The role of the Brucella abortus adhesin bapA in the infection of host cells

MERWAISS F; UGALDE JE; CZIBENER C

Capital Federal

Congreso; Brucellosis 2011 International Research Conference; 2011

Asociación Argentina de Microbiología

To date almost nothing is known on the molecular determinats that allow Brucella to attach and invade host cells or to trespass epithelial barries. To identify novel virulence factors we performed a bioinformatic screening searching the genome for regions that deviate from the average G+C content with the premise that the acquisition of horizontally transmitted regions that have occurred recently in evolution have not homogenized with the rest of the genome and, hence, have a significant difference in the G+C content compared with the rest of the genome. Using this approach we identified a cluster of genes (Bab1_2009-Bab_2012) which codes for four open reading frames. Three of them have no homology to anything in databases and one (Bab1_2009) has a bacterial immunoglobulin like domain present in several bacterial adhesins including the intimin of the enterohemorragic Escherichia coli. To determine the role this region might play in the interaction with the host cell we constructed deletion mutants in the entire region and in the Bab1_2009 gene and determined intracelullar replication in professional and non-professional phagocytes and attachment and invasion efficiency. The results indicate that both mutants have a significant defect in the attachment to host cells. Additionally, over-expression of Bab1_2009 results in a strain with a dramatic increase in attachment measured in intracellular replication and adhesion/invasion assays. For these reasons we have named this gene bapA (for Brucella Adhesion Protein A). To further gain insight into the cellular targets of BapA we produced in E. coli the bacterial immunoglobulin like domain flagged and histidine tagged and generated a mouse antiserum. Incubation of the wild-type but not the bapA mutant strain with this antiserum prior to infection sifnificantly reduces the internalization. On the other hand, incubation of HeLa cells with the recombinant protein induces a profound actin rearrengment that culminates with the deattachment of the cells and death suggesting that the protein, either competes with adhesion proteins, or signals the cells and fires a response that induces the observed phenotypes. In sum, we have identified a new B. abortus adhesin and are currently in the process of identifying its cellular receptor.