Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells

Abstract

Null cyclic beta-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic beta-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response.

FIG. 1

TLC of cyclic β-1,2-glucans accumulated by different B. abortus strains. Cyclic β-1,2-glucans were extracted and submitted to TLC as described in Materials and Methods. Lanes: 1, B. abortus 2308; 2, B. abortus 2308 cgs mutant (BvI129 strain); 3, B. abortus strain BvI129 complemented with plasmid pBA19; 4, B. abortus S19; 5, B. abortus S19 cgs mutant (BAI129 strain); 6, B. abortus strain BAI129 complemented with plasmid pBA19. ∗ and ∗∗, migration of charged and neutral B. abortus cyclic β-1,2-glucans, respectively.

FIG. 2

Western blot of different B. abortus strains. Whole-cell lysates of different B. abortus strains were electrophoresed and transferred, and LPS O antigen was detected as described in Materials and Methods. Lanes: 1, B. abortus 2308 (wild-type strain); 2, B. abortus 2308 BvI129 (cgs mutant); 3, B. abortus S19 (vaccinal strain); 4, B. abortus S19 BAI129 (cgs mutant); 5, B. abortus RB51.

FIG. 3

Brucella persistence in spleens of mice inoculated with different strains of B. abortus. Mice were inoculated intraperitoneally as described in Materials and Methods with 10⁴ CFU of B. abortus. (A) Shaded bars, B. abortus 2308 (wild-type strain); open bars, B. abortus 2308 BvI129 (cgs mutant). (B) Shaded bars, B. abortus S19 (vaccinal strain); open bars, B. abortus S19 BAI129 cgs mutant. At different times postinfection (2, 4, or 12 weeks) five mice per group were killed and their spleens were removed. The numbers of CFU in spleen tissues were determined as indicated in Materials and Methods.

FIG. 4

Intracellular replication of different strains of B. abortus. HeLa cells were inoculated with 5 × 10⁷ CFU of different strains of B. abortus. After 2 h of incubation at 37°C, cells were washed and streptomycin and gentamicin were added as described in Materials and Methods. Numbers of CFU were determined at the indicated times. Each determination is the average of two independent experiments carried out in duplicate. In all cases, the standard error for each point was less than 5%. Symbols: ●——●, B. abortus 2308; ●----●, B. abortus BvI129 (cgs mutant strain); ○——○, B. abortus S19; ○····○; B. abortus BAI129 (cgs mutant strain); ▴——▴, B. abortus BvI129 (pBA19); ▴----▴, B. abortus BAI129(pBA19).

FIG. 5

Quantitation of IFN-γ in spleen of infected mice. BALB/c mice were infected with 10⁴ CFU of B. abortus S19 (shaded bars) or B. abortus cgs mutant (open bars). At different times postinfection, spleens were removed and total RNA was extracted and analyzed separately. (A) Quantitation of IFN-γ mRNA was carried out by RT-PCR as described in Materials and Methods. Error bars indicate standard deviations. (B) Agarose gel of the amplicon products from three mice at each time point. After the gel dried, radioactivity was detected by autoradiograph.

FIG. 6

Pattern of immunoglobulins elicited by B. abortus S19 or the B. abortus BAI129 cgs mutant. KELA results for pooled sera from five mice inoculated with 7 × 10⁸ CFU of B. abortus S19 (A) or B. abortus BAI129 (B) obtained at 1, 2, or 4 weeks postinfection are shown. IgG1 plus IgG2a give total KELA units.