Abstract
Pore forming proteins are a broad class of pathogenic proteins secreted by organisms as virulence factors due to their ability to form pores on the target cell membrane. Bacterial pore forming toxins (PFTs) belong to a subclass of pore forming proteins widely implicated in bacterial infections. Although the action of PFTs on target cells have been widely investigated, the underlying membrane response of lipids during membrane binding and pore formation has received less attention. With the advent of superresolution microscopy as well as the ability to carry out molecular dynamics (MD) simulations of the large protein membrane assemblies, novel microscopic insights on the pore forming mechanism have emerged over the last decade. In this review, we focus primarily on results collated in our laboratory which probe dynamic lipid reorganization induced in the plasma membrane during various stages of pore formation by two archetypal bacterial PFTs, cytolysin A (ClyA), an α-toxin and listeriolysin O (LLO), a β-toxin. The extent of lipid perturbation is dependent on both the secondary structure of the membrane inserted motifs of pore complex as well as the topological variations of the pore complex. Using confocal and superresolution stimulated emission depletion (STED) fluorescence correlation spectroscopy (FCS) and MD simulations, lipid diffusion, cholesterol reorganization and deviations from Brownian diffusion are correlated with the oligomeric state of the membrane bound protein as well as the underlying membrane composition. Deviations from free diffusion are typically observed at length scales below ∼130 nm to reveal the presence of local dynamical heterogeneities that emerge at the nanoscale—driven in part by preferential protein binding to cholesterol and domains present in the lipid membrane. Interrogating the lipid dynamics at the nanoscale allows us further differentiate between binding and pore formation of β- and α-PFTs to specific domains in the membrane. The molecular insights gained from the intricate coupling that occurs between proteins and membrane lipids and receptors during pore formation are expected to improve our understanding of the virulent action of PFTs.
Highlights
A large class of bacterial pathogens have evolved to infect target cells by releasing pore forming membrane disruptive proteins whose sole purpose is to compromise metabolic and signaling pathways leading to cell lysis (Morton et al, 2019)
We summarize key findings which probe the manner in which lipid dynamics is altered, using fluorescence microscopic techniques such as confocal and stimulated emission depletion (STED) imaging, fluorescence correlation spectroscopy (FCS), Förster Resonance Energy Transfer (FRET) and molecular dynamics (MD) simulations ranging from all-atom to coarse grained methods
We have extensively reviewed the changes observed on lipid dynamics and membrane reorganization that occurs during the pore formation the β-Pore-forming toxins (PFTs), listeriolysin O (LLO) and the α-PFT, cytolysin A (ClyA)
Summary
A large class of bacterial pathogens have evolved to infect target cells by releasing pore forming membrane disruptive proteins whose sole purpose is to compromise metabolic and signaling pathways leading to cell lysis (Morton et al, 2019). PFTs are broadly classified as α- and β-toxins based on the secondary structure of the membrane inserted motifs that constitute the transmembrane pores as illustrated in Figures 1A,B respectively These differences in secondary structure influence the pore structure, protein-lipid interactions and pore formation pathways. Depending on the PFT, membrane binding is activated by specific receptors present on the mammalian cell membrane (Dal Peraro and van der Goot, 2016) These receptors could be specific lipids, cholesterol or proteins depending on the toxin. The repair processes involve active or passive membrane remodeling events such as exocytosis and endocytosis in order to rid the membrane of the damaged toxin associated sites on the plasma membrane (Husmann et al, 2009; Keyel et al, 2011; Atanassoff et al, 2014) These processes which involve large membrane deformations are intrinsically connected to the underlying membrane fluidity. The review provides a current view on biomembrane response towards PFT attack and its implications in plasma membrane repair and cellular signaling mechanisms
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