In Vivo Targets of Pasteurella Multocida Toxin.

Many Pasteurella multocida strains are carried as commensals, while some cause disease in animals and humans. Some type D strains cause atrophic rhinitis in pigs, where the causative agent is known to be the Pasteurella multocida toxin (PMT). PMT activates three families of G-proteins-Gq/11, G12/13, and Gi/o-leading to cellular mitogenesis and other sequelae. The effects of PMT on whole animals in vivo have been investigated previously, but only at the level of organ-specific pathogenesis. We report here the first study to screen all the organs targeted by the toxin by using the QE antibody that recognizes only PMT-modified G-proteins. Under our experimental conditions, short-term treatment of PMT is shown to have multiple in vivo targets, demonstrating G-alpha protein modification, stimulation of proliferation markers and expression of active ß-catenin in a tissue- and cell-specific manner. This highlights the usefulness of PMT as an important tool for dissecting the specific roles of different G-alpha proteins in vivo.

Promoter orientation of the immunomodulatory Bacteroides fragilis capsular polysaccharide A (PSA) is off in individuals with inflammatory bowel disease (IBD).

Bacteroides fragilis is a member of the normal microbiota of the lower gastrointestinal tract, but some strains produce the putative tumourigenic B. fragilis toxin (BFT). In addition, B. fragilis can produce multiple capsular polysaccharides that comprise a microcapsule layer, including an immunomodulatory, zwitterionic, polysaccharide A (PSA) capable of stimulating anti-inflammatory interleukin-10 (IL-10) production. It is known that the PSA promoter can undergo inversion, thereby regulating the expression of PSA. A PCR digestion technique was used to investigate B. fragilis capsular PSA promoter orientation using human samples for the first time. It was found that approximately half of the B. fragilis population in a healthy patient population had PSA orientated in the 'ON' position. However, individuals with inflammatory bowel disease (IBD) had a significantly lower percentage of the B. fragilis population with PSA orientated 'ON' in comparison with the other patient cohorts studied. Similarly, the putative tumourigenic bft-positive B. fragilis populations were significantly associated with a lower proportion of the PSA promoter orientated 'ON'. These results suggest that the proportion of the B. fragilis population with the PSA promoter 'ON' may be an indicator of gastrointestinal health.

G-Alpha Subunit Abundance and Activity Differentially Regulate ß-Catenin Signaling.

Heterotrimeric G proteins are signal transduction proteins involved in regulating numerous signaling events. In particular, previous studies have demonstrated a role for G-proteins in regulating ß-catenin signaling. However, the link between G-proteins and ß-catenin signaling is controversial and appears to depend on G-protein specificity. We describe a detailed analysis of a link between specific G-alpha subunits and ß-catenin using G-alpha subunit genetic knockout and knockdown approaches. The Pasteurella multocida toxin was utilized as a unique tool to activate G-proteins, with LiCl treatment serving as a ß-catenin signaling agonist. The results show that Pasteurella multocida toxin (PMT) significantly enhanced LiCl-induced active ß-catenin levels in HEK293T cells and mouse embryo fibroblasts. Evaluation of the effect of specific G-alpha proteins on the regulation of ß-catenin showed that Gq/11 and G12/13 knockout cells had significantly higher levels of active and total ß-catenin than wild-type cells. The stimulation of active ß-catenin by PMT and LiCl was lost upon both constitutive and transient knockdown of G12 and G13 but not Gq Based on our results, we conclude that endogenous G-alpha proteins are negative regulators of active ß-catenin; however, PMT-activated G-alpha subunits positively regulate LiCl-induced ß-catenin expression in a G12/13-dependent manner. Hence, G-alpha subunit regulation of ß-catenin is context dependent.

The actions of Pasteurella multocida toxin on neuronal cells.

Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(1₋3), Gα(q), 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 Gα(q) 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.

Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q), G(12) and G(i). PMT has been shown by others to lead to the deamidation of recombinant Gα(i) at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q) and G(i) families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11), a member of the G(q) family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13) was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s) was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i), but not Gα(q). We also show that PMT inhibits the GTPase activity of G(q).

New genotoxin shows diversity of bacterial attack mechanisms.

Bacteria make a wide range of toxic products that interact with eukaryotic cellular machinery in a precise way. These toxins interfere with key eukaryotic processes, such as cellular signalling components, and some directly attack the genome. Nougayrède and colleagues have recently identified a novel hybrid peptide-polyketide compound from Escherichia coli that leads to DNA damage. This novel compound is produced by pathogenic and, most interestingly, commensal isolates. Although it is not yet clear how the peptide-polyketide compound functions at the molecular level, it is possible that it contributes to bacterial pathogenesis and bacterially induced carcinogenesis.

Histidine Residues at the Active Site of the Pasteurella multocida Toxin.

We have investigated histidine residues near the active site of the mitogenic Pasteurella multocida toxin. Mutation of H1202 or H1228 had little effect, while the effect of mutation on H1223 depended on the amino acid substituted. Mutation of H1205 caused complete loss of activity, indicating its importance in PMT activity.

Opinion: Bacterial toxins and cancer--a case to answer?

Since the discovery that Helicobacter pylori infection leads to gastric cancer, other chronic bacterial infections have been shown to cause cancer. The bacterial and host molecular mechanisms remain unclear. However, many bacteria that cause persistent infections produce toxins that specifically disrupt cellular signalling to perturb the regulation of cell growth or to induce inflammation. Other bacterial toxins directly damage DNA. Such toxins mimic carcinogens and tumour promoters and might represent a paradigm for bacterially induced carcinogenesis.

Identification and characterization of the Pasteurella multocida toxin translocation domain.

The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 - residues 402-423, H2 - 437-457) linked by a hydrophilic loop (PMT-TL - 424-436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helix-loop-helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion.

The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation.

Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture. It is an intracellularly acting toxin that stimulates several signal transduction pathways. The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates. The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements. Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains. The location of all three domains has been confirmed directly. Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain. Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain. Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin. Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process. We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation. Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling. Natural or experimental infection of animals with toxigenic P. multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone. The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone. PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin. These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation. Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro. In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells.

Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation.

UNLABELLED: The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP-2 and -4 expression. These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation. INTRODUCTION: The signal transduction pathways controlling osteoblast differentiation are not well understood. In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro. MATERIALS AND METHODS: Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation. Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy. RESULTS: PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression. There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation. PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK. Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation. Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and -4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation. CONCLUSIONS: These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation. Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone.

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage.

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.

Pasteurella multocida toxin facilitates inositol phosphate formation by bombesin through tyrosine phosphorylation of G alpha q.

The intracellularly acting Pasteurella multocida toxin (PMT) is a potent mitogen that stimulates Gq-dependent formation of inositol trisphosphate. We show that PMT, a nontoxic mutant of PMT (PMTC1165S), and bombesin each stimulate time-dependent phosphorylation of G alpha q at tyrosine 349. Although PMT and PMTC1165S each cause phosphorylation of G alpha q, only the wild-type toxin activates Gq. Pretreatment of cells with wild-type or mutant PMT potentiated the formation of inositol phosphates stimulated by bombesin equally. These data show that PMT potentiates bombesin receptor signaling through tyrosine phosphorylation of Gq and distinguishes between the two proposed models of Gq activation, showing that tyrosine phosphorylation is not linked to receptor uncoupling.

How bacteria could cause cancer: one step at a time.

Helicobacter pylori highlighted the potential for bacteria to cause cancer. It is becoming clear that chronic infection with other bacteria, notably Salmonella typhi, can also facilitate tumour development. Infections caused by several bacteria (e.g. Bartonella spp., Lawsonia intracellularis and Citrobacter rodentium) can induce cellular proliferation that can be reversed by antibiotic treatment. Other chronic bacterial infections have the effect of blocking apoptosis. However, the underlying cellular mechanisms are far from clear. Conversely, several bacterial toxins interfere with cellular signalling mechanisms in a way that is characteristic of tumour promoters. These include Pasteurella multocida toxin, which uniquely acts as a mitogen, and Escherichia coli cytotoxic necrotizing factor, which activates Rho family signalling. This leads to activation of COX2, which is involved in several stages of tumour development, including inhibition of apoptosis. Such toxins could provide valuable models for bacterial involvement in cancer, but more significantly they could play a direct role in cancer causation and progression.

Role of the dermonecrotic toxin of Bordetella bronchiseptica in the pathogenesis of respiratory disease in swine.

Bordetella bronchiseptica is one of the etiologic agents causing atrophic rhinitis and pneumonia in swine. It produces several purported virulence factors, including the dermonecrotic toxin (DNT), which has been implicated in the turbinate atrophy seen in cases of atrophic rhinitis. The purpose of these experiments was to clarify the role of this toxin in respiratory disease by comparing the pathogenicity in swine of two isogenic dnt mutants to their virulent DNT(+) parent strains. Two separate experiments were performed, one with each of the mutant-parent pairs. One-week-old cesarean-derived, colostrum-deprived pigs were inoculated intranasally with the parent strain, the dnt mutant strain, or phosphate-buffered saline. Weekly nasal washes were performed to monitor colonization of the nasal cavity, and the pigs were euthanized 4 weeks after inoculation to determine colonization of tissues and to examine the respiratory tract for pathology. There was evidence that colonization of the upper respiratory tract, but not the lower respiratory tract, was slightly greater for the parent strains than for the dnt mutants. Moderate turbinate atrophy and bronchopneumonia were found in most pigs given the parent strains, while there was no turbinate atrophy or pneumonia in pigs challenged with the dnt mutant strains. Therefore, production of DNT by B. bronchiseptica is necessary to produce the lesions of turbinate atrophy and bronchopneumonia in pigs infected with this organism.