Abstract
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). After trafficking along the endocytic and secretory pathways, BCVs 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 proteins remain 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 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. In bone-marrow derived macrophages infected with B. abortus, ENO-1 associates to BCVs in a BPE123-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 α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus.
Highlights
The intracellular pathogen Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonotic disease (Pappas et al, 2005; Moreno, 2014)
The same samples were analyzed by Western Blot with anti ENO-1 antibodies and a band with molecular weight compatible with alpha-enolase was detected (Figure 1C), validating the identity of the differential band identified as ENO-1
Type IV secretion systems are membrane-associated protein complexes used by many Gram-negative pathogenic bacteria including Brucella spp., Legionella pneumophila, Coxiella burnetii, Bartonella spp., Helicobacter pylori, Bordetella pertussis, and Rickettsia prowazekii, to translocate effector proteins that either hijack or interfere with host cell pathways (Llosa et al, 2009; Voth and Heinzen, 2009; de Jong and Tsolis, 2012; Voth et al, 2012; Isaac and Isberg, 2014; Siamer and Dehio, 2015)
Summary
The intracellular pathogen Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonotic disease (Pappas et al, 2005; Moreno, 2014). Brucella infection causes abortion and sterility in animals, and undulating fever and debilitating disorders in humans. Brucellae are able to replicate in a wide range of mammalian cell types, including epithelial cells, fibroblasts, microglia, and endothelial cells. Bacteria survive and replicate within these professional phagocytic cells prior to their dissemination to placental trophoblasts (in pregnant females), reproductive tract and the mononuclear phagocyte system, where they persist to establish a long-term infection in the host and eventually produce cardiovascular, hepatic, neurologic and osteoarticular disease (Adams, 2002; Atluri et al, 2011)
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