Pasteurella multocida toxin is a major virulence factor of Pasteurella multocida, which causes pasteurellosis in men and animals and atrophic rhinitis in rabbits and pigs. The Ϸ145 kDa protein toxin stimulates various signal transduction pathways by activating heterotrimeric G proteins of the G␣ q, G␣i, and G␣12/13 families by using an as yet unknown mechanism. Here, we show that Pasteurella multocida toxin deamidates glutamine-205 of G␣ i2 to glutamic acid. Therefore, the toxin inhibits the intrinsic GTPase activity of G␣ i and causes persistent activation of the G protein. A similar modification is also evident for G␣ q, but not for the closely related G␣ 11, which is not a substrate of Pasteurella multocida toxin. Our data identify the ␣-subunits of heterotrimeric G proteins as the direct molecular target of Pasteurella multocida toxin and indicate that the toxin does not act like a protease, which was suggested from its thiol protease-like catalytic triad, but instead causes constitutive activation of G proteins by deamidase activity.bacterial protein toxin ͉ Gi protein ͉ posttranslational modification ͉ transglutaminase

Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP-N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. (1)H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

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The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase C␤. In this study different toxin fragments were investigated. A C-terminal fragment encompassing amino acids 581 through 1285 (PMT581C) was constructed, which was inactive toward intact embryonic bovine lung (EBL) cells after addition to culture medium but caused reorganization of the actin cytoskeleton and rounding up of cells when introduced into the cells by electroporation. As the holotoxin, the toxin fragment PMT581C induced an increase in total inositol phosphate levels after introduction into the cell by electroporation. A C-terminal fragment shorter than PMT581C as well as N-terminal fragments were inactive. Exchange of cysteine-1165 for serine in the holotoxin resulted in a complete loss of the ability to increase inositol phosphate levels. Correspondingly, the mutated toxin fragment PMT581C.C1165S was inactive after cell introduction by electroporation, suggesting an essential role of Cys-1165 in the biological activity of the toxin.The bacterium Pasteurella multocida is the causative agent of atrophic rhinitis in animals and causes wound infections in humans. PMT is sufficient to induce all major symptoms of atrophic rhinitis in animals (3, 10). PMT appears to act on the ␣-subunit of the G q,11 family of heterotrimeric G proteins (25), which stimulates phosphatidylinositol hydrolysis, resulting in an increase in inositol phosphate and diacylglycerol levels (21) and a mobilization of intracellular calcium pools. Moreover, the toxin is a potent mitogen for several cell types (4, 13, 16) and stimulates anchorage-independent DNA synthesis and growth in soft agar in Rat1 fibroblasts (9). The mitogenic effect of PMT on HEK293 cells seems to be caused by stimulation of the mitogen-activated protein kinase pathway via G q,11 -dependent transactivation of the epidermal growth factor receptor (20). In Swiss 3T3 cells, this mitogenic effect of PMT is transient and is followed by a blockade of the cell cycle progression (23).In cultured cells, PMT induces pronounced cytoskeletal changes. Treatment of Swiss 3T3 fibroblasts results in stress fiber formation and focal adhesion assembly (4, 11), which has been proposed to be caused by an activation of the small GTP-binding protein Rho (11). Similarly, stress fiber formation and an increased permeability of endothelial monolayers of human umbilical vein endothelial cells induced by PMT are dependent on the activation of the Rho-signaling pathway (5). A different cytopathic effect of PMT is observed in Vero and embryonic bovine lung (EBL) cells, which is characterized by rounding up of cells (15,17).PMT consists of 1,285 amino acid residues and the primary structure of the N terminus of the toxin has 24 and 27% identity at the amino acid level to the N terminus of the cytotoxic necrotizing factors CNF1 and CNF2, respectively, of Escherichia coli. CNF1 and CNF2 are deamidases acting on Rho proteins (7,18). The catalytic domain of CNF1 is localized to the C-terminal part of...

Two-pore channels (TPCs) are localized in endo-lysosomal compartments and assumed to play an important role for vesicular fusion and endosomal trafficking. Recently, it has been shown that both TPC1 and 2 were required for host cell entry and pathogenicity of Ebola viruses. Here, we investigate the cellular function of TPC1 using protein toxins as model substrates for distinct endosomal processing routes. Toxin uptake and activation through early endosomes but not processing through other compartments were reduced in TPC1 knockout cells. Detailed co-localization studies with subcellular markers confirmed predominant localization of TPC1 to early and recycling endosomes. Proteomic analysis of native TPC1 channels finally identified direct interaction with a distinct set of syntaxins involved in fusion of intracellular vesicles. Together, our results demonstrate a general role of TPC1 for uptake and processing of proteins in early and recycling endosomes, likely by providing high local Ca2+ concentrations required for SNARE-mediated vesicle fusion.