Identification of novel gene products associated with virulence of Brucella abortus using gene discovery and targeted approaches.

摘要

Brucella abortus, the causative agent of brucellosis, results in fetal abortions in domestic livestock and wildlife, and is also a potential pathogen for use as a bioterrorist agent against humans. To date, little is known about the virulence determinants and pathogenesis in vivo of this organism because it appears to lack many common virulence factors found in other gram-negative facultative intracellular pathogens. Additionally, Brucella spp. have evolved to survive in different in vivo host niches, the mechanisms by which this specificity is defined are still uncertain. To address these deficiencies, we conducted experiments using gene discovery methodologies and targeted genetic approaches to identify novel virulence loci that might serve important roles in the disease processes of brucellosis.;Using the IVIAT methodology (In Vivo Induced Antigen Technology), we have identified ten in vivo induced (IVI) genes whose products are immunogenic in Wyoming elk infected with B. abortus. From the ten loci, three genes were selected for further study. The three selected genes are functionally diverse, encoding various products including; a metabolic enzyme, an ion transporter component, and an outer membrane protein. Two additional genes identified by targeted genetic studies were also selected for evaluation, these genes are predicted to encode; a metabolic enzyme and an auto-secreted protein. Interestingly, the auto-secreted protein was found to be reactive with antibody from elk sera. All five genes were cloned and expressed as recombinant His-tagged proteins in E. coli, purified, and used to immunize mice in a murine brucellosis model. Analyses of the immune animals' response to infection indicated that one of the five IVI antigens elicited a powerful immune response. Remarkably, malate dehygrogenase, a metabolic enzyme induced a Th2-like response in the mice that promoted clearance of B. abortus S19 from animals. Further study of these IVI genes may help the development of a new recombinant vaccine for the prevention of brucellosis.