Genomic Insertion of a Heterologous Acetyltransferase Generates a New Lipopolysaccharide Antigenic Structure in Brucella abortus and Brucella melitensis

Abstract

Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus <i>Brucella</i>. In <i>Brucella abortus</i> and <i>Brucella melitensis</i>, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a <i>N</i>-formyl-perosamine homopolymer <i>O</i>-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the <i>Brucella</i> O-PS can be structurally and antigenically modified using <i>wbdR</i>, the acetyl-transferase gene involved in <i>N</i>-acetyl-perosamine synthesis in <i>Escherichia coli</i> O157:H7. <i>Brucella</i> constructs carrying plasmidic <i>wbdR</i> expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting <i>wbdR</i> into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of <i>wbdR</i> constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both <i>N</i>-formyl and <i>N</i>-acetyl-perosamine. Moreover, deletion of the <i>Brucella</i> formyltransferase gene <i>wbkC</i> in <i>wbdR</i> constructs generated bacteria producing only <i>N</i>-acetyl-perosamine homopolymers, proving that <i>wbdR</i> can replace for <i>wbkC</i>. Absorption experiments with immune sera revealed that the <i>wbdR</i> constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that <i>B. abortus</i> and <i>B. melitensis wbdR</i> constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The <i>wbdR</i> constructs showed resistance to polycations similar to that of the wild-type strains but displayed increased sensitivity to normal serum similar to that of a <i>per</i> R mutant. In mice, the <i>wbdR</i> constructs produced chronic infections and triggered antibody responses that can be differentiated from those evoked by the wild-type strain in S-LPS ELISAs. These results open the possibilities of developing brucellosis vaccines that are both antigenically tagged and lack the diagnostic epitopes of virulent field strains, thereby solving the diagnostic interference created by current vaccines against <i>Brucella</i>.