Joachim H. C. Orth

Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation

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 ≈145 kDa protein toxin stimulates various signal transduction pathways by activating heterotrimeric G proteins of the Gαq, Gαi, and Gα12/13 families by using an as yet unknown mechanism. Here, we show that Pasteurella multocida toxin deamidates glutamine-205 of Gαi2 to glutamic acid. Therefore, the toxin inhibits the intrinsic GTPase activity of Gαi and causes persistent activation of the G protein. A similar modification is also evident for Gαq, but not for the closely related Gα11, which is not a substrate of Pasteurella multocida toxin. Our data identify the α-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.

Pasteurella multocida Toxin-induced Activation of RhoA Is Mediated via Two Families of Gα Proteins, Gαq and Gα12/13

Pasteurella multocida toxin (PMT) is a potent mitogen, which is known to activate phospholipase Cβ by stimulating the α-subunit of the heterotrimeric G protein Gq. PMT also activates RhoA and RhoA-dependent pathways. Using YM-254890, a specific inhibitor of Gq/11, we studied whether activation of RhoA involves G proteins other than Gq/11. YM-254890 inhibited PMT or muscarinic M3-receptor-mediated stimulation of phospholipase Cβ at similar concentrations in HEK293m3 cells. In these cells, PMT-induced RhoA activation and enhancement of RhoA-dependent luciferase activity were partially inhibited by YM-254890. In Gαq/11-deficient fibroblasts, PMT induced activation of RhoA, increase in RhoA-dependent luciferase activity, and increase in ERK phosphorylation. None of these effects were influenced by YM-254890. However, RhoA activation by PMT was inhibited by RGS2, RGS16, lscRGS, and dominant negative G13GA, indicating involvement of Gα12/13 in the PMT effect on RhoA. In Gα12/13 gene-deficient cells, PMT-induced stimulation of RhoA, luciferase activity, and ERK phosphorylation were blocked by YM-254890, indicating the involvement of Gq. Infection with a virus harboring the gene of Gα13 reconstituted the increase in RhoA-dependent luciferase activity by PMT even in the presence of YM-254890. The data show that YM-254890 is able to block PMT activation of Gαq and indicate that, in addition to Gαq, the Gα12/13 G proteins are targets of PMT.

Biological activity of a C-terminal fragment of Pasteurella multocida toxin.

ABSTRACT The protein toxin of Pasteurella multocida PMT is a potent mitogen and activator of phospholipase Cβ. 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.

Activation of Gαi and Subsequent Uncoupling of Receptor-Gαi Signaling by Pasteurella multocida Toxin

Bacterial protein toxins are powerful tools for elucidating signaling mechanisms in eukaryotic cells. A number of bacterial protein toxins, e.g. cholera toxin, pertussis toxin (PTx), or Pasteurella multocida toxin (PMT), target heterotrimeric G proteins and have been used to stimulate or block specific signaling pathways or to demonstrate the contribution of their target proteins in cellular effects. PMT is a major virulence factor of P. multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PMT modulates various signaling pathways, including phospholipase Cβ and RhoA, by acting on the heterotrimeric G proteins Gαq and Gα12/13, respectively. Here we report that PMT is a powerful activator of Gi protein. We show that PMT decreases basal isoproterenol and forskolin-stimulated cAMP accumulation in intact Swiss 3T3 cells, inhibits adenylyl cyclase activity in cell membrane preparations, and enhances the inhibition of cAMP accumulation caused by lysophosphatidic acid via endothelial differentiation gene receptors. PMT-mediated inhibition of cAMP production is independent of toxin activation of Gαq and/or Gα12/13. Although the effects of PMT are not inhibited by PTx, PMT blocks PTx-catalyzed ADP-ribosylation of Gi. PMT also inhibits steady-state GTPase activity and GTP binding of Gi in Swiss 3T3 cell membranes stimulated by lysophosphatidic acid. The data indicate that PMT is a novel activator of Gi, modulating its GTPase activity and converting it into a PTx-insensitive state.

A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins

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. 1H-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.

Modulation of host cell gene expression through activation of STAT transcription factors by Pasteurella multocida toxin.

The Pasteurella multocida toxin (PMT) is highly mitogenic and has potential carcinogenic properties. PMT causes porcine atrophic rhinitis that is characterized by bone resorption and loss of nasal turbinates, but experimental nasal infection also leads to excess proliferation of bladder epithelial cells. PMT acts intracellularly and activates phospholipase C-linked signals and MAPK pathways via the heterotrimeric Gαq and Gα12/13 proteins. We found that PMT induces activation of STAT proteins, and we identified STAT1, STAT3, and STAT5 as new targets of PMT-induced Gαq signaling. Inhibition of Janus kinases completely abolished STAT activation. PMT-dependent STAT phosphorylation remained constitutive for at least 18 h. PMT caused down-regulation of the expression of the suppressor of cytokine signaling-3, indicating a novel mechanism to maintain activation of STATs. Moreover, stimulation of Swiss 3T3 cells with PMT increased transcription of the cancer-associated STAT-dependent gene cyclooxygenase-2. Because constitutive activation of STATs has been found in a number of cancers, our findings offer a new mechanism for a carcinogenic role of PMT.

Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins.

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gαi2, it was demonstrated that the toxin deamidates an essential glutamine residue of the Gαi2 subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G‐protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gαq/11, Gαi1,2,3, and Gα12/13 of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G‐protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.—Orth, J. H. C., Fester, I., Siegert, P., Weise, M., Lanner, U., Kamitani, S., Tachibana, T, Wilson, B. A., Schlosser, A., Horiguchi, Y., Aktories, K. Substrate specificity of Pasteurella multocida toxin for α subunits of heterotrimeric G proteins. FASEB J. 27, 832–842 (2013). www.fasebj.org

Pasteurella multocida toxin activates Gβγ dimers of heterotrimeric G proteins

The mitogenic Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida, which causes Pasteurellosis in man and animals. The toxin activates the small GTPase RhoA, the MAP kinase ERK and STAT proteins via the stimulation of members of two G protein families, G(q) and G(12/13). PMT action also results in an increase in inositol phosphates, which is due to the stimulation of PLCbeta via Galpha(q). Recent studies indicate that PMT additionally activates Galpha(i) to inhibit adenylyl cyclase. Here we show that PMT acts not only via Galpha but also through Gbetagamma signaling. Activation of Gbetagamma by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) gamma and formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)) as indicated by the recruitment of a PIP(3)-binding pleckstrin homology (PH) domain-containing protein to the plasma membrane. Moreover, it is demonstrated that Gbetagamma is necessary for PMT-induced signaling via Galpha. Mutants of Galpha(q) incapable of binding or releasing Gbetagamma are not activated by PMT. Similarly, sequestration of Gbetagamma inhibits PMT-induced Galpha-signaling.

Adrenergic Repression of the Epigenetic Reader MeCP2 Facilitates Cardiac Adaptation in Chronic Heart Failure.

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Pasteurella multocida toxin

The major virulence factor of Pasteurella multocida responsible for atrophic rhinitis, pneumonia-like respiratory disease, and dermonecrosis is a monomeric 1285 amino acid protein toxin (PMT, Pasteurella multocida toxin) produced primarily by capsular type D and some capsular type A strains. Recently, the molecular mechanism of PMT was defined as the specific deamidation of a crucial glutamine residue in the α-subunit of heterotrimeric G proteins. As a consequence of PMT action, the G proteins are constitutively activated, which results in modulation of multiple downstream signaling pathways leading to pleiotropic cellular effects, including mitogenesis and proliferation, as well as the manipulation of cell differentiation and other cell fate decisions involved in adipogenesis, osteogenesis, and immunity. This chapter reviews what is currently known about the structure, molecular mechanism, and substrate specificity of PMT, as well as its interaction with and effects on mammalian cells and its role in pathogenesis.