5. Functional Characterization of two substrates of the Brucella abortus Type-IV secretion system

HERRMANN, C. K.; COMERCI D. J

Chicago

Conferencia; 65th Annual Brucellosis Research Conference; 2012

Brucellosis Research Society

In a previous work we performed a combined in silico-in vivo screening and found three proteins that are translocated into the host cell in a VirB-dependent manner: BPE043, a hypothetical coiled-coil protein; BPE005, a putative cyclic nucleotide-binding protein and BPE275, a member of the rhomboid protease family. To unveil the function of these proteins, we generated B. abortus clean deletion mutants of those genes and analyzed their phenotype. Our results revealed that two mutants displayed different but remarkable defects in virulence. The first one, B. abortus Dbpe005 showed a significant increase in LAMP-1 acquisition that affected the biogenesis of the replicative BCV and thus, bacteria survival and intracellular replication. The second one, B. abortus Dbpe275 displayed reduced adhesion to host cell which resulted in a reduced invasion efficiency. However, the intracellular replication stages were not affected in this mutant. In order to determine the importance of BPE275 activity in the infection process, we generated an expression vector containing a single-nucleotide mutation of BPE275 on the active site of the protease domain (Ser153 à Ala153). This plasmid was then transferred to B. abortus Dbpe275 and the complementation phenotype was analyzed. Our experiments showed that Ser153 is essential for BPE275 suggesting that the protease activity is required for adhesion/internalization of B. abortus, highlighting the importance of VirB and effector proteins for the Brucella-host cell interaction.