Different approaches to identify virulence-associated transcriptional networks in the facultative intracellular pathogen Brucella

SIEIRA R; MARTIN-CALDARERI J; RUIZ V; ZORREGUIETA A

Rosario

Congreso; IX Congreso de Microbiología General; 2013

Brucella spp. are Gram-negative, facultative intracellular bacteria responsible for brucellosis, a zoonotic disease that affects a wide range of mammals including humans. Pathogenicity of Brucella relies on its ability to survive and replicate within phagocytic and non-phagocytic cells of the eukaryotic host. This is achieved by a series of mechanisms that allow Brucella to attach, internalize, and express different virulence factors that contribute to avoid lysosomal degradation and to promote the biogenesis of the intracellular replication niche. Here, we describe our ongoing projects which are focused on identifying regulatory networks involving Quorum Sensing (QS)-elements and genes related with intracellular survival of Brucella. By using molecular biology, biochemical, and bioinformatic approaches, we constructed a map of protein-DNA interactions that define a network which links virulence determinants (virB), a metabolic operon (hut), autotransporter proteins involved in the attachment of Brucella to the eukaryotic host cell (btaE), and putative transcription factors (syrB2). Reporter gene analyses revealed that some of these interactions constitute functional DNA-binding sites for transcriptional regulators which coordinate the expression of these targets. In addition to these findings, we are also interested in increasing our knowledge of this network by deciphering the regulon of VjbR, a QS-related regulator that plays a major role in the pathogenicity of Brucella. This regulator specifically interacts with the virB promoter, and is also known to be directly or indirectly involved in the control of expression of hundreds of additional genes. In order to identify additional VjbR target-DNA sequences, our current goal is to apply high-throughput technologies based on our knowledge of laboratory conditions that maximize expression of the VjbR protein and mimic the environmental intracellular cues that  Brucella encounters within the host cell. Such conditions require the convergence of different signals, which involve starvation, a defined metabolic state of the bacterium, and pH values that Brucella necessary has to face during the acidification of the intraphagosomal environment.