Action Of Pasteurella Multocida Toxin Research Articles

Involvement of Osteocytes in the Action of Pasteurella multocida Toxin.

Pasteurella multocida toxin (PMT) causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. It has been reported that the toxin deamidates and activates heterotrimeric G proteins, resulting in increased differentiation of osteoclasts and blockade of osteoblast differentiation. So far, the action of PMT on osteocytes, which is the most abundant cell type in bone tissue, is not known. In MLO-Y4 osteocytes, PMT deamidated heterotrimeric G proteins, resulting in loss of osteocyte dendritic processes, stress fiber formation, cell spreading and activation of RhoC but not of RhoA. Moreover, the toxin caused processing of membrane-bound receptor activator of NF-κB ligand (RANKL) to release soluble RANKL and enhanced the secretion of osteoclastogenic TNF-α. In a co-culture model of osteocytes and bone marrow cells, PMT-induced osteoclastogenesis was largely increased as compared to the mono-culture model. The enhancement of osteoclastogenesis observed in the co-culture was blocked by sequestering RANKL with osteoprotegerin and by an antibody against TNF-α indicating involvement of release of the osteoclastogenic factors from osteocytes. Data support the crucial role of osteocytes in bone metabolism and osteoclastogenesis and identify osteocytes as important target cells of PMT in progressive atrophic rhinitis.

The actions of Pasteurella multocida toxin on neuronal cells

Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(1-3), Gαq, Gα11, Gα12 and Gα13 by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gαq and Gα11 could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration–inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP2 hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected.M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y2 receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y2 receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.

Pasteurella multocida toxin (PMT) upregulates CTGF which leads to mTORC1 activation in Swiss 3T3 cells

Pasteurella multocida toxin (PMT) is a mitogenic protein that hijacks cellular signal transduction pathways via deamidation of heterotrimeric G proteins. We previously showed that rPMT activates mTOR signaling via a Gαq/11/PLCβ/PKC mediated pathway, leading in part to cell proliferation and migration. Herein, we show that mTOR and MAPK, but not membrane-associated tyrosine kinases, are activated in serum-starved 3T3 cells by an autocrine/paracrine substance(s) secreted into the conditioned medium following rPMT treatment. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gαq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in rPMT-treated serum-starved 3T3 cells relative to untreated cells. These results were further confirmed using RT-PCR and Western blot analyses. In accord with rPMT-induced mTOR activation, upregulation of CTGF protein was observed in WT MEF, but not in Gαq/11 double knockout MEF cells. Although CTGF expression is regulated by TGFβ, rPMT did not activate TGFβ pathway. In addition, MEK inhibitors U0126 or PD98059, but not mTOR specific inhibitors, rapamycin and Torin 1, inhibited rPMT-induced upregulation of CTGF. Importantly, CTGF overexpression in serum-starved 3T3 cells using adenovirus led to phosphorylation of ribosomal protein S6, a downstream target of mTOR. However, despite the ability of CTGF to activate the mTOR pathway, upregulation of CTGF alone could not induce morphological changes as those observed in rPMT-treated cells. Our findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.

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 PLCβ via Gα q. Recent studies indicate that PMT additionally activates Gα i to inhibit adenylyl cyclase. Here we show that PMT acts not only via Gα but also through Gβγ signaling. Activation of Gβγ by PMT causes stimulation of phosphoinositide 3-kinase (PI3K) γ 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 Gβγ is necessary for PMT-induced signaling via Gα. Mutants of Gα q incapable of binding or releasing Gβγ are not activated by PMT. Similarly, sequestration of Gβγ inhibits PMT-induced Gα-signaling.

Action of Pasteurella multocida toxin on Gαq is persistent and independent of interaction with G-protein-coupled receptors

Pasteurella multocida toxin (PMT) activates Gαq and facilitates stimulation of inositol phosphate accumulation induced by agonists via Gq-coupled membrane receptors. Here, we studied the effects of PMT on agonist-induced GTPγS binding to Gq in cell membranes and a role of G-protein-coupled receptors in the action of PMT. Pre-treatment of Swiss 3T3 cells with PMT increased bombesin or vasopressin-induced GTPγS-binding in cell membranes by about 50 to 150%. Increase in agonist-stimulated GTPγS-binding caused by PMT pretreatment was specific for Gαq and not observed with Gα11. PMT-induced effects on GTPγS-binding were persistent after removing the toxin or in the presence of anti-PMT antibody. Stimulation of agonist-induced GTPγS-binding by PMT was independent of phosphorylation of the C-terminal tyrosine356 of Gαq. Activation of phospholipase C by PMT occurred via Gαq which was fused to the α1b-adrenoceptor and also with a C-terminally deleted Gαq, which is not able to interact with G protein-coupled membrane receptors. The data indicate that activation of Gαq by PMT is persistent and independent of a functional interaction of Gq with G-protein-coupled receptors.

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of ?-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.

Pasteurella multocida toxin as a tool for studying Gq signal transduction.

Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C (PLC) signal transduction through its selective action on the Galphaq subunit. This review summarizes what is currently known about the molecular action of PMT on Gq and the resulting cellular effects. Examples are presented illustrating the use of PMT as a powerful tool for dissecting the molecular mechanisms involving pertussis toxin (PT)-insensitive heterotrimeric G proteins.

Pleiotropic Effects of Pasteurella multocida Toxin Are Mediated by Gq-dependent and -independent Mechanisms: INVOLVEMENT OF Gq BUT NOT G11

Pasteurella multocida toxin (PMT) is a highly potent mitogen for a variety of cell types. PMT has been shown to induce various cellular signaling processes, and it has been suggested to function through the heterotrimeric G-proteins G(q)/G(11). To analyze the role of G(q)/G(11) in the action of PMT, we have studied the effect of the toxin in Galpha(q)/Galpha(11) double-deficient fibroblasts as well as in fibroblasts lacking only Galpha(q) or Galpha(11). Interestingly, formation of inositol phosphates in response to PMT was exclusively dependent on Galpha(q) but not on the closely related Galpha(11). Although Galpha(q)/Galpha(11) double-deficient and Galpha(q)-deficient cells did not respond with any production of inositol phosphates to PMT, PMT was still able to induce various other cellular effects in these cells, including the activation of Rho, the Rho-dependent formation of actin stress fibers and focal adhesions, as well as the stimulation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. These data show that PMT leads to a variety of cellular effects that are mediated only in part by the heterotrimeric G-protein G(q).

Pasteurella multocida toxin: the mitogenic toxin that stimulates signalling cascades to regulate growth and differentiation

Abstract Pasteurella multocida toxin (PMT) is an unusual toxin that acts as a mitogen by stimulating various intracellular signalling cascades. Pathways downstream of the G-protein G q and also downstream of the Rho proteins are activated. Thus PMT action stimulates phospholipase C leading to activation of protein kinase C, an increase in inositol phosphates, and a rise in intracellular calcium. Rho activation of the Rho kinase leads to cytoskeletal reorganisation, tyrosine phosphorylation of the focal adhesion kinase, and activation of the Src proto-oncogene. In addition, signalling through the Ras-MAP kinase signalling pathway is also initiated. PMT is an intracellularly acting toxin, and functional domains that carry out different aspects of its function have been described. The intracellular target of the toxin is currently not known. PMT also acts to inhibit differentiation, in particular of bone cells, where it prevents the formation of mineralised bone nodules in vitro. The toxin is the causative agent of a porcine disease that is characterised by bone resorption. Injection of very low doses of toxin leads to proliferative effects, but at higher doses is lethal. The possible effect of PMT-induced perturbation of signal transduction pathways is discussed.

Effects of Pasteurella multocida toxin on porcine bone marrow cell differentiation into osteoclasts and osteoblasts.

The effect of Pasteurella multocida toxin (PMT) on porcine osteoclast and osteoblast differentiation was studied using in vitro cell culture systems. When grown in the presence of Vitamin D3, isolated porcine bone marrow cells formed multinucleated cells with features characteristic of osteoclasts. Exposure of bone marrow cells to Vitamin D3 and PMT during growth resulted in formation of increased numbers and earlier appearance of osteoclasts compared to controls. Ultrafiltered medium form PMT-treated cells likewise increased osteoclast numbers, suggesting that a soluble mediator may be involved in the action of PMT. When cell cultures were treated with fluorescein-labeled PMT, fluorescence was found within the cytoplasm of small, round cells that did not resemble either osteoclasts or osteoclastic precursor cells. Cultures of porcine bone marrow cells exposed to dexamethasone, ascorbic acid, and beta-glycerophosphate developed into osteoblastic cells that formed multilayered, mineralized nodules. Exposure of osteoblastic cultures to low concentration of PMT resulted in retarded cell growth, formation of decreased numbers of nodules and minimal to no mineralization in the nodules; higher concentration of PMT resulted in increased cellular debris and poor growth of cells, with no nodule formation. These findings suggest that PMT may induce turbinate atrophy in pigs by increasing osteoclast numbers and inhibiting osteoblastic bone formation. The effect of PMT on osteoclastic differentiation and growth may not be due to a direct effect on preosteoclastic cells, but rather due to alterations in the soluble mediator secretion by marrow stromal cells.

Interconversion of GRP78/BiP. A novel event in the action of Pasteurella multocida toxin, bombesin, and platelet-derived growth factor.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin.

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.