Abstract
Pasteurella multocida toxin (PMT) has been hypothesized to cause activation of a GTP-binding protein (G-protein)-coupled phosphatidylinositol-specific phospholipase C (PLC) in intact cells. We used voltage-clamped Xenopus oocytes to test for direct PMT-mediated stimulation of PLC by monitoring the endogenous Ca2+-dependent C1- current. Injection of PMT induced an inward, two-component Cl- current, similar to that evoked by injection of IP3 through intracellular Ca2+ mobilization and Ca2+ influx through voltage-gated Ca2+ channels. These PMT-induced currents were blocked by specific inhibitors of Ca2+ and Cl- channels, removal of extracellular Ca2+, or chelation of intracellular Ca2+. Specific antibodies directed against an N-terminal, but not a C-terminal, peptide of PMT inhibited the toxin-induced currents, implicating that the N terminus of PMT is important for toxin activity. Injection with specific antibodies against PLCbeta1, PLCbeta2, PLCbeta3, or PLCgamma1 identified PLCbeta1 as the primary mediator of the PMT-induced Cl- currents. Injection with guanosine 5'-O-(2-(thio)diphosphate), antibodies to the common GTP-binding region of G-protein alpha subunits, or antibodies to different regions of G-protein beta subunits established the involvement of a G-protein alpha subunit in PMT-activation of PLCbeta1. Injection with specific antibodies against the alpha-subunits of G(q/11), G(s/olf), G(i/o/t/z), or G(i-1/i-2/i-3) isoforms confirmed the involvement of Gq/11alpha. Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of G(q)alpha in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G(q)alpha cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G(q)alpha.
Highlights
Infections of Pasteurella multocida are associated with such severe diseases as pasteurellosis, dermonecrosis resulting from bite wounds, and the irreversible bone atrophy of progressive atrophic rhinitis [1]
The reported mitogenic response caused by P. multocida toxin (PMT) on intact cells has been hypothesized to be the result of activation of a cellular phosphatidylinositol-specific phospholipase C (PLC) [10, 11], which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol
To identify the intracellular targets involved in the PMT-induced IP3 signaling pathway, we tested the effects of specific antibodies against Gpan␣, Gq/11␣ (C-terminal and N-terminal), Gi/o/t/z␣, Gi-1/i-2/i-3␣ isoforms, Gs/olf␣, Gpan (C-terminal, internal, and N-terminal), PLC1, PLC2, PLC3, PLC␥1, an N-terminal peptide of PMT, or a C-terminal peptide of PMT on the PMT-induced ClϪ currents
Summary
Infections of Pasteurella multocida are associated with such severe diseases as pasteurellosis, dermonecrosis resulting from bite wounds, and the irreversible bone atrophy of progressive atrophic rhinitis [1]. In Gq␣-overexpressing oocytes, even at alone failed to elicit a response (representative of 2), whereas co-injection of a mixture of anti-toxA28–42 and 10 ng of IP3 evoked the characteristic Ca2ϩ-dependent ClϪ currents (representative of 2). Antibodies against PLC␥1 and PLC2 did not block (p Ͼ 0.4 for both) the Ca2ϩ-dependent ClϪ current induced by PMT; anti-PLC2 appeared to cause a 25% enhancement of the response.
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