NtrYX, a redox activated two component system in Brucella: signal transduction and transcriptional networks

FERNÁNDEZ, IGNACIO; CARRICA, MARIELA DEL CARMEN; PARIS, GASTON; GOLDBAUM, FERNANDO

Heidelberg

Simposio; EMBO|EMBL Symposium: New Approaches and Concepts in Microbiology; 2013

EMBO|EMBL

Brucella is a genus of facultative intracellular bacteria responsible for a zoonotic disease called Brucellosis. Given that its replicative niche is a microaerobic environment, Brucella needs to sense oxygen availability in order to generate an adaptive response. Our group has identified an oxygen/redox sensor called NtrY that is involved in the adaptation of Brucella to microaerobiosis and is important for its ability to replicate in macrophages. NtrY is a membrane-bound histidine kinase with cytosolic HAMP and PAS domains. In a recent article we demonstrated that NtrY binds heme and the activity of the protein increases upon reduction of its cofactor. Our current work allowed us to establish that the isolated PAS domain interacts with heme (therefore it is the sensor domain) and performing titration experiments we could obtain a Kd= 40μM for that binding process. Previously we had shown that NtrY phosphorylates NtrX, its cognate response regulator, which belongs to the NtrC-like response regulators subfamily. Our recent experiments demonstrated that NtrX is a stable dimer in solution able to form higher oligomers and binds ATP and ADP with comparable affinity. Gel shift assays indicate that NtrX binds to a promoter sequence within its operon. Using transcriptional fusions, we could determine that NtrX autoregulates its expression, stimulating the transcription from two different promoters.We also published a connection between the PrrBA and NtrYX systems, in which PrrA enhances the transcription of NtrYX. In order to investigate possible links with other signaling cascades, we performed qRT-PCR using an NtrYX mutant strain. This approach allowed us discover that PhoP is upregulated when NtrY is absent, and that NtrBC is under the control of NtrY. These findings represent the first steps into the description of a transcriptional network that involves NtrYX signaling system in the adaptation to microaerobiosis, a central aspect in the pathogenesis of Brucella.