Like other multicellular organisms, the model nematode C. elegans responds to infection by inducing the expression of defense genes. Among the genes upregulated in response to a natural fungal pathogen is nlp-29, encoding an antimicrobial peptide. In a screen for mutants that fail to express nlp-29 following fungal infection, we isolated alleles of tpa-1, homologous to the mammalian protein kinase C (PKC) delta. Through epistasis analyses, we demonstrate that C. elegans PKC acts through the p38 MAPK pathway to regulate nlp-29. This involves G protein signaling and specific C-type phospholipases acting upstream of PKCdelta. Unexpectedly and unlike in mammals, tpa-1 does not act via D-type protein kinases, but another C. elegans PKC gene, pkc-3, functions nonredundantly with tpa-1 to control nlp-29 expression. Finally, the tribbles-like kinase nipi-3 acts upstream of PKCdelta in this antifungal immune signaling cascade. These findings greatly expand our understanding of the pathways involved in C. elegans innate immunity.

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.