Dissection of virulence-associated transcriptional networks in Brucella: seeking for new regulatory proteins through bioinformatic, biochemical and molecular approaches.

GABRIELA SYCZ; ANGELES ZORREGUIETA; HERNÁN BONOMI; ROCÍO TAU; RODRIGO SIEIRA

Ciudad Autónoma de Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS

In facultative intracellular bacteria of the genus Brucella, significant progress has been made in identifying virulence factors. However, the genetic program orchestrating the Brucella intracellular adaptation process is still poorly understood. In this regard, our group recently identified the regulon of VjbR, a transcription factor known to play a major role in the pathogenesis of Brucella. Our ChIP-seq and RNA-seq analyses showed that VjbR controls bacterial functions relevant for survival during the initial stages of the intracellular infection, and revealed that VjbR indeed acts as a global regulator exhibiting a large amount of binding sites across the Brucella genome. However, further analysis of our data indicated that the VjbR transcriptional network is highly complex. For instance, we found that VjbR failed to bind to many genomic positions containing conserved VjbR-binding motifs, which suggested the possible existence of competitors that prevent binding of VjbR to specific promoters under the assayed conditions. To explore this possibility, we developed a bioinformatic method that allowed us to identify conserved sequences adjacent to VjbR-binding motifs that failed to bind VjbR in vivo, which could act as binding sites for competitor transcription factors. EMSA analysis of one of such specific promoters showed that in the absence of competitors, VjbR alone was able to interact in vitro with its target DNA sequence. Moreover, in Brucella crude extracts we detected two proteins able to bind to the analyzed promoter. Using biochemical and molecular methods we isolated one of these proteins, wich was identified as a transcription factor known to coactivate expression in other direct targets of VjbR. Determination of the second DNA-binding protein, however, will requiere further work. In summary, here we present a pipeline suitable to identify possible competitor and/or coactivator transcription factors in complex transcriptional regulatory networks.