Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

Abstract

The locations of the catalytic and receptor-binding domains of the Pasteurella multocida toxin (PMT) were investigated. N-and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed ␣/␤ domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

“…3A). The substitution of Cys 1165 with serine, glycine, or arginine abolishes the PMT activities (15,17,18), which was confirmed in this study (Fig. 3B).…”

“…Previous studies indicated that the intracellular toxic activity of PMT was attributable to its C terminus (15,17). To confirm this finding, we introduced the recombinant C-PMT (residues 569-1,285) directly into Swiss 3T3 cells with the aid of HVJ liposomes and examined the cells for elevated levels of DNA synthesis and PLC activity, which have usually been determined for the quantitative measurement of PMT toxicity (2,4,9).…”

“…PMT consists of a single polypeptide chain of 1,285 aa. Several lines of evidence indicate that the N-terminal region of the toxin binds to target cells and that the C-terminal region carries the intracellularly active moiety, the nature of which remains unknown (15)(16)(17)(18). The N-terminal region is partly homologous to Escherichia coli cytotoxic necrotizing factors, CNF1 and CNF2 (19,20).…”

Action of Pasteurella multocida Toxin Depends on the Helical Domain of Gαq

“…It is generally accepted that the toxin is structured according to a typical AB toxin. Initial studies revealed that the N terminus of PMT is involved in the binding and in translocation of the toxin into target cells (6), whereas the biologically active domain is located in the C-terminal part of the protein (6,7). This concept is in line with a significant sequence similarity of PMT at its N terminus with the N-terminal part of the cytotoxic necrotizing factor of Escherichia coli that is also involved in binding and translocation.…”

Characterisation and comparison of Pasteurella multocida isolated from different species in the Czech Republic: capsular PCR typing, ribotyping and dermonecrotoxin production

“…multocida isolates can also be differentiated based on their ability to produce dermonecrotoxin (DNT). DNT exhibits osteolytic (DiGiacomo et al, 1993;Sternerkock et al, 1995) and mitogenic activity (Lax and Grigoriadis, 2001;Pullinger et al, 2001) which results into serious lesions (Chrisp and Foged, 1991;Schimmel et al, 1992). DNT was recorded mainly in isolates originating from pigs (Nielsen et al, 1986).…”