Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model

Abstract

Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-D-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manB(core) to distinguish it from the wbk manB(O-Ag). The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.

FIG. 1.

Surface properties of B. abortus 2308 Nal^r and derived R mutants in the genes per, wbkA, wa**, and manB_core (mutants 9.49, 2.17, 80.16, and 55.30, respectively) probed with polymyxin B, phages R/C and Wb (specific for R and S brucellae, respectively), and anti-LPS MAbs of the indicated specificities.

FIG. 2.

Physical map of the B. melitensis 16M genome regions (A to C) in which the genes homologous to B. abortus per, wbkA, wa**, and manB_core are located. The map is based on the complete genome sequence of B. melitensis 16M (GenBank accession numbers AE008917 and AE008918). Open arrows represent ORFs related to LPS biosynthesis (the names of the genes involved are shown). Solid triangles indicate the sites of the mini-Tn5 insertions in B. abortus mutants.

FIG. 3.

Exposure of the major Omps on the surface of B. abortus 2308 Nal^r and derived R mutants assessed as the reactivity of whole cells with specific MAbs.

FIG. 4.

SDS-PAGE (A) and Western blot analysis (B to F) of the LPS of strain 2308 Nal^r (1) and the per (2), wbkA (3), wa*** (4), and manB_core (5) mutants. The antibodies used were those in polyclonal sera to O-polysaccharide (B) and to the per (C) and manB_core (D) mutants or MAbs to outer core (Baro-1 [E]) and inner core (Baro-2 [F]). Lanes Ra and Rd of panel A contained the LPS of serovar Minnesota Ra and Rd mutants.

FIG. 5.

HPTLC analysis of lipid A heterogeneity of the LPS of B. abortus 2308 Nal^r (1) and of the per (2), wbkA (3), wa** (4), and manB_core (5) mutants. Lane Ec contained lipid A of E. coli ATCC 35218. hexa, Hexa-acylated; penta, pentra-acylated; tetra, tetra-acylated.

FIG. 6.

Infection kinetics in the spleens of BALB/c mice inoculated with the mini-Tn5 R B. abortus mutants and B. abortus RB51 (vertical bars represent the SDs). Symbols: ○, wbkA; ▿, per; , manB_core; ⋄, wa**; ▴, RB51.

FIG. 7.

Antibody response to surface antigens in mice inoculated with mini-Tn5 R B. abortus mutants and B. abortus RB51. Horizontal dashed lines mark the antibody levels in the blood of control mice 15 days after infection with either B. abortus 2308 or B. ovis PA. Day 0 values are from noninoculated mice. Symbols: ○, wbkA; ▿, per; , manB_core; ⋄, wa**; ▴, RB51.

FIG. 8.

Partial structure of O. intermedium strain LMG3301 core and its lipid A disaccharide.