TRANSPOSON-DERIVED BRUCELLA-ABORTUS ROUGH MUTANTS ARE ATTENUATED AND EXHIBIT REDUCED INTRACELLULAR SURVIVAL

The O antigen of Brucella abortus has been described as a major virule nce determinant based an the attenuated survival of fortuitously isola ted rough variants, However, the lack of genetic definition of these m utants and the virulence of naturally occurring rough species, Brucell a ovis and Brucella canis, has confused interpretation, To better char acterize the role of O antigen in virulence and survival, transposon m utagenesis was used to generate B. abortus rough mutants defective in Cd-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion mas used to verify insertion in genes encoding lipopoly saccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected dif ferences among the mutants were observed. In an effort to define the b asis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. AII elf the B. abortus rough mutants exhibited a 4 - to 5-log-unit increase, compared to the smooth parental strain, in s ensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent wi th an inability of O antigen to exclude relatively small molecules. Se nsitivity to polymyxin B, which was employed as a model cationic, amph ipathic peptide similar to defensins found in phagocytic cells, reveal ed survival differences among the rough mutants similar to those obser ved in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin Hf and reduced survival in mice. This mutant was characte rized by a truncated rough LIPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomu tase (pmm), suggesting that this activity may be required for the synt hesis of a full-length core polysaccharide in addition to O antigen. B . abortus O antigen appears to be essential for extra-and intracellula r survival in mice.