IDENTIFICATION OF A PHOSPHATASE AS AN INTERACTING PARTNER OF THE Brucella abortus EFFECTOR BPE123

REVORA V; MARCHESINI M.I; COMERCI D. J

Mar del Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2015

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Brucella abortus is an intracellular pathogen whose virulence depends on a type IV secretion system (VirB). This system translocates proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche where it actively replicates. BPE123 (BAB2_0123) is a protein of 17 kDa highly conserved in all sequenced Brucella species, and it is translocated into the host cell cytoplasm in a VirB-dependent manner. In order to identify proteins that interact with BPE123, that may constitute a VirB substrates or proteins required for the translocation process, we have performed a bacterial two hybrid screen. We demonstrate that BPE123 interacts with a phosphatase named SerB (BAB1_1410), classified as a member of the phosphoserine phosphatase subgroup of the HAD (haloacid dehalogenase) family of hydrolases. These results were further confirmed by co-immunoprecipitation experiments. SerB was expressed in Escherichia coli as a histidine-tagged fusion protein (His_SerB) and the purified protein exhibited a phosphatase activity towards p-nitrophenyl phosphate (Km= 1.53 mM). Optimal activity was observed at pH of 7, with a strong preference for Mg2+ over Mn2+. His_SerB activity was inhibited by sodium pyrophosphate and EDTA. Further experiments are in progress to determine whether SerB is a VirB substrate itself and to analyze its role during infection.