Transposon-Derived Brucella abortus Rough Mutants Are Attenuated and Exhibit Reduced Intracellular Survival

Abstract

The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential for extra- and intracellular survival in mice.

Internal affairs: investigating theBrucellaintracellular lifestyle

“…The LPS O-polysaccharide crucially impacts outcome of infection with B. abortus, B. melitensis and B. suis. Strains bearing mutations in several genes involved in various stages of the LPS biosynthesis pathway such as the mannosyltransferase wbdA (Kohler et al, 2002) or the phosphomannomutase manB (Allen et al, 1998;Foulongne et al, 2000;Kohler et al, 2002) and whose mutation generally result in a 'Rough' phenotype are avirulent in cellular or animal infection models (Roop et al, 1991;Cheville et al, 1992;Winter et al, 1996;Allen et al, 1998;Elzer et al, 1998;Godfroid et al, 1998;McQuiston et al, 1999;Foulongne et al, 2000;Hong et al, 2000;Ugalde et al, 2000;Kohler et al, 2002;Lestrate et al, 2003). The degree of this attenuation depends on the particular gene and pathway affected.…”

The Brucella abortus host factor I (HF‐I) protein contributes to stress resistance during stationary phase and is a major determinant of virulence in mice

“…Smooth-type lipopolysaccharide (LPS) was demonstrated using crystal violet dye exclusion (Alton et al, 1975) by B. abortus colonies on Schaedler agar (SA). This phenotypic property is important with regard to subsequent studies in vivo as the intact O-side chain of the LPS appears to be a major determinant of virulence for B. abortus (Allen et al, 1998), and B. abortus has the capacity to exhibit smooth to rough transition.…”

Identification and characterization of in vivo attenuated mutants of Brucella melitensis

“…To analyse the phenotype of the mutants further, in vitro assays were performed. Expression of complete LPS molecule is known to be required for full virulence of Brucella (Allen et al, 1998;Godfroid et al, 1998;McQuiston et al, 1999). Therefore, crystal violet colony staining was used to detect rough mutants (White, 1951).…”