Genetic and Immunological Analyses of a Brucella abortus Protein Exhibiting Lectin-like Properties
Brucella abortus is a facultative, intracellular zoonotic pathogen, which can cause undulant fever in humans and abortion in cattle. Despite all of the progress in brucellosis research, there are still many unanswered questions regarding the molecular mechanisms involved in the pathogenesis of Brucella infections. To better understand the Brucella antigens involved in virulence and/or immunity, genetic and immunologic characterization of a 16 kDa protein of B. abortus was performed. Using PCR methods, the gene encoding the 16 kDa protein was cloned and sequenced. PCR and Southern blot analysis revealed that the gene is conserved among the 6 nomen species of Brucella. Overexpression of this protein in B. abortus vaccine strain RB51 was achieved using Brucella groE and sodC promoters as well as its own promoter. Protection and clearance studies were performed in mice to determine the role of this protein in Brucella immunity and pathogenesis. Inoculation with either strain RB51 overexpressing the 16 kDa protein or a DNA vaccine encoding the 16 kDa protein gene failed to provide significant protection. No difference was noted between the splenic clearance of B. abortus strain 2308 and its recombinant overexpressing the 16 kDa protein. A mutant of strain 2308 (2308D16) was created by disrupting the 16 kDa proteinâs gene with a chloramphenicol resistance cassette. Western blot analysis indicated that the O antigen profile of strain 2308D16 differed from that of strain 2308. Mice cleared strain 2308D16 faster than strain 2308 indicating the potential attenuation of the disruption mutant. Purified 16 kDa protein was obtained by overexpressing it in E. coli via the pRSET expression system. Western blotting results initially identified this protein as an immunoglobulin-binding protein. Hemagglutination assay revealed that the 16 kDa protein exhibits lectin-like properties. Preliminary studies using hemagglutination inhibition identified mannose as a possible sugar to which the 16 kDa protein can interact. The lectin-like properties exhibited by the 16 kDa protein appears to influence smooth lipopolysaccharide production, and thereby may be involved in virulence.
Advisor:Dr. Thomas E. Toth; Dr. Stephen M. Boyle; Dr. Gerhardt G. Schurig; Dr. Nammalwar Sriranganathan
School:Virginia Polytechnic Institute and State University
School Location:USA - Virginia
Source Type:Master's Thesis
Keywords:biomedical sciences and pathobiology
Date of Publication:02/16/2000