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.

FIG. 1

Deduced amino acid sequence of Tn5-flanking DNA from the B. abortus rough mutants aligned with amino acid sequences from GenBank. (A) Alignment of CA180 ORF product with Rhizobium sp. strain NoeI and Vibrio cholerae PMM; (B) alignment of CA353 ORF product with V. cholerae RfbD-like gene product and V. cholerae oxidoreductase (oxido.); (C) alignment of CA533 ORF product with V. cholerae RfbV and an outer membrane protein (OMP) precursor of V. cholerae; (D) alignment of CA613 ORF product with RfbA of P. aeruginosa and TrsF of Y. enterocolitica. Conserved amino acids are indicated by vertical lines, and similar amino acids are indicated by colons. Numbers at the beginnings and ends of sequences indicate the positions of published amino acids. Dashes indicate spaces inserted to improve alignment. Alignments were performed with CLUSTAL V. See the text for complete descriptions of the genes.

FIG. 1

Deduced amino acid sequence of Tn5-flanking DNA from the B. abortus rough mutants aligned with amino acid sequences from GenBank. (A) Alignment of CA180 ORF product with Rhizobium sp. strain NoeI and Vibrio cholerae PMM; (B) alignment of CA353 ORF product with V. cholerae RfbD-like gene product and V. cholerae oxidoreductase (oxido.); (C) alignment of CA533 ORF product with V. cholerae RfbV and an outer membrane protein (OMP) precursor of V. cholerae; (D) alignment of CA613 ORF product with RfbA of P. aeruginosa and TrsF of Y. enterocolitica. Conserved amino acids are indicated by vertical lines, and similar amino acids are indicated by colons. Numbers at the beginnings and ends of sequences indicate the positions of published amino acids. Dashes indicate spaces inserted to improve alignment. Alignments were performed with CLUSTAL V. See the text for complete descriptions of the genes.

FIG. 2

Immunoblot of whole-cell lysates from B. abortus smooth and rough strains electrophoresed on a 12% (wt/vol) gel, transferred to a polyvinylidene difluoride membrane, and reacted with anti-O-antigen antibody (MAb 39). Lane 1, S2308; lane 2, S19; lane 3, RB51; lane 4, CA613; lane 5, CA533; lane 6, CA353; lane 7, CA180. Positions of molecular mass markers (in kilodaltons) are shown on the left.

FIG. 3

Immunoblot of whole-cell lysates from B. abortus smooth and rough strains electrophoresed on a 12% (wt/vol) gel, transferred to a polyvinylidene difluoride membrane, hydrolyzed in 10% acetic acid, and reacted with anti-lipid A antibody (MAb 177). Lane 1, molecular mass markers (sizes in kilodaltons are shown on the left); lane 2, S2308; lane 3, S19; lane 4, RB51; lane 5, CA613; lane 6, CA533; lane 7, CA353; lane 8, CA180.

FIG. 4

Complement-mediated killing of B. abortus strains in bovine sera. Complement-mediated killing was performed as described in Materials and Methods. Bacterial counts are reported as log CFU/ml (fresh sera) − log CFU/ml (heat-inactivated sera). The results represent means from four independent assays (P < 0.0001).

FIG. 5

Percent survival of B. abortus strains in bovine peripheral blood macrophages. Macrophage-mediated killing was performed as described in Materials and Methods. Results are expressed as percent survival in comparison to that of S2308 and represent the averages from three independent trials (P < 0.05).

FIG. 6

Bactericidal effect of PmB on B. abortus strains. PmB-mediated killing was performed as described in Materials and Methods. Average results of three assays are expressed as percentages of the brucellae surviving in wells incubated in the absence of PmB. Asterisks indicate statistically significant results as noted in the text. Symbols: ▪, S2308; •, RB51; ▴, CA180; ⧫, CA353; □, CA533; ○, CA613.