Trapping of Vibrio cholerae cytolysin in the membrane-bound monomeric state blocks membrane insertion and functional pore formation by the toxin

Abstract

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytolytic toxin that belongs to the family of β barrel pore-forming protein toxins. VCC induces lysis of its target eukaryotic cells by forming transmembrane oligomeric β barrel pores. The mechanism of membrane pore formation by VCC follows the overall scheme of the archetypical β barrel pore-forming protein toxin mode of action, in which the water-soluble monomeric form of the toxin first binds to the target cell membrane, then assembles into a prepore oligomeric intermediate, and finally converts into the functional transmembrane oligomeric β barrel pore. However, there exists a vast knowledge gap in our understanding regarding the intricate details of the membrane pore formation process employed by VCC. In particular, the membrane oligomerization and membrane insertion steps of the process have only been described to a limited extent. In this study, we determined the key residues in VCC that are critical to trigger membrane oligomerization of the toxin. Alteration of such key residues traps the toxin in its membrane-bound monomeric state and abrogates subsequent oligomerization, membrane insertion, and functional transmembrane pore-formation events. The results obtained from our study also suggest that the membrane insertion of VCC depends critically on the oligomerization process and that it cannot be initiated in the membrane-bound monomeric form of the toxin. In sum, our study, for the first time, dissects membrane binding from the subsequent oligomerization and membrane insertion steps and, thus, defines the exact sequence of events in the membrane pore formation process by VCC.

Keywords: Membrane; Membrane Protein; Pore-forming Toxin; Protein Assembly; Protein Structure; Vibrio cholerae Cytolysin; bacterial Toxin.

FIGURE 1.

Characterization of the VCC variants harboring single point mutations at the oligomerization interface. A, amino acid sequence alignment of VCC and its closely related cytolysins/hemolysins from three other Vibrio species. The locations of the conserved residues, Asp-214, Arg-330 and Phe-581, in VCC are indicated. Other conserved residues, Lys-269 (interacting with Asp-214), Ser-380 and Ala-218 (interacting with Arg-330), and Val-197 (interacting with Phe-581) at the interprotomer interface of the VCC oligomer, are also marked. B, structural model of the transmembrane oligomeric form of VCC showing the locations of the conserved residues (as shown in A) at the interface of the two neighboring protomers. C, SDS-PAGE/Coomassie staining of the purified form of the wild-type and mutant VCC variants. Proteins were treated in presence of SDS-PAGE sample buffer with (B) or without boiling (UB). Protein standards are shown in lane M. D, intrinsic tryptophan fluorescence emission spectra of the VCC variants. Fluorescence emission intensities are shown in terms of counts per second (cps). E, far-UV CD spectra of the VCC variants. Far-UV CD signals are shown in terms of ellipticity measured in millidegrees (mdeg).

FIGURE 2.

Binding and pore-forming activity of the VCC variants on human erythrocytes. A, pore-forming activity of the VCC variants monitored by estimating the hemolytic activity of the proteins (100 nm) against human erythrocytes. B, binding of the VCC variants (75 nm) with human erythrocytes determined by a flow cytometry-based assay. Solid line, WT VCC; dashed line, mutant VCC variants as indicated at top; shaded curve, control.

FIGURE 3.

Binding and membrane permeabilization activity of the VCC variants on Asolectin-cholesterol liposome membranes. A, pore-forming activity of the VCC variants (1 μm) as determined by estimating the calcein release from the Asolectin-cholesterol liposomes. B, binding of the VCC variants (1 μm) to the Asolectin-cholesterol liposome vesicles as determined by pulldown-based assay. Pellet fractions containing liposome-bound proteins and the supernatant (Sup) fraction containing unbound proteins were analyzed by SDS-PAGE/Coomassie staining. Protein standards are shown in lane M. C and D, binding of the VCC variants to the membrane lipid bilayer of Asolectin-cholesterol as determined by SPR-based assay. Various concentrations (10 different concentrations from 100 nm to 1 μm) of wild-type and mutant proteins were injected over the membrane lipid bilayer of Asolectin-cholesterol liposomes generated on the SPR sensor chips. Subsequently, buffer was injected for a definite time period, at the end of which residual sensogram response units were monitored to estimate the extent of irreversible binding of the VCC variants to the liposome membrane. An overlay of the binding sensograms shows steady-state binding of the VCC variants (C). Analysis of the end point response units shows a concentration-dependent increase in irreversible binding of the VCC variants toward the Asolectin-cholesterol membrane lipid bilayer (D).

FIGURE 4.

Oligomerization ability of the VCC variants. A, SDS-stable oligomer formation by the VCC variants in the human erythrocytes membranes. Erythrocyte membrane-bound proteins were pelleted by ultracentrifugation and probed by immunoblotting. Samples treated with SDS-PAGE sample buffer without boiling (UB) allowed detection of the SDS-stable oligomers. F581A-VCC showed an ∼0.25-fold reduced oligomer formation compared with WT-VCC. No SDS-stable oligomer formation was noticed for R330A-VCC. D214A-VCC showed a marginally reduced oligomer formation compared with that of WT-VCC. WT-VCC was included in each of the immunoblot analyses as a control. Oligomer band intensities were compared using the Gel Analysis tool within the ImageJ program (National Institutes of Health, http://imagej.nih.gov/ij/). B, SDS-stable oligomer formation by the VCC variants in the presence of Asolectin-cholesterol liposomes. Proteins were treated in the presence of liposomes and probed by immunoblotting. Samples treated with SDS-PAGE sample buffer without boiling allowed detection of the SDS-stable oligomers. F581A-VCC showed an ∼0.4-fold reduced oligomer formation compared with WT-VCC. No significant extent of SDS-stable oligomer formation was observed for R330A-VCC. D214A-VCC showed a marginally reduced oligomer formation compared with WT-VCC. WT-VCC was included as a control in each of the immunoblot analyses. Oligomer band intensities were compared as described in A. C, R330A-VCC failed to generate SDS-stable oligomers in Asolectin-cholesterol liposomes even after prolonged incubation for 6 h, whereas WT-VCC, D214A-VCC, and F581A-VCC formed a considerable amount of oligomers in the liposome membranes. VCC variants were incubated with the liposomes, and liposome-bound proteins were pelleted by ultracentrifugation and analyzed by SDS-PAGE/Coomassie staining. Samples treated with SDS-PAGE sample buffer without boiling allowed detection of the SDS-stable oligomers. D, BS³ cross-linking of SDS-labile prepore oligomers formed in the Asolectin-cholesterol liposome membrane. Liposome-bound proteins were subjected to BS³ cross-linking and analyzed by SDS-PAGE/Coomassie staining. For R330A-VCC, a cross-linked oligomer could not be detected, whereas for WT-VCC, SDS-labile prepore oligomers could be trapped by BS³ cross-linking. Bands of oligomeric and monomeric species are marked with double and single arrowheads, respectively.

FIGURE 5.

Membrane insertion of the pore-forming stem loop of the VCC variants probed by a FRET-based assay. A–C, W318F-VCC was taken as a control for the FRET-based assay. W318F-VCC showed wild type-like intrinsic tryptophan fluorescence emission (A) and far-UV CD spectra (B). Fluorescence emission intensities are shown in terms of counts per second (cps). Far-UV CD signals are shown in terms of ellipticity measured in millidegrees (mdeg). Also, W318F-VCC displayed a similar extent of hemolytic activity (C) and liposome permeabilization (C, inset) compared with that of WT-VCC. D, incubation of WT-VCC in the presence of DPH-labeled Asolectin-cholesterol liposomes triggered a time-dependent increase in the tryptophan-to-DPH FRET signal, presumably because of an efficient FRET from the Trp-318 located within the stem region of the protein to the membrane-embedded DPH. This notion was confirmed by the observation that mutation of W318F in VCC completely blocked the time-dependent increase in the tryptophan-to-DPH FRET signal. For D214A-VCC and F581A-VCC, the efficiency of the process was compromised modestly. For R330A-VCC, no significant time-dependent increase in the FRET signal was observed, suggesting a severe blockade of the membrane insertion step for the mutant.