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Mutation of a putative ADP-ribosylation motif in the Pasteurella multocida toxin does not affect mitogenic activity
P N Ward1, T E Higgins, A C Murphy
1Agricultural and Food Research Council, Institute for Animal Health, Compton, Berkshire, UK.
Abstract:
Pasteurella multocida toxin (PMT) is a potent mitogen for Swiss 3T3 fibroblasts and cytotoxic to embryonic bovine lung cells. Site-directed mutagenesis was used to investigate the functional significance of a three amino acid motif in PMT that is present in five other bacterial protein toxins which exhibit ADP-ribosyl transferase activity. Crude lysates of mutant clones were fully cytotoxic for embryonic bovine lung cells. Purified mutant toxin was also as effective at stimulating inositol phosphate turnover and nucleic acid synthesis as wild type toxin. We conclude that this motif has no functional significance in Pasteurella multocida toxin.
Insights
The Pasteurella multocida toxin (PMT) motif is not essential for its cytotoxic or mitogenic activity. Mutagenesis studies confirmed wild-type toxin function, indicating the motif
Area of Science:
- Bacteriology
- Molecular Biology
- Cell Biology
Background:
- Pasteurella multocida toxin (PMT) is a known virulence factor.
- PMT exhibits mitogenic and cytotoxic effects on mammalian cells.
- A conserved three amino acid motif in PMT is also found in other bacterial ADP-ribosyl transferase toxins.
Purpose of the Study:
- To investigate the functional significance of a specific three amino acid motif in PMT.
- To determine if this motif is essential for PMT's mitogenic and cytotoxic activities.
Main Methods:
- Site-directed mutagenesis was employed to alter the three amino acid motif in PMT.
- Mutant PMT clones were analyzed for cytotoxicity against embryonic bovine lung cells.
- Purified mutant toxin was assessed for its ability to stimulate inositol phosphate turnover and nucleic acid synthesis.
Main Results:
- Crude lysates of mutant PMT clones retained full cytotoxicity.
- Purified mutant toxin demonstrated efficacy comparable to wild-type PMT in stimulating inositol phosphate turnover.
- Purified mutant toxin showed similar nucleic acid synthesis stimulation as wild-type PMT.
Conclusions:
- The investigated three amino acid motif is not functionally significant for Pasteurella multocida toxin.
- PMT's mitogenic and cytotoxic activities do not rely on this specific conserved motif.