Abstract
Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus Brucella. In Brucella abortus and Brucella melitensis, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a N-formyl-perosamine homopolymer O-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the Brucella O-PS can be structurally and antigenically modified using wbdR, the acetyl-transferase gene involved in N-acetyl-perosamine synthesis in Escherichia coli O157:H7. Brucella constructs carrying plasmidic wbdR expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting wbdR into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of wbdR constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both N-formyl and N-acetyl-perosamine. Moreover, deletion of the Brucella formyltransferase gene wbkC in wbdR constructs generated bacteria producing only N-acetyl-perosamine homopolymers, proving that wbdR can replace for wbkC. Absorption experiments with immune sera revealed that the wbdR constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that B. abortus and B. melitensis wbdR constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The wbdR 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 per R mutant. In mice, the wbdR 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 Brucella.
Highlights
The Gram-negative bacteria of the genus Brucella are the etiological agents of brucellosis, a zoonosis that causes abortions and infertility in domestic livestock and wildlife and a grave and debilitating disease in humans
The reactivity of the monoclonal antibody 42D2 to the A>M epitope was negative with all smooth lipopolysaccharide (S-LPS) obtained in the B. melitensis background (Supplementary Figure S7B). These results show that expression of wbdR in a B. melitensis background results in the synthesis of O-specific polysaccharide (O-O-PS-core oligosaccharide (PS)) carrying N-acetylperosamine with epitopic modifications that parallel those observed in the B. abortus background
The results presented in this work demonstrate that wbdR, the acetyl-transferase gene involved in the synthesis of the N-acetyl-perosamine of the heteropolymeric O-PS repeating unit of E. coli O157:H7, can replace for the autochthonous N-formyl-transferase gene wbkC in representative strains of B. abortus and B. melitensis biovars 1
Summary
The Gram-negative bacteria of the genus Brucella are the etiological agents of brucellosis, a zoonosis that causes abortions and infertility in domestic livestock and wildlife and a grave and debilitating disease in humans. In at least B. abortus, B. suis, and B. melitensis, the O-PS is involved in the resistance to killing by non-immune serum, reduces the access of complement and bactericidal peptides to OM targets and its loss causes attenuation (Lapaque et al, 2005; González et al, 2008) These O-PS properties could be accounted for by topological effects of O-PS length such as a steric hindrance of soluble elements of innate immunity and of non-specific adhesion to host cells (Lapaque et al, 2005; González et al, 2008), it is possible that N-formyl-perosamine is needed for virulence. Taking into account the prominent role of this section of the S-LPS in serological diagnosis
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