The host alpha-enolase interacts with a Brucella abortus TFSS sustrate

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

Chicago

Conferencia; 68th Annual Brucellosis Research Conference; 2015

Brucellosis International Research Association

Brucella abortus invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCV mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System VirB is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the virb system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated effector proteins remains unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (Bab2-0123)is a B. abortus Virb/translocated effector protein recently identified by our group, and here we demonstrate that it is translocated in a two step process, requiring a periplasmic intermediate. BPE123 is a hypothetical protein whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. Microscopy studies further confirmed BPE123-ENO-1 interaction, ENO-1 relocalization was detected upon ectopic expression of BPE123 in Hela cells, where both proteins localized to reticular structures. In bone-marrow derived macrophages infected with B. abortus, ENO'1 associated to BCVs in a BPE-123 dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO'1 depletion by siRNA impaired B. abortus intracellular replication in Hela cells, confirming a role for alpha-enolase during the infection process. Taken together, these results indicate that teh T4SS effector BPE123 interacts with the host multifunctional protein ENO-1, which in turn is important for B. abortus intracellular replication.