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Anthrax toxin-induced rupture of artificial lipid bilayer membranes
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Image of FIG. 1.
FIG. 1.

A schematic illustration of two putative mechanisms for PA channel-mediated LF and EF transport into the cytoplasm. One model suggests that LF and EF thread through the pore. The results shown here suggest that anthrax toxin complexes (i.e., LF or EF bound to the PA channel) rupture membranes. A previous study demonstrated that LF in the complex is enzymatically active.

Image of FIG. 2.
FIG. 2.

Interaction of animal-harvested anthrax toxin with artificial lipid bilayer membranes. (a) The relationship of anthrax toxin was initially strongly rectifying (•). The degree of rectification decreased with increasing time after sample addition (10 min (□), and 90 min (▵). Inset: schematic illustration of LF bound to a PA channel in an artificial lipid bilayer membrane. (b) Ionic current time series recordings and video micrographs () at = 180 mV demonstrate that anthrax toxin harvested from infected rabbits causes the membrane to become unstable and eventually rupture (†). The grey rods in the micrographs are Ag/AgCl electrodes, and the solutions contained 100 mM KCl, 5 mM MES at pH 7.2. Capsule material was present in the isolated fractions of anthrax toxin.

Image of FIG. 3.
FIG. 3.

The effect of pH gradients on the PA channel instantaneous relationship in the absence and presence of LF (a)–(d). The and compartments were buffered at either pH 7.2 or 5.5, as indicated in each panel. The LF concentration in the chamber was either zero (□) or 1 nM (•). Normalized current for the LF-free (e) and 1 nM LF data (f) highlights relative changes due to pH. Colorization indicates the pH condition as used in (a)–(d), normalized at 10 mV and 120 mV, respectively. The error bars, which represent the standard deviation ( ≥ 7), are generally smaller than the symbols.

Image of FIG. 4.
FIG. 4.

Time course of the PA channel conductance when (a) LF was removed before or (b) maintained [LF] = 1 nM during -side acidification. () The conductance equivalent of ≈60 PA channels was reconstituted into a planar bilayer membrane at pH 7.2|7.2 (). Then, 1 nM LF was added to the chamber (). () The pH 5.5|7.2 gradient was formed by perfusing the chamber with pH 5.5 buffer that contained either [LF] = 0 () or [LF] = 1 nM (). () The neutral pH condition (pH 7.2|7.2) () was restored by perfusing the chamber with pH 7.2 buffer. If LF was present, the chamber was first purfused with pH 5.5 buffer () then pH 7.2 buffer (). The ionic current was monitored for ≈20 min after each perfusion and ∼60 min after the final perfusion. The applied potential was = +50 mV. Breaks in the current recordings correspond to ≈30 s pulses at = −50 mV. The periodic noise corresponds to magnetic stirring during perfusion. (c) Instantaneous measurements and the current rectification ratio () taken at the beginning ( ) and end ( ,•) of the ionic current series confirm that the complex became essentially irreversibly bound only when maintained [LF] = 1 nM during -side acidification. Similar results were obtained with EF ().

Image of FIG. 5.
FIG. 5.

Rupture events of planar membranes caused by complexes of either essentially irreversibly bound recombinant (a) LF:PA channel and (b) EF:PA channel. Membrane rupture was also achieved by exposing the channel to the addition of the polycations (c) 28 kg/mol poly--lysine, and (d) 56 kg/mol poly(allylamine hydrocholoride). Chirality and / addition did not inhibit the membrane rupture capability (). The membranes rupture events are denoted by (†). The solutions on either side of the membrane contained 100 mM KCl and 5 mM MES at pH 7.2. Membrane rupture with irreversibly bound recombinant proteins was also achievable at pH 5.5|7.2 (). The applied potential was || = 120 mV for recombinant proteins or || = 50 mV for the polycations.

Image of FIG. 6.
FIG. 6.

Hypothetical pH-induced changes to the putative binding pocket for the PA channel and LF. () A top-down view for part of the theoretical model of the PA channel and the crystal structure of LF oriented to illustrate the proposed binding site. The colored regions correspond to the subunits shown in the right panel. () A “folded-open” representation of the LF:PA channel binding pocket that includes a PA dimer and LF. The space-fill region emphasizes the residues at the binding site. The electrostatic potentials were computed at (a) pH 7.2, (b) pH 5.5, and (c) their difference. Negative and positive electrostatic surface potentials are denoted by and , respectively.

Image of FIG. 7.
FIG. 7.

Protein topology of the disordered N-terminus of EF/LF and the lumen of the PA channel β-barrel. () Extended chain representation of the first 30 disordered amino acids (right to left) from the N-terminus of LF. () The lumen of the β-barrel from a model of its structure. () Extended chain representation of the first 30 disordered amino acids (right to left) from the N-terminus of EF. PyMol was used to generate the topological representation based on sequences of EF, LF, and whole model of (PA). Residues are colored by the following classifications: – His, – Basic, – Acidic, – Hydrophobic. Blue and Red denote N and O moieties, respectively. The bar represents the membrane spanning region of the β-barrel.

Image of FIG. 8.
FIG. 8.

The pH dependence of the LF:PA channel binding constant as estimated from the channel conductance at = +70 mV. The symbols correspond to the pH conditions described in Fig. 3 (pH 7.2|7.2 □; pH 5.5|7.2 ○). The solid lines are the least-squares best fits of a simple binding equation for 1:1 stoichiometry (i.e., 1/(1 + [LF]/ ), where is the reaction dissociation constant. () The pH dependence of . The pH in the chamber was 7.2. The dashed line is to guide the eye. The error bars represent the standard deviation from n = 3–5 membranes.


Generic image for table
Table I.

Calculated pKa values of residue side chains in the PA channel lumen.


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Anthrax toxin-induced rupture of artificial lipid bilayer membranes