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Brucella  spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in mammals.  Brucella  strains containing the O-polysaccharide in their cell wall structure exhibit a smooth phenotype whereas the strains devoid of the polysaccharide show rough phenotype.  B. abortus  strain RB51 is a stable rough attenuated mutant which is used as a licensed live vaccine for bovine brucellosis. Previous studies have shown that the  wboA  gene, which encodes a glycosyltransferase required for the synthesis of O-polysaccharide, is disrupted in  B. abortus  RB51 by an  IS711  element. Although complementation of strain RB51 with a functional  wboA  gene results in O-polysaccharide synthesis in the cytoplasm, it does not result in smooth phenotype. The aim of this study was to determine if overexpression of  Brucella  WbkA or WbkE, two additional putative glycosyltransferases essential for O-polysaccharide synthesis, in strain RB51 would result in the O-polysaccharide synthesis and smooth phenotype. Our results demonstrate that overexpression of  wbkA  or  wbkE  gene in RB51 does not result in O-polysaccharide expression as shown by Western blotting with specific antibodies. However,  wbkA , but not  wbkE , overexpression leads to the development of a clumping phenotype and the production of exopolysaccharide(s) containing mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Moreover, we found that the clumping recombinant strain displays increased adhesion to polystyrene plates. The recombinant strain was similar to strain RB51 in its attenuation characteristic and in its ability to induce protective immunity against virulent  B. abortus  challenge in mice.

Overexpression of Brucella putative glycosyltransferase WbkA in B. abortus RB51 leads to production of exopolysaccharide