Abstract
The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.
Highlights
Brucella abortus invades both phagocytic and non-phagocytic cells [1,2,3,4,5,6] residing inside a membrane-bound compartment called the Brucella-containing vacuole (BCV)
Mass spectrometry analysis of BCV proteins separated by twodimensional (2D) gel electrophoresis revealed the presence of the eukaryotic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Bacteria can be hidden from host cell defences to persist within the host, and can take advantage of the membrane reservoir delivered by the endoplasmic reticulum (ER) to replicate
Summary
Brucella abortus invades both phagocytic and non-phagocytic cells [1,2,3,4,5,6] residing inside a membrane-bound compartment called the Brucella-containing vacuole (BCV) Bacteria ensure their survival and replication within host cells by avoiding fusion with lysosomes and by controlling interactions with the endoplasmic reticulum (ER) [1,5]. The Brucella replicative organelle has been, until now, characterized by the presence of ER chaperones such as calnexin, calreticulin, the translocator sec61b, and the ER resident enzyme protein disulfide-isomerase PDI [1,5,11] Aside from these ER resident proteins, no other eukaryotic or prokaryotic proteins have been associated with the BCV membrane as yet, identification of these proteins is essential to understand how Brucella maintains interactions with the ER and keeps replicating within this compartment. Further work on GAPDH revealed a role for GAPDH and the small GTPase Rab 2 in the intracellular replication of B. abortus
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