DIFFERENT APPROACHES TO IDENTIFY VIRULENCE-ASSOCIATED TRANSCRIPTIONAL NETWORKS IN THE FACULTATIVE INTRACELLULAR PATHOGEN Brucella

RODRIGO SIEIRA; JOHANNA MARTIN CALDARERI; VERÓNICA RUIZ-RANWEZ; ANGELES ZORREGUIETA

Rosario, Santa Fé

Congreso; IX Congreso de la Sociedad Argentina de Microbiología General; 2013

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