Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding

Abstract

Perfringolysin O (PFO) is the prototype for the cholesterol-dependent cytolysins, a family of bacterial pore-forming toxins that act on eukaryotic membranes. The pore-forming mechanism of PFO exhibits an absolute requirement for membrane cholesterol, but the complex interplay between the structural arrangement of the PFO C-terminal domain and the distribution of cholesterol in the target membrane is poorly understood. Herein we show that PFO binding to the bilayer and the initiation of the sequence of events that culminate in the formation of a transmembrane pore depend on the availability of free cholesterol at the membrane surface, while changes in the acyl chain packing of the phospholipids and cholesterol in the membrane core, or the presence or absence of detergent-resistant domains do not correlate with PFO binding. Moreover, PFO association with the membrane was inhibited by the addition of sphingomyelin, a typical component of membrane rafts in cell membranes. Finally, addition of molecules that do not interact with PFO, but intercalate into the membrane and displace cholesterol from its association with phospholipids (e.g., epicholesterol), reduced the amount of cholesterol required to trigger PFO binding. Taken together, our studies reveal that PFO binding to membranes is triggered when the concentration of cholesterol exceeds the association capacity of the phospholipids, and this cholesterol excess is then free to associate with the toxin.

Figure 1

Cholesterol dependence of PFO binding to liposomes composed of cholesterol and various phospholipids. Changes in the PFO Trp emission intensity are shown as a function of the cholesterol content of the different liposomes. The net F/F₀ was calculated as described in Experimental Procedures. (A) DOPC, ◆; SOPC, ■; POPC, ●. (B) DSPC, ▲; DPPC, ■; DMPC, ○. The cholesterol dependence curve for POPC (●) is shown for reference. Error bars indicate the standard deviation observed for three independent measurements per data point.

Figure 2

Cholesterol dependence of PFO oligomerization on POPC- or DOPC-cholesterol liposomes. The amount of cholesterol present in the PC-liposomes is shown below each lane. Lanes A–F show PFO oligomerization with POPC/cholesterol liposomes, while lanes G–L contain DOPC/cholesterol liposomes. The NBD-PFO monomer and oligomer bands were visualized with a BioRad Molecular Imager FX as described in the Experimental Procedures.

Figure 3

Effect of cholesterol concentration and phospholipid unsaturation on lipid order in liposomes as measured by DPH anisotropy. DPH was incorporated into the PC/cholesterol membranes as described in Experimental Procedures. DPPC, ▲; DSPC, ○; POPC, ●; DOPC, ■.

Figure 4

Detergent insolubility of various PC/cholesterol liposomes. Liposomes with trace amounts of [³H]cholesterol and [¹⁴C] PC were extracted with TX-100 at 0 °C. Radioactivity in the pellet fractions was measured after ultracentrifugation, and the percent of radioactivity in the pellet was plotted as a function of cholesterol concentration. (A) Fraction of [³H]cholesterol found in pellet. (B) Fraction of [¹⁴C]PC found in pellet. In both (A) and (B), the PC species are the following: DOPC, ○; POPC, ●; and DPPC, ■.

Figure 5

PFO binding to DOPC/SM/cholesterol membranes. (A) Changes in the Trp emission intensity of PFO are shown as a function of the percentage of SM in the nonsterol fraction of the membranes. The net F/F₀ was calculated as described in Experimental Procedures. Error bars indicate the standard deviation observed for three independent measurements per data point. (B) NBD-PFO oligomerization on DOPC/SM/cholesterol membranes. After incubation of PFO with the membranes, samples were solubilized with sample buffer and separated by 1.5% SDS–AGE. The NBD-PFO monomer and oligomer bands were visualized with a BioRad Molecular Imager FX as described in the Experimental Procedures. In both (A) and (B), the membranes were composed of 50 mol% cholesterol and various proportions of DOPC and SM comprising the remaining 50 mol% lipid in the nonsterol fraction. For example, when SM is at 50% in the nonsterol fraction, there is an equimolar amount of SM and DOPC (25 mol% each). (C). Formation of detergent-resistant domains in DOPC/SM/cholesterol membranes. The liposomes were composed of 40 mol% cholesterol and various proportions of DOPC and SM comprising the remaining 60 mol% lipid in the nonsterol fraction. Liposomes with trace amounts of [³H] cholesterol were extracted with TX-100 at 0 °C. Radioactivity in the pellet fractions was measured after ultracentrifugation, and the percent of radioactivity in the pellet was plotted as a function of the percentage of SM in the nonsterol fraction of the membrane.

Figure 6

NBD-PFO oligomerization on membranes containing cholesterol and epicholesterol. The liposomes contained 52 mol% POPC and 48 mol% sterol. The mol% of the individual sterols comprising the sterol fraction of the liposome is indicated below each lane in the above figure. After incubation of PFO with the membranes, samples were solubilized with sample buffer and separated by 1.5% SDS–AGE. The NBD-PFO monomer and oligomer bands were visualized with a BioRad Molecular Imager FX as described in the Experimental Procedures.