Membrane Permeabilization Induced by Cytolytic δ-Endotoxin CytA fromBacillus thuringiensisvar.israelensis

Abstract

CytA is a member of a functionally defined family of insecticidal delta-endotoxins occurring in parasporal crystals of Bacillus thuringiensis var. israelensis. We investigated the ability of CytA to permeabilize the membrane and release fluorescence marker molecules from unilamellar lipid vesicles. Both protoxin (27 kDa) and proteolytically activated toxin (24 kDa) were very effective in permeabilization of unilamellar lipid vesicles: concentrations as low as several nanomolar produced a significant effect. The toxin was about 2-3 times more effective than the protoxin. The concentration of CytA required for the same extent of calcein release in large unilamellar vesicles (LUV) was 5-10 times lower than that in small unilamellar vesicles (SUV). Both small (calcein) and large (fluorescein-dextrans, MW 3000 and 10 000) molecules were released from the vesicles by CytA with comparable single-exponential kinetics. The release was an all-or-none event, i.e., each vesicle either released all of its contents or remained completely intact. Binding of CytA to lipid membranes did not show appreciable cooperativity, the apparent binding constant (Kapp) being on the order of 10(5) M-1. The plots of kinetics of release vs bound protein/ lipid ratio and the differential effects of CytA on LUV vs SUV indicate that at least 140 toxin molecules or 311 protoxin molecules must bind to an LUV before the latter starts losing its integrity. The necessity of adsorption of this relatively large number of toxin molecules to trigger permeabilization, together with the lack of discrimination in the size of the released marker molecules, suggests that the effect of CytA is a general, detergent-like, perturbation of the membrane rather than creation of small, well-defined, proteinaceous channels.

Compaction of the Escherichia coli nucleoid caused by Cyt1Aa

“…Two hypotheses were reported in the literature describing the mechanism of membrane disruption by Cyt1Aa -perforation (Gazit et al, 1997) or detergent-like (Butko et al, 1996(Butko et al, , 1997. Membrane fluidity (measured by anisotropy of DPH fluorescence; Binenbaum et al, 1999) did not change following induction of cyt1Aa (Fig.…”

Interaction of wheat α‐thionin with large unilamellar vesicles

“…We can estimate the surface area of each vesicle from the number of lipid molecules in the outer monolayer and the area occupied by each lipid. If we assume that 54% of the total lipids (83,000) are in the outer monolayer and that the area per lipid is 0.7 nm2 (Butko et al, 1996), we obtain a value of approximately 3 1,000 nm2. This means that each peptide molecule would cover an area of about 45 nm2.…”

“…2; Table 1). If we consider that a vesicle with a mean diameter of 100 nm consists of approximately 83,000 lipid molecules (Butko et al, 1996), a protein to lipid molar ratio of 0.0083 represents an average value of about 700 a-thionin molecules per vesicle. This seems to be the minimum amount of peptide that must be bound to the vesicle in order to obtain 100% release.…”

“…israelensis (Butko et al, 1996(Butko et al, , 1997. Moreover, attempts have been made to design peptides with detergent-like properties for the solubilization of integral membrane proteins (Schafmeister et al, 1994) for which the term "peptitergents" has been coined.…”

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

“…Gross liposome disruption by ⌬CR_ PrP was ruled out by analyzing liposome integrity using cryo-EM. This was necessary because such membrane-disrupting effects have been described for other transmembrane proteins or peptides (52)(53)(54). Additional support for the formation of pores within the lipid bilayer was obtained by NBD fluorescence quenching assays.…”