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Identification and characterization of the Pasteurella multocida toxin translocation domain

Lookup NU author(s): Professor Jeremy Lakey

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Abstract

The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coll cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted Hydrophobic helices (H1 - residues 402-423, H2 - 437-457) linked by a hydrophilic loop (PMT-TL - 424-436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helix-loop-helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion.


Publication metadata

Author(s): Baldwin MR, Lakey JH, Lax AJ

Publication type: Article

Publication status: Published

Journal: Molecular Microbiology

Year: 2004

Volume: 54

Issue: 1

Pages: 239-250

ISSN (print): 0950-382X

ISSN (electronic): 1365-2958

Publisher: Wiley-Blackwell

URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04264.x

DOI: 10.1111/j.1365-2958.2004.04264.x

PubMed id: 15458419


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