The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication

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.

TheBrucella abortusPhosphoglycerate Kinase Mutant Is Highly Attenuated and Induces Protection Superior to That of Vaccine Strain 19 in Immunocompromised and Immunocompetent Mice

“…In parallel with these studies, we show here that PGK enzyme, involved in the downstream pathway of glycolysis, is critical for intramacrophagic growth and bacterial virulence in vivo. Recently, Fugier et al (14) have shown that inhibition of host cell GAPDH and enolase, both involved in glycolysis, resulted in reduced intracellular replication of Brucella. It is possible that Brucella is also using the host cell glycolysis to its advantage, as a source of energy.…”

Mechanism of glyceraldehyde‐3‐phosphate dehydrogenase inactivation by tyrosine nitration

“…Recent investigations have begun to illuminate these roles. These include endocytosis and membrane fusion, 17 translational control, 18 nuclear tRNA transport, 19 signaling, 20 pathogen virulence, 21 and cell death. 22 It is not clear how GAPDH can be targeted for these specific functions in the cell, but is believed to involve posttranslational modification of the protein.…”

A Brucella abortus cstA mutant is defective for association with endoplasmic reticulum exit sites and displays altered trafficking in HeLa cells

“…This is not very surprising since previous data reported proliferation outside ER-derived compartments (Arenas et al, 2000;Bellaire et al, 2005 (ERGIC) or autophagosomes, as proposed previously (Pizarro-Cerdá et al, 1998). ERGIC would be an interesting candidate since it was already shown that (i) Rab2, a Rab GTPase associated with ERGIC, is recruited on the BCVs and that (ii) the GAPDH/COPI/Rab2/PKC i/l complex, involved in retrograde transport between the ER and Golgi apparatus, is required for bacterial proliferation (Fugier et al, 2009). Preliminary data indicate that although the wild-type strain mainly colocalizes with an ERGIC marker (ERGIC53) at 12 h post-infection, though markedly less at 20 h post-infection, the DcstA mutant is not more frequently associated with this marker (data not shown), suggesting that DcstA does not accumulate and proliferate in ERGIC.…”