Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida

Smyth, Martin G.

doi:10.1016/s0378-1097(99)00456-5

Cited by 6 publications

(15 citation statements)

References 15 publications

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“…CNF1 and PMT share 24% homology in the first 500 amino acids of their respective N termini and show the highest degree of similarity in regions that are proposed to encompass transmembrane domains in both toxin molecules (amino acids 199 to 420 of CNF1 and amino acids 245 to 467 of PMT) (14,36,40,41). Despite this protein homology, recent work has indicated that the intracellular activity of PMT lies within the N terminus of the toxin.…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

2001

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Cytotoxic necrotizing factor type 1 (CNF1) of uropathogenic Escherichia coli belongs to a family of bacterial toxins that target the small GTP-binding Rho proteins that regulate the actin cytoskeleton. Members of this toxin family typically inactivate Rho; however, CNF1 and the highly related CNF2 activate Rho by deamidation. Other investigators have reported that the first 190 amino acids of CNF1 constitute the cellular binding domain and that the CNF1 enzymatic domain lies within a 300-amino-acid stretch in the C terminus of the toxin. Amino acids 53 to 75 appear to be critical for cell receptor recognition, while amino acids Cys866 and His881 are considered essential for deamidation activity. To delineate further the functional domains of CNF1, we generated 16 monoclonal antibodies (MAbs) against the toxin and used them for epitope mapping studies. Based on Western blot immunoreactivity patterns obtained from a series of truncated CNF1 proteins, this panel of MAbs mapped to epitopes located throughout the toxin, including the binding and enzymatic domains. All MAbs showed reactivity to CNF1 by Western and dot blot analyses. However, only 7 of the 16 MAbs exhibited crossreactivity with CNF2. Furthermore, only three MAbs demonstrated the capacity to neutralize toxin in either HEp-2 cell assays (inhibition of multinucleation) or 5637 bladder cell assays (inhibition of cytotoxicity). Since CNF1 epitopes recognized by neutralizing MAbs are likely to represent domains or regions necessary for the biological activities of the toxin, the epitopes recognized by these three MAbs, designated JC4 (immunoglobulin G2a [IgG2a]), BF8 (IgA), and NG8 (IgG2a), were more precisely defined. MAbs JC4 and BF8 reacted with epitopes that were common to CNF1 and CNF2 and located within the putative CNF1 binding domain. MAb JC4 recognized an epitope spanning amino acids 169 to 191, whereas MAb BF8 mapped to an epitope between amino acids 135 and 164. Despite the capacity of both MAbs to recognize CNF2 in Western blot analyses, only MAb BF8 neutralized CNF2. MAb NG8 showed reactivity to a CNF1-specific epitope located between amino acids 683 and 730, a region that includes a very small portion of the putative enzymatic domain. Taken together, these findings identify three new regions of the toxin that appear to be critical for the biological activity of CNF1.

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Section: Discussionmentioning

confidence: 99%

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

2001

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“…Pasteurella multocida toxin is an intracellularly acting toxin Smyth et al ., 1995;1999). It is thought to bind to a ganglioside cell-surface receptor, and enter cells by endocytosis (Pettit et al ., 1993), in order to modify its target.…”

Section: Introductionmentioning

confidence: 99%

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Pullinger

Bevir

Lax

2003

Molecular Microbiology

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SummaryToxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA , previously suggested it was horizontally acquired, because it had a low G + + + + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.

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“…Its action is also inhibited by neutralizing antibody or methylamine added early but not late after toxin. PMT undergoes a conformational change at low pH, which affects its protease sensitivity and circular dichroism spectra (46,47). This suggests that PMT may be trafficked and perhaps processed through a low-pH compartment.…”

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confidence: 99%

Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001

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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.

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Reduced pH causes structural changes in the potent mitogenic toxin of Pasteurella multocida

Cited by 6 publications

References 15 publications

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

Epitope Mapping of Monoclonal Antibodies Capable of Neutralizing Cytotoxic Necrotizing Factor Type 1 of Uropathogenic Escherichia coli

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

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