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Updated: Sep 26, 2026
Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major
Published on: October 28, 2022
His1205 and His1223 are essential for the activity of the mitogenic Pasteurella multocida toxin
Joachim H C Orth1, Dagmar Blöcker, Klaus Aktories
1Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.
Abstract:
Pasteurella multocida produces a 146-kDa protein toxin (PMT), which activates multiple cellular signal-transduction pathways, resulting in the activation of PLCbeta, Rho, JNK, and ERK. In addition to an essential cysteine residue at position 1165, PMT contains several histidine residues in the catalytically important C-terminal part of the protein. To elucidate the role of the histidine residues, we treated PMT with the histidine-modifying substance diethyl pyrocarbonate (DEPC). DEPC inhibited PMT in a time- and concentration-dependent manner, suggesting that one or several histidine residues are essential for the biological activity of PMT. In experiments in which PMT was directly delivered into the cytosol of EBL cells by electroporation, we show that DEPC treatment inhibits the catalytically important histidine residues. Leucine substitutions of eight individual histidine residues in the C-terminal catalytic domain of PMT were constructed, and the effect on the biological activity of PMT was analyzed by determining PLCbeta, Rho, and ERK activation. Substitution of two histidine residues, H1205 and H1223, led to inactivation of the resulting PMT proteins, indicating that H1205 and H1223 play an important role in biological activity of the toxin. In addition, we show that the mutant toxins appear to be correctly folded, as judged by protease digestion. The precise function of H1205 and H1223 is not yet known. However, treatment of PMT with the cation chelating substance 1,10-phenantroline led to inactivation of the toxin, indicating that the essential histidine residues and cysteine 1165 might be involved in metal ion binding.
Insights
This study reveals that histidine residues H1205 and H1223 are crucial for the Pasteurella multocida toxin's (PMT) activity, impacting cellular signaling pathways. These findings suggest their potential role in metal ion binding, essential for PMT function.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pasteurella multocida toxin (PMT) is a 146-kDa protein that disrupts cellular signaling pathways, including PLCbeta, Rho, JNK, and ERK.
- PMT possesses an essential cysteine at position 1165 and several histidine residues in its catalytic C-terminal domain.
Purpose of the Study:
- To investigate the role of histidine residues in the catalytic activity of PMT.
- To identify specific histidine residues critical for PMT's biological function.
Main Methods:
- Treatment of PMT with diethyl pyrocarbonate (DEPC), a histidine-modifying agent.
- Electroporation of PMT into EBL cells to assess direct cytosolic effects.
- Site-directed mutagenesis of histidine residues to leucine and analysis of toxin activity.
- Protease digestion to evaluate the structural integrity of mutant PMT proteins.
Main Results:
- DEPC inhibited PMT activity in a time- and concentration-dependent manner, indicating essential histidine residues.
- Mutagenesis revealed that substitutions at H1205 and H1223 inactivated PMT, highlighting their importance.
- Mutant toxins retained correct folding, as confirmed by protease digestion assays.
- 1,10-phenanthroline treatment inactivated PMT, suggesting histidine residues and Cys1165 may bind metal ions.
Conclusions:
- Histidine residues H1205 and H1223 are critical for the catalytic activity of Pasteurella multocida toxin.
- The findings suggest a potential role for these histidine residues, along with Cys1165, in metal ion binding, which is vital for PMT's function.
