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Infection and Immunity, January 1999, p. 80-87, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

Brenda A. Wilson,* Virgilio G. Ponferrada, Jefferson E. Vallance, and Mengfei Ho

Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435

Received 12 May 1998/Returned for modification 29 July 1998/Accepted 19 October 1998

We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqalpha protein that is coupled to phosphatidylinositol-specific phospholipase Cbeta 1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268-1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Col. Glenn Hwy., Dayton, OH 45435. Phone: (937) 775-4803. Fax: (937) 775-3730. E-mail: bwilson{at}wright.edu.

Infection and Immunity, January 1999, p. 80-87, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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