Pasteurella multocida toxin (PMT) is a major virulence factor associated with progressive atrophic rhinitis, respiratory disease in animals, and dermonecrosis, respiratory disease, and bacteremia in humans resulting from bite wounds or exposure to infected animals. PMT is a 1285 amino acid protein that can act on multiple cell types. It enters mammalian cells via receptor-mediated endocytosis and activates intracellular signal transduction events, including phospholipid hydrolysis, calcium mobilization, protein phosphorylation, DNA synthesis, and cytoskeletal rearrangements, which cause cell proliferation. We have demonstrated that the PMT-mediated stimulation of phospholipase C activity occurs through transient, but irreversible PMT action on the Gq protein. We have proposed a model for PMT action. We also characterized a number of the Gq-dependent pathways using PMT, and our results have led us to hypothesize that the pleiotropic effects of PMT on different cells is due to the diverse roles that the Gq target plays in the different cell types. In addition, we determined that the N-terminus of PMT is important for intracellular activity and that both N- and C-termini are important for binding and entry into mammalian cells. Our hypothesis is that PMT entry is mediated through multiple binding determinants on the toxin protein and through multiple receptors. Our long-range goals are to understand the structure and mechanism of action of PMT at the molecular and biochemical level, to facilitate future therapeutic intervention in P. multocida disease and to increase our preparedness against potential bacterial toxin-related threats involving similar mechanisms, as well as to increase our understanding of the molecular signaling events involved in Gq-dependent signaling. To achieve our goals, we propose the following Aims: (1) To elucidate the molecular mechanism by which PMT acts on the Gq-protein, by determining whether the effect of PMT on Gq-protein is caused by covalent modification or by direct or indirect protein interaction. (2) To determine the biochemical basis for the effect of PMT on Gq-coupled signal transduction, (I) by determining the effect of PMT on Gq activity and (II) by determining the effect of PMT on downstream Gq-signaling pathways. (3) To define the functional domain(s) of PMT responsible for binding eukaryotic cell receptors and translocating the intracellular activity domain into the cytosol. (4) To characterize the cellular receptor(s) and to elucidate the internalization pathway(s) utilized by PMT to gain entry into cells.
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