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Updated: Dec 24, 2025
Deciphering the Molecular Mechanism and Function of Pore-Forming Toxins Using Leishmania major
Published on: October 28, 2022
Molecular and Functional Analysis of Pore-Forming Toxin Monalysin From Entomopathogenic Bacterium Pseudomonas
Saori Nonaka1, Emil Salim1,2, Koki Kamiya3,4
1Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan.
Abstract:
Pseudomonas entomophila is a highly pathogenic bacterium that infects insects. It is also used as a suitable model pathogen to analyze Drosophila's innate immunity. P. entomophila's virulence is largely derived from Monalysin, a β-barrel pore-forming toxin that damages Drosophila tissues, inducing necrotic cell death. Here we report the first and efficient purification of endogenous Monalysin and its characterization. Monalysin is successfully purified as a pro-form, and trypsin treatment results in a cleaved mature form of purified Monalysin which kills Drosophila cell lines and adult flies. Electrophysiological measurement of Monalysin in a lipid membrane with an on-chip device confirms that Monalysin forms a pore, in a cleavage-dependent manner. This analysis also provides a pore-size estimate of Monalysin using current amplitude for a single pore and suggests lipid preferences for the insertion. Atomic Force Microscope (AFM) analysis displays its structure in a solution and shows that active-Monalysin is stable and composed of an 8-mer complex; this observation is consistent with mass spectrometry data. AFM analysis also shows the 8-mer structure of active-Monalysin in a lipid bilayer, and real-time imaging demonstrates the moment at which Monalysin is inserted into the lipid membrane. These results collectively suggest that endogenous Monalysin is indeed a pore-forming toxin composed of a rigid structure before pore formation in the lipid membrane. The endogenous Monalysin characterized in this study could be a desirable tool for analyzing host defense mechanisms against entomopathogenic bacteria producing damage-inducing toxins.
Insights
Researchers purified Monalysin, a toxin from Pseudomonas entomophila, and confirmed its pore-forming activity dependent on cleavage. This active toxin damages insect cells and tissues, providing a tool to study innate immunity.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas entomophila is a pathogenic bacterium used to study insect immunity.
- Monalysin, a beta-barrel pore-forming toxin, is key to P. entomophila's virulence, causing tissue damage and necrosis in Drosophila.
Purpose of the Study:
- To achieve the first efficient purification of endogenous Monalysin.
- To characterize the purified toxin's structure, pore-forming activity, and mechanism of action.
Main Methods:
- Purification of endogenous Monalysin from P. entomophila.
- Electrophysiological measurements using an on-chip device to assess pore formation.
- Atomic Force Microscopy (AFM) to analyze toxin structure and membrane insertion.
- Mass spectrometry for structural confirmation.
Main Results:
- Monalysin was successfully purified as a pro-form and activated by trypsin cleavage.
- Activated Monalysin demonstrated pore formation in lipid membranes in a cleavage-dependent manner.
- AFM revealed Monalysin as a stable 8-mer complex, both in solution and within lipid bilayers.
- Real-time imaging confirmed Monalysin's insertion into lipid membranes.
Conclusions:
- Endogenous Monalysin is a pore-forming toxin with a rigid structure prior to membrane insertion.
- Cleavage is essential for Monalysin's pore-forming activity and toxicity.
- Purified Monalysin serves as a valuable tool for investigating host defense mechanisms against bacterial toxins.
