Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine

Abstract

Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.

Figure 1

Genetic organization of regions encoding LPS genes that are susceptible to mutation with a subsequent generation of R mutants. (A) GI-2 region of B. melitensis and the scar and circular intermediate (wbo genes are omitted in the circular intermediate) resulting from the int- promoted excision; (B)wbkA region and the corresponding scar and circular intermediate resulting from homologous recombination between ISBm1 transposases; (C) the wbkFD region of B. melitensis and B. canis (wbkD is annotated as capD in B. canis); (D)wbo locus in B. melitensis and B. abortus RB51 and (E)manBA_core locus in B. melitensis chromosome II with its corresponding mutants showing the deletion and point mutations in manB_core gene (asterisks). The position of the primers used in the PCR analyses is indicated as P-number of the primer. The ORF annotations are based on the B. melitensis 16 M genome [GenBank: AE008917 and AE008918] and B. canis chromosome I [GenBank: CP000872].

Figure 2

Genetic and phenotypic characterization of spontaneous Rev 1 R mutants. (A) Southern blot analysis of B. melitensis Rev 1 spontaneous R variants with IS711 (left panel), wbkA (center panel) and GI2 BMEI0999 (right panel) as probes. Note that, for the IS711 probe and B. melitensis Rev 1 and ∆wbkA but not ∆GI-2, the signal at 4.2 kb corresponds to two bands (predicted MW 4.2 and 4.14 kb). For simplicity, fragments with molecular masses lower than 3.8 kb were omitted. (B) Western blot of purified R-LPS and S-LPS, Rev 1, wbkA and GI-2 R mutants and the corresponding complemented strains probed with anti-LPS antibodies.

Figure 3

Stabilization of regions GI-2 and wbkA by deletion of appropriate section of GI-2 phage-related integrase (Δint), wbkA-flanking IS (ΔISBm1) and both (Rev 2). (A) PCR detection of the chromosomal scar and circular intermediate resulting from (GI-2 excision (left panel) and wbkA excision (right panel). (B) S-R dissociation rates.

Figure 4

Genetic stabilization in Rev 2 does not alter the in vitro growth and vaccine properties of Rev 1 reference vaccine. (A) In vitro growth kinetics; (B) Residual virulence (log CFU/spleen); (C) Spleen weights; and (D) Efficacy against a virulent B. melitensis virulent challenge, in BALB/c mice. Statistical comparison of means was performed by one-way ANOVA and Fisher’s PLSD test: ^aP < 0.005 vs. unvaccinated control (PBS group).