Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication

Abstract

Smooth lipopolysaccharide (LPS) of Brucella abortus has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. While the protective properties of LPS against complement are well accepted, there is still some controversy about the capacity of rough mutants to replicate intracellularly. The B. abortus phosphoglucomutase gene (pgm) was cloned, sequenced, and disrupted. The gene has a high index of identity to Agrobacterium tumefaciens pgm but is not part of the glycogen operon. A B. abortus null mutant lacks LPS O antigen but has an LPS core with an electrophoretic profile undistinguishable from that of the wild-type core, suggesting that glucose, galactose, or a derivative of these sugars may be part of the linkage between the core and the O antigen. This mutant is unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication. This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the cell against complement-mediated lysis, but is not involved in intracellular survival.

FIG. 1

Comparison of the B. abortus phosphoglucomutase protein with the A. tumefaciens and A. thaliana proteins. Conserved amino acids are indicated by black boxes. The alignment was performed with the MegAlign program.

FIG. 2

PAGE of B. abortus LPS. (A) PAGE with 12% acrylamide. Lane 1, wild-type LPS; lane 2, B. abortus B2211 pgm mutant LPS. (B) Tricine-PAGE. Lane 1, wild-type LPS; lane 2, B. abortus B2211 pgm mutant LPS. Gels were silver stained.

FIG. 3

Intracellular multiplication of the B. abortus wild-type strain and the B. abortus B2211 pgm mutant in HeLa cells. Cells were infected, and at the indicated times postinfection (p.i.) the number of intracellular bacteria was determined as described in Materials and Methods.

FIG. 4

Virulence in mice. Mice were infected as described in Materials and Methods. (A) Recovery of viable bacteria from spleens at 15 days postinfection. (B) Weights of spleens of infected mice at 15 days postinfection. Error bars indicate standard deviations.

FIG. 5

Effect of PmB on B. abortus strains B2211 (pgm mutant) and 2308. (A) Bactericidal effect. PmB-mediated killing was performed as described in Materials and Methods, and results are expressed as percentages of survival. (B) Inhibition of growth. The assay was carried out as described in Materials and Methods. Results are expressed as percentages of CFU recovered at the indicated concentrations of PmB. Error bars indicate standard deviations.