Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.

The OspF family of phosphothreonine lyase, including SpvC from Salmonella, irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination. We determined crystal structures of SpvC and its complex with a phosphopeptide substrate. SpvC adopts a unique fold of alpha/beta type. The disordered N terminus harbors a canonical D motif for MAPK substrate docking. The enzyme-substrate complex structure indicates that recognition of the phosphotyrosine followed by insertion of the threonine phosphate into an arginine pocket places the phosphothreonine into the enzyme active site. This requires the conformational flexibility of pT-X-pY, which suggests that p38 (pT-G-pY) is likely the preferred physiological substrate. Structure-based biochemical and enzymatic analysis allows us to propose a general acid/base mechanism for beta elimination reaction catalyzed by the phosphothreonine lyase. The mechanism described here provides a structural understanding of MAPK inactivation by a family of pathogenic effectors conserved in plant and animal systems and may also open a new route for biological catalysis.

The secretin GspD of the type II secretion system (T2SS) forms a channel across the outer membrane in Gram-negative bacteria to transport substrates from the periplasm to the extracellular milieu. The lack of an atomic-resolution structure of the GspD channel hinders the investigation of substrate translocation mechanism of T2SS. Here we report cryo-EM structures of two GspD channels (∼1 MDa), from Escherichia coli K12 and Vibrio cholerae, at ∼3 Å resolution. The structures reveal a pentadecameric channel architecture, wherein three rings of GspD N domains form the periplasmic channel. The secretin domain constitutes a novel double β-barrel channel, with at least 60 β-strands in each barrel, and is stabilized by S domains. The outer membrane channel is sealed by β-strand-enriched gates. On the basis of the partially open state captured, we proposed a detailed gate-opening mechanism. Our structures provide a structural basis for understanding the secretin superfamily and the mechanism of substrate translocation in T2SS.