Molecular and Functional Analysis of Pore-Forming Toxin Monalysin From Entomopathogenic Bacterium Pseudomonas entomophila.

Saori Nonaka,Aki Hori,Rangga Meidianto Asri,Takumi Nishiuchi,Emil Salim,Koki Kamiya,Takayuki Kuraishi,Firzan Nainu,Ayu Masyita,Shoji Takeuchi,Noriyuki Kodera

doi:10.3389/fimmu.2020.00520

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.

Highlights

The innate immune system is the front line of defense against microbial infection in metazoan animals [1]
Innate immune cells can sense infectious threats either by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [1,2,3]: pathogen-specific molecules such as peptidoglycans utilized by microbes as an essential substance of their life [1], or host-derived molecules that are normally kept inside in their cells but released because of the tissue damage by infection [2], respectively
To examine whether P. entomophila extract acts as source for the purification of endogenous Monalysin, we tested whether P. entomophila extract had Monalysin-derived cytotoxic activity

Summary

Introduction

The innate immune system is the front line of defense against microbial infection in metazoan animals [1]. Innate immune cells can sense infectious threats either by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [1,2,3]: pathogen-specific molecules such as peptidoglycans utilized by microbes as an essential substance of their life [1], or host-derived molecules that are normally kept inside in their cells but released because of the tissue damage by infection [2], respectively. The Toll pathway is responsible for infectious threats from fungi or Gram-positive bacteria, and it senses fungal β-glucans or bacterial Lysine (Lys)-type peptidoglycans with a pattern recognition receptor PGRP-SA/GNBP1 complex or GNBP3 in the hemolymph [6,7,8,9]. The whole picture of damage-induced innate immunity is far from understood

Methods

Results

Conclusion