Structure-Function Relationship of Lipid A in Campylobacter Rectus Campylobacter rectus is a grarn-negative, anaerobic, oxidase-positive, motile rod-shaped bacterium which has been implicated as a potential pathogen of chronic adult periodontitis and periodontal disease of immunosuppressed patients such as those suffering from diabetes mellitus, AIDS, and Crohn's disease. Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, is a virulence factor in those bacteria associated with periodontal disease as it is able to penetrate gingival epithelial tissue and stimulate bone resorptive activity. Furthermore, it has been established that the endotoxic properties of LPS are mediated by their lipid A component. Chemical and biological characterization studies have shown that lipid A, at 37.2% of the dry weight, is the single most abundant component of LPS in C. rectus. We hypothesize that the lipid A and its associated core oligosaccharide may be important in the elicitation of prostoglandins observed in the host upon infection with C. rectus. It is the intent of this project to determine the structure of the lipid A in C. rectus in order to provide insight into relationships between its chemical structure and biological functions. To achieve this goal, batch cultures of C. rectus were grown in mycoplasma-formate-fumarate broth, and the LPS isolated using a cold MgC12-ethanol precipitation. From 1.85 g of cells (dry weight), 160.7 mg of LPS was recovered; a yield of 8.7%. Thepresence of LPS was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). C. rectus LPS banding patterns, typical of those established by Gillespie, et. al. (I & I, 1988), were observed when the gels were stained with Coomassie blue and silver stain. A Coomassie blue protein assay using bovine serum albumin (BSA) standards revealed no significant protein within the LPS sample, while UV spectrometry at A260-280 determined no nucleic acid contamination. Following a Bligh-Dyer extraction using a solution of chloroform: methanol (2:1), thin layer chromatography revealed LPS sample free of phospholipids. 2-keto-3-deoxyoctonate (KDO), a sugar unique to LPS, was quantified by a colorimetric assay at A548, and found to be present at 0.45% of the dr weight of LPS. In subsequent procedures, lipid A will be extracted by hydrolysis of LPS with 1% (volvol) acetic acid and the structure elicited by 13C- and 31P-NMR. The structure of the lipid A component of C. rectus resulting from this study will provide insight into relationships between the structure and endotoxic functions. Results will also have taxonomic relevance to establish if this species shows homology with the unique features of the lipid A structure described in Campylobacter jejuni.

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National Institute of Dental & Craniofacial Research (NIDCR)
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State University of New York at Buffalo
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