Characterization of the Brucella abortus rfb gene locus and the role of O-antigen in the resistance to complement lysis and macrophage defense mechanisms

Abstract

In Gram-negative organisms, the lack of 0-antigen expression on the lipopolysaccharide molecule produces a rough phenotype which is associated with attenuation. Transposon mutagenesis of Brucella abortus with Tn5 generated several rough (rfb) mutants which do not express detectable 0antigen on their outer membrane surface. Initial sequencing of Tn5 flanking DNA revealed interruptions within genes encoding proteins with homology to gene products of the r)$ locus which participates in 0-antigen biosynthesis. Complement and macrophages represent two major host defense mechanisms controlling extracellular and intracellular killing of invading organisms. The rfb mutants were equally sensitive to bovine complement. Bactericidal assays, including sensitivity to polymixin B, hydrogen peroxide, and murine and bovine macrophages, revealed distinct differences in sensitivity among the rib mutants. Similar differences in sensitivity among the rfb mutants were also recorded in the BALB/c mouse model. The difference in attenuation among the rfb mutants, and the inability to complement the mutation, may be due to pleiotropic effects. This data suggests that the survival of Brticella is controlled by intracellular killing mechanisms.