BRUCELLA ABORTUS BEARS AN ATYPICAL RIBOFLAVIN PATHWAY THAT IS INVOLVED IN ITS VIRULENCE

BONOMI, H.R.; MARCHESINI, I.; COMERCI, D.; UGALDE, R.; GOLDBAUM, F.A.

Mar del Plata, Argentina

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

  Riboflavin (vitamin B2) is an essential metabolite that is used in the synthesis other vital cofactors. Plants, fungi and prokaryotes can synthesize riboflavin while animals must obtain it from the diet. Lumazine Synthase (LS) catalyzes the penultimate step of the biosynthesis. Brucella has two paralog genes coding for LS named ribH1 and ribH2. B. abortus spp. mutant strains lacking ribH1 or ribH2 show no auxotrophy for riboflavin. However, only ribh2- shows an important attenuation in its virulence and resistance to oxidative stress. We failed in many attempts to obtain a double mutant, suggesting a lethal phenotype. However, in the presence of a rescue plasmid containing a wild type copy of ribH1, the genomic double mutant could be obtained. Conducting Plasmid-Shuffling experiments with this late strain we clearly showed that both RibH1 and RibH2 have LS activity in vivo. We also generated an active site mutant W22A of RibH2 and proved that it is unable to complement the LS activity in vivo. Auxotrophy for riboflavin is also demonstrated in the ribh1-/ribh2- mutant strain. TLC was used to assess flavin cellular content in different strains. Virulence and oxidative stress experiments with different mutant and complemented strains were conducted in order to show the connection between flavins and virulence mechanisms.