Biochemical and functional studies on RibH2 of Brucella spp.

BONOMI, H.; MARCHESINI, M. I.; ZYLBERMAN V; KLINKE, S.; DIEGO JOSE COMERCI; UGALDE, R. A.; GOLDBAUM, F. A.

Rosario, Santa Fe

Congreso; XLII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research(SAIB); 2006

Brucella spp. have two paralog genes coding for Lumazine Synthases (LS) named RibH1 and RibH2. Previous work shows that RibH1 is the active enzyme while RibH2 has negligible LS activity. A B.abortus strain lacking RibH2 showed attenuated virulence and enhanced sensitivity to H2O2. These results indicate that this protein is a virulence factor and our aim is to find its molecular mechanism. Intents to generate a double ribh1/ribh2 mutant suggest that at least one of these genes must be present for viability. Plasmidic vectors have been constructed with the ribh2 promoter transcriptionally fused to lacZ in order to determine when this gene is transcribed during macrophage infection and what conditions up- or down-regulate its expression in culture. We have also determined that RibH2 binds the RedOx cofactors riboflavin and hemin with high affinity and specificity in vitro. RibH2 from Mesorhizobium loti, a closely related bacterium, showed binding to hemin in vitro as well. To assess the connection between riboflavin and hemin with virulence we generated a mutant W22A incapable of binding riboflavin to use it in complementation experiments. Hemin-binding mutants will also be done. To appraise the roll of RibH2 in NO detoxification we are also conducting survival experiments with wt, ribh2- and complemented B.abortus in activated macrophages and in culture in the presence of a NO donor