BIOCHEMICAL CHARACTERIZATION OFCYCLOPHILINS in Brucella

MURUAGA E,; BUFFA, GN.; ROSET MS; G. BRIONES

Congreso; SAIB 2018; 2018

Brucella is an intracellular bacterial pathogenthat causes the worldwide zoonotic disease brucellosis.Brucellavirulence relieson its ability to transition to an intracellular lifestyle within host cells.Comparative proteomic studies identified two overexpressed proteins during B. abortusintracellular life characterized as potential cyclophilins by sequenceanalysis, called CypA and CypB. Cyclophilins are enzymes that catalyzecis/trans isomerization of peptide bonds that involve prolines(PPIase).Brucella?s cyclophilins play an important role in stress adaptationand virulence. While CypA shares homologies with cyclophilins of Gram-negativebacteria, CypB has primary protein structure characteristics of eukaryoticcyclophilins, thus, inhibible by Cyclosporine A (CsA). The relation withpathogenesis coupled with the sequence similarity to eukaryotic cyclophilins isstrongly suggestive of CypB being deployed byB. abortusas an effector thatmimics the host Cyclophilins. We analyzed PPIase activity and inhibition by CsAof recombinant proteins and evaluatedin vitroandin vivochaperone role performingNdeI residual activity assay and a bacterial stress survival comparison,respectively. Additionally, we examined formation of cyclophilin oligomersusing SDS-PAGE, gel filtration and bacterial two-hybrid system. Our resultsshowed that recombinant CypA and CypB present PPIase and chaperone activitiesand that PPIase activity of CypB is more inhibited by CsA than PPIase activityof CypA, supporting in silico information of activity and homology. We observedthat CypB dimerizedin vitroandin vivo, while CypA did not, suggesting distinct functions