the glyceraldehyde-3-phosphate dehydrogenase and the small gtpase rab 2 are crucial for brucella replicationglyceraldehyde-3-phosphate脱氢酶和小gtpase rab 2对布鲁氏菌复制至关重要.pdf

The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication Emilie Fugier1,2,3, Suzana P. Salcedo1,2,3, Chantal de Chastellier 1,2,3, Matthieu Pophillat1,2,3, Alexandre Muller1,2,3, Vilma Arce-Gorvel1,2,3, Patrick Fourquet1,2,3, Jean-Pierre Gorvel1,2,3* ´ ´ ´ 1 Aix Marseille Universite, Faculte des Sciences de Luminy, Centre d’Immunologie de Marseille-Luminy (CIML), UMR6546, Marseille, France, 2 Institut National de la Sante et ´ de la Recherche Medicale (INSERM), U631, Marseille, France, 3 Centre National de la Recherche Scientifique (CNRS), UMR6102, Marseille, France 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 i, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in