King's College London

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, G(q), 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 Galpha(q), and that this is one potential molecular target for the toxin. Galpha(q) 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 Galpha(q), tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha(q) phosphorylation without leading to its activation. Phosphorylation of Galpha(q) triggered by the inactive mutant potentiates activation of G(q) via a GPCR, demonstrating that phosphorylation of G(q) 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.