BIOCHEMICAL CHARACTERIZATION OFCYCLOPHILINS IN Brucella

BRIONES GABRIEL; BUFFA GABRIELA NATALIA; MURUAGA EMANUEL JAVIER; ROSET MARA SABRINA

Congreso; 54th Annual Meeting Agentine Society for Biochemistry and Molecular Biology LIV Reunion Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis.Brucella virulence relies on its ability totransition to an intracellular lifestyle within host cells. Comparative proteomic studies identified two overexpressed proteins during B.abortus intracellular life characterized as potential cyclophilins by sequence analysis, called CypA and CypB. Cyclophilins are enzymes thatcatalyze cis/trans isomerization of peptide bonds that involve prolines (PPIase).Brucella?s cyclophilins play an important role in stressadaptation and virulence. While CypA shares homologies with cyclophilins of Gram-negative bacteria, CypB has primary protein structurecharacteristics of eukaryotic cyclophilins, thus, inhibible by Cyclosporine A (CsA). The relation with pathogenesis coupled with the sequencesimilarity to eukaryotic cyclophilins is strongly suggestive of CypB being deployed byB. abortusas an effector that mimics the hostCyclophilins. We analyzed PPIase activity and inhibition by CsA of recombinant proteins and evaluatedin vitroandin vivochaperone roleperforming NdeI residual activity assay and a bacterial stress survival comparison, respectively. Additionally, we examined formation ofcyclophilin oligomers using SDS-PAGE, gel filtration and bacterial two-hybrid system. Our results showed that recombinant CypA and CypBpresent PPIase and chaperone activities and that PPIase activity of CypB is more inhibited by CsA than PPIase activity of CypA, supporting insilico information of activity and homology. We observed that CypB dimerizedin vitroandin vivo, while CypA did not, suggesting distinctfunctions