A combined in silico-in vivo screening strategy to identify Brucella abortus effector proteins

HERRMANN, C. K.; UGALDE, R. A.; DIEGO JOSE COMERCI

San Miguel de Tucumán

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).; 2009

SAIBBM

Brucella abortus is a facultative intracellular pathogen that replicates inside mammalian phagocytes. A type IV secretion system is known to be essential to subvert lysosome fusion and to create an organelle that supports replication, probably by means of effector protein translocation. Our main goal is to identify and characterize these proteins using an in silico approach. We searched B. abortus 2308 genome for proteins with certain properties that could make them good candidates for secretion, including: shared homology with eukaryotic domains, conserved genes in á-proteobacteria, special location in the genome, shared homology with known effectors in related species and presence of certain secondary structures known for their role in protein-protein interaction. As a result, we found 85 proteins, each containing at least one of the mentioned properties. These proteins were cloned in a pBBR4 derived vector specially designed to make fusions to the catalytic domain of the Bordetella pertussis Adenilate cyclase, a reporter system that proved to be successful for our goal. These constructs are currently under screening to determine those proteins that are actually translocated into the host cell. Fusion proteins positives for translocation under wild type genetic background that results negatives under an isogenic VirB null background will be identified as potential VirB effectors.