The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.
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 iota, 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.
Fugier
Emilie
E
Aix Marseille Université, Centre d'Immunologie de Marseille-Luminy, UMR6546, Marseille, France.
Salcedo
Suzana P
SP
de Chastellier
Chantal
C
Pophillat
Matthieu
M
Muller
Alexandre
A
Arce-Gorvel
Vilma
V
Fourquet
Patrick
P
Gorvel
Jean-Pierre
JP
D000818:Animals; D002003:Brucella abortus / Q000166:cytology* / Q000254:growth & development; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000382:microbiology; D002470:Cell Survival; D004721:Endoplasmic Reticulum / Q000382:microbiology; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry / Q000378:metabolism*; D054884:Host-Pathogen Interactions / Q000502:physiology; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D055571:Secretory Pathway / Q000502:physiology; D014617:Vacuoles / Q000737:chemistry / Q000201:enzymology / Q000382:microbiology; D020693:rab2 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D008565:Membrane Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D020693:rab2 GTP-Binding Protein