Identification of a phosphatase as an interacting partner of the Brucella abortus effector BPE123

REVORA, VIRGINIA; MARCHESINI, MARÍA INÉS; COMERCI, DIEGO JOSÉ

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

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