms, evading or subverting the immune response, and promoting of host tissue damage. We are pursuing this goal in the following ways.
- Analysis of LPS core composition and biosynthesis:
- Early studies of the chemical composition of Fusobacterium LPS revealed several different chemotypes. However, the exact genetic and biochemical character of this important molecule are poorly understood. We are actively working to purify and biochemically characterize the structure of the LPS core from ATCC10953.
- Generation of isogenic mutants:
- An important step toward understanding the role of LPS in virulence will be the construction of isogenic mutants. Unfortunately, the genetics of Fusobacterium are still rather rudimentary. We are attempting to create a suicide vector for F. nucleatum. This plasmid will allow us to introduce site-specific non-polar mutations into LPS core genes. The resulting strains will have truncated LPS molecules of known structure. The effect of these mutations upon viability and pathogenicity will be of great interest.
- Determination of importance of LPS in virulence:
- Once we have constructed isogenic mutants, we will be in a position to test them for alterations in pathogenicity. We plan to use a variety of assays for these analyses. Cell-free studies will measure differences in the ability to bind to host proteins (such as fibrinogen) and survival in serum. Cell culture assays will be used to determine differences in binding, internalization, and mitogenicity using murine fibroblasts and lymphocytes. Finally, I envision several small-scale mouse studies to compare promising LPS mutants to wild type F. nucleatum with regard to promotion of bone resorption in experimental oral infections.
- Genetic diversity of LPS in Fusobacterium:
- The complete genome of Fusobacterium nucleatum ssp. nucleatum (ATCC25586) has been reported. In addition, two other partial sequences (F. nucleatum ssp. polymorphum - ATCC10953- and F. nucleatum ssp. vincentii - ATCC49256) are nearing completion. Comparison of these sequences reveals that the LPS core genes are highly conserved. However, the organization and content of the O-antigen loci are very different. We are currently collaborating with Dr. Rudolph Gmür in Switzerland to determine the organization and distribution of LPS genes throughout the Fusobacterium clade.
- Possible correlation of virulence and LPS chemotypes:
- It may be that certain forms of LPS are associated with more severe clinical outcomes. We will attempt to correlate the distribution of specific LPS genes (corresponding to specific glycosyl structures) to disease severity. One reason that Fusobacterium is often dismissed as an important player in periodontal disease is its apparent ubiquity. These studies may provide evidence for sub-structure within the F. nucleatum clade with respect to pathogenicity.
