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Infection and Immunity, December 2001, p. 7839-7850, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7839-7850.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

Gillian D. Pullinger,1,* R. Sowdhamini,2,dagger and Alistair J. Lax1

Department of Oral Microbiology, Kings College London, Guy's Hospital, London SE1 9RT,1 and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW,2 United Kingdom

Received 10 July 2001/Returned for modification 24 August 2001/Accepted 6 September 2001

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 alpha /beta domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

* Corresponding author. Mailing address: Department of Oral Microbiology, Floor 28, Guy's Tower, Guy's Hospital, London SE1 9RT, United Kingdom. Phone: 44-020-7955-5000, ext. 5612. Fax: 44-020-7955-2847. E-mail: gillian.pullinger{at}kcl.ac.uk.

dagger Present address: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560 065, India.

Infection and Immunity, December 2001, p. 7839-7850, Vol. 69, No. 12
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.12.7839-7850.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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