Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes

Abstract

Cholesterol distribution in the cell is maintained by both vesicular and non-vesicular sterol transport. Non-vesicular transport is mediated by the interaction of membrane-embedded cholesterol and water-soluble proteins. Small changes to the lipid composition of the membrane that do not change the total cholesterol content, can significantly affect how cholesterol interacts with other molecules at the surface of the membrane. The cholesterol-dependent cytolysin Perfringolysin O (PFO) constitutes a powerful tool to detect cholesterol in membranes, and the use of PFO-based probes has flourished in recent years. By using a non-lytic PFO derivative, we showed that the sensitivity of the probes for cholesterol can be tuned by modifications introduced directly in the membrane-interacting loops and/or by modifying residues away from the membrane-interacting domain. Through the use of these biosensors on live RAW 264.7 cells, we found that changes in the overall cholesterol content have a limited effect on the average cholesterol accessibility at the surface of the membrane. We showed that these exquisite biosensors report on changes in cholesterol reactivity at the membrane surface independently of the overall cholesterol content in the membrane.

Missing elimination via membrane vesicle shedding contributes to the diminished calcium sensitivity of listeriolysin O

“…Considering vesicle shedding as a pure membrane repair process should be carefully reassessed. On the other hand, CDCs are also used as model tools in other biology fields 22 , as cholesterol-binding probes 23 . Thus, understanding the differences in the membrane behavior of CDC toxins is also critically important for the interpretation of the experimental results, obtained with their help.…”

Interaction of Cholesterol with Perfringolysin O: What Have We Learned from Functional Analysis?

“…Binding of a water-soluble PFO monomer to the membrane is diffusional and electrostatic interactions may play a role since it has been observed that the introduction or elimination of charged residues alters binding [ 12 , 16 , 24 , 25 , 49 ]. While on the membrane surface, insertion of non-polar and aromatic amino acids, and/or specific interactions with membrane lipids, help to anchor the protein to the membrane [ 50 ].…”

“…For example, T490 in the PFO-cholesterol model is predicted to be involved in a complex H-bonding network involving both its side chain and backbone carbonyl and the side chains of T460 and E458 (in its protonated form) from the undecapeptide. The static model, however, does not provide a simple explanation for the adjacent L491S mutation that does not significantly change the cholesterol-binding of PFO [ 25 ]. This implies a newly found H-bonding role for the Ser side chain at this position that, in contrast with the reduced affinity observed for the L491A substitution, conserved the affinity of PFO for cholesterol.…”

“…The L3 is located on the far edge of D4, away from a nascent cavity formed by the undecapeptide, L1, and L2 ( Figure 1 ). Modifications introduced into L3 have been shown either to have a negligible effect on cholesterol interaction, or to decrease the amount of cholesterol required for binding [ 13 , 16 , 25 ]. These results suggest that L3 plays a limited role in cholesterol recognition, and its effect on binding may be related to nonspecific interactions with the membrane that stabilize the bound form of the monomer (e.g., decreasing the k off ).…”

The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes

“…HeLa cells incubated with P. aeruginosa were washed to remove unbound bacteria, and permeabilized using a Cys-less derivative of perfringolysin O (rPFO). rPFO is a pore-forming toxin that only perforates mammalian cell membranes due to its specificity for cholesterol (30,31). We used antibodies against glyceraldehyde-phosphate dehydrogenase (GAPDH) and Na ϩ /K ϩ -ATPase to detect cytosolic and plasma membrane fractions, respectively (Fig.…”