A new Brucella abortus oxygen/redox sensor: the NtrY/X two component system

CARRICA MARIELA; MARTI MARCELO; FERNANDEZ IGNACIO; PARIS GASTON; GOLDBAUM FERNANDO

Buenos Aires

Congreso; Brucellosis 2011, International Research Conference Including the 64th Brucellosis Research Conference; 2011

Brucella success as phatogen is due to its ability to adapt to the harsh environmental conditions found in the mammalian host, such as oxygen depletion. Brucella encounters low oxygen tension after bacterial ingestion during the passage through the gastro-intestinal tract via Peyer´s patches, during intramacrophagic multiplication in phagosomes, and during multiplication in granulomatous lesions localised in liver, spleen or brain. The response of many bacteria to environmental stimuli is mediated by two component regulatory systems. In particular, the NtrY/NtrX two component system has been described in other microorganisms as a nitrogen fixation and metabolism regulator and it was shown to be involved in the intracellular multiplication of Brucella suis. The signal sensed by the histidine kinase NtrY is unknown. We report the biochemical characterization of NtrY from Brucella abortus and the effect of the mutation of the gene that encodes it in the bacteria. We found that NtrY has a Per-ARNT-SIM (PAS) domain that binds heme as co-factor. The heme iron can be reduced to Fe2+ and is rapidly oxidized to Fe3+ in presence of oxygen. By UV-visible spectroscopy analysis we show that nitric oxide and carbon monoxide are ligands of NtrY. Importantly, we demonstrate that the oxidation state of the heme iron modulates NtrY autokinase activity: in anoxygenic reduced ferrous form, NtrY has high autokinase activity, and it rapidly decreases when the protein is exposed to oxygen and the heme iron is oxidized. Also, by qPCR experiments, we showed that ntrY gene expression is increased in anaerobic growth conditions. We constructed a Brucella abortus ntrY mutant strain and showed that it grows slower than the wild type parental strain and that its capacity to replicate inside macrophages and mice is affected. These results suggest that the NtrY/X two component system could be involved in the environmental sensing of Brucella abortus and in the adaptation to its replicative niche