5. Brucella abortus Exploits Host Cell Alpha-Enolase Via The Virb Effector Bpe123 To Promote Intracellular Replication

MORRONE SEIJO S.; GUAIMAS F.; MARCHESINI M.I; COMERCI D. J

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB).; 2017

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Brucella abortus, the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). Brucella Type IV Secretion System (VirB) is a major virulence factor which translocate effector proteins from the BCV into host cell cytoplasm, thus modulating several signaling pathways to favor bacterial intracellular survival and replication. BPE123 is a B.abortus VirB sustrate previusly identified by our group. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolasa (ENO1) was identified as a potential interaction partner of BPE123. This interaction was confirmed in vivo by immunoprecipitation assay and by confocal microscopy analysis of ENO1 redistribution in the presence of BPE123. Aditionally, quantitative analysis of confocal micrographs of macrophages infected with B. abortus showed that ENO1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 might also be occurring during the intracellular phase of B. abortus. Interestingly, down-regulation of the expression of ENO1 in HeLa cells infected with B. abortus affected its intracellular replication, demostrating a role of ENO1 in Brucella intracellular lifestyle. To investigated if the interaction between BPE123 and ENO1 affect the catalityc activity of ENO1, activity assays were performed and demostrated that ENO1 activity is enhanced not only in HeLa cells ectopically expresing BPE123 but also in B. abortus-infected THP-1 macrophages. These results suggests that interaction between BPE123 and ENO1 induces structural and/or functional changes accounting for the activation of host cell alpha-enolase during the infection process. Further experiments are underway to study the changes of the kinetic parameters of ENO1 in the presence of BPE123.