INSTITUTO DE INVESTIGACIONES BIOTECNOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
BIOCHEMICAL CHARACTERIZATION OF CYCLOPHILINS in Brucella
EMANUEL MURUAGA; MARA ROSET; GABRIELA BUFFA; GABRIEL BRIONES
Congreso; 54th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2018
Brucellais an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis.Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Comparative proteomic studies identified two overexpressed proteins duringB. abortusintracellular life characterized as potential cyclophilins by sequence analysis, called CypA and CypB. Cyclophilins are enzymes that catalyze cis/trans isomerization of peptide bonds that involve prolines (PPIase).Brucella?s cyclophilins play an important role in stress adaptation and virulence. While CypA shares homologies with cyclophilins of Gram-negative bacteria, CypB has primary protein structure characteristics of eukaryotic cyclophilins, thus, inhibible by Cyclosporine A (CsA). The relation with pathogenesis coupled with the sequence similarity to eukaryotic cyclophilins is strongly suggestive of CypB being deployed byB. abortus as an effector that mimics the host Cyclophilins. We analyzed PPIase activity and inhibition by CsA of recombinant proteins and evaluatedin vitroandin vivochaperone role performing NdeI residual activity assay and a bacterial stress survival comparison, respectively. Additionally, we examined formation of cyclophilin oligomers using SDS-PAGE, gel filtration and bacterial two-hybrid system. Our results showed that recombinant CypA and CypB present PPIase and chaperone activities and that PPIase activity of CypB is more inhibited by CsA than PPIase activity of CypA, supporting in silico information of activity and homology. We observed that CypB dimerizedin vitroandin vivo, while CypA did not, suggesting distinct functions.