Overexpression of wbkF gene in Brucella abortus RB51WboA leads to increased O-polysaccharide expression and enhanced vaccine efficacy against B. abortus 2308, B. melitensis 16M, and B. suis 1330 in a murine brucellosis model

Brucella abortus RB51 is an attenuated, stable, spontaneous rough mutant derived in the laboratory from the virulent strain B. abortus 2308. Previous studies discovered that the wboA gene, which encodes a glycosyltransferase required for synthesis of the O-polysaccharide, is disrupted in strain RB51 by an IS711 element. However, complementation of strain RB51 with a functional wboA gene (strain RB51WboA) does not confer it a smooth phenotype but results in low levels of cytoplasmic O-polysaccharide synthesis. In this study, we asked if increasing the potential availability of bactoprenol priming precursors in strain RB51WboA would increase the levels of O-polysaccharide synthesis and enhance the protective efficacy against virulent Brucella challenge. To achieve this, we overexpressed the wbkF gene, which encodes a putative undecaprenyl-glycosyltransferase involved in bactoprenol priming for O-polysaccharide polymerization, in strain RB51WboA to generate strain RB51WboAKF. In comparison to strain RB51WboA, strain RB51WboAKF expressed higher levels of O-polysaccharide, but was still attenuated and remained phenotypically rough. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 isotype. Splenocytes from mice vaccinated with strain RB51WboAKF secreted higher levels of antigen-specific IFN-gamma and TNF-alpha and contained more numbers of antigen-specific IFN-gamma secreting CD4(+) and CD8(+) T lymphocytes when compared to those of the RB51 or RB51WboA vaccinated groups. Immunization with strain RB51WboAKF conferred enhanced protection against virulent B. abortus 2308, B. melitensis 16M and B. suis 1330 challenge when compared to the currently used vaccine strains. Our results suggest that strain RB51WboAKF has the potential to be a more efficacious vaccine than its parent strain in natural hosts.