Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes

Abstract

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

Mammalian membrane trafficking as seen through the lens of bacterial toxins

“…Many other toxins, including Shiga toxin (Falguières et al, 2001;Kovbasnjuk, Edidin, & Donowitz, 2001) and pore-forming toxins (Abrami et al, 1998b;Waheed et al, 2001), were also shown to preferentially attach to microdomains, highlighting a shared mechanism for cellular activity/entry. Decades later, toxins continue to be exploited to probe for membrane compartmentalization (Dumitru et al, 2018;Maekawa, Yang, & Fairn, 2016;Russo et al, 2018). Their binding domains, conjugates, or derivatives have been used to not only monitor lipid distribution and trafficking but as probes that directly reorganise surface lipids, therefore driving the formation of membrane domains.…”

Quantifying lipid changes in various membrane compartments using lipid binding protein domains

“…This secreted pore-forming toxin is a member of the cholesterol-dependent cytolysins. It binds to cholesterol of the extracellular leaflet of the PM followed by oligomerization eventually leading to cell lysis [72]. It has a cholesterol binding domain (D4) that is non-toxic and is sufficient to target the toxin to cholesterol-rich domains.…”

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes—The Update

“…Moreover, one big issue is that filipin and fluorophore-labeled cholesterols cannot distinguish cholesterol in the cytosolic leaflets and luminal (exofacial) leaflets of cellular membranes ([38]; Figure 2). Perfringolysin O (PFO, theta toxin) is a protein secreted by the Gram-positive anaerobe Clostridium perfringens and binds to cholesterol in the exofacial leaflets of the PM resulting in pore formation and cell lysis [44]. PFO structurally possesses four domains and, among them, domain 4 (D4) recognizes the hydroxyl group at the position 3 of cholesterol ([45,46]; Figure 1 and Figure 3A).…”

“…D4 was shown to be sufficient for binding of PFO to cholesterol [47,48]. By introducing a variety of mutations into PFO or D4, which removes its cytotoxicity or increase the affinity to cholesterol, researchers have successfully developed cholesterol biosensors that can distinguish transbilayer distribution of cholesterol in cellular membranes ([44]; Figure 3 and Figure 4). This review will focus on PFO-derived cholesterol biosensors, especially D4 mutants, and show the latest findings, which will open new doors for novel cholesterol biology.…”