Identification of the genes encoding for the immunologic proteins of Bacteroides forsythus
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Bacteroides forsythus is strongly linked epidemioIogically to periodontitis and to advanced as well as recurrent periodontitis. However very few studies have investigated the role of virulence determinants in B. forsythus that might contribute to its pathogenesis. This is partly due to its fastidious growth requirements. In order to overcome this limitation, a genomic library of B. forsythus ATCC 43037 was screened with pooled human antiserum from both healthy individuals and from patients who exhibiited various periodontal diseases. Fifteen immunoreactive plaques were identified and rescued as phagemids. Escherichia coli containing these phagemids were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Westem blot analysis using the pooled human antisera. Three phagemids (7/30, 5/28 and 1/30) which exhibited immunoreactivity by Westem blot analysis were further analysed by EZ: :TN transposon mutagenesis and subcIoning to Iocalize the open reading frame (ORF) responsible for its immunoreactivity. The ORF responsible for the immunoreactivity of 7/30 was localized and the 49-kDa protein was expressed using the pET vector system. E. coli over expressing the 49-kDa protein was then screened with individual human antisera from both healthy and diseased individuals by Western blot analysis and the results were inconclusive. The 49-kDa protein appears to have very low homoIogy with an IgA protease in E. coli but its putative role in B. forsythus pathogenesis needs further investigation. The ORF responsible for the immunoreactivity of 5/28 was localized to a 2.5-kb DNA insert but the ORF responsible for the immunoreactivity could not be discerned even after determining the DNA sequence of this fragment. The ORF responsible for the immunoreactivity of 1/30 was localized to a 3.2-kb DNA fragment and the protein was 120 kDa in molecular weight. This protein appears to have regions that are homoIogous to a variety of proteins in the databases, including internalin D, outer membrane fluffing adhesin protein, ATP binding protein and surface antigen from other bacteria. This 120-kDa protein appears to contain putative adhesive and virulence properties identified in other bacteria based on database analysis, and therefore merits further investigation. SDS-PAGE and Western blot analyses of E. coli containing phagemids isolated from immunoreactive plaques identified three phagemids that were analysed in this study, a strategy using a non-denaturing method to screen these phagemids may be necessary to identify additional immunoreactive proteins in the future.
Thesis (M.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 2001 (Pediatric Dentistry).PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Includes bibliographic references (leaves 78-88).
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