Membrane topology of VacA cytotoxin from H. pylori.

The interaction of VacA with membranes involves: (i) a low pH activation that induces VacA monomerization in solution, (ii) binding of the monomers to the membrane, (iii) oligomerization and (iv) channel formation. To better understand the structure-activity relationship of VacA, we determined its topology in a lipid membrane by a combination of proteolytic, structural and fluorescence techniques. Residues 40-66, 111-169, 205-266, 548-574 and 723-767 were protected from proteolysis because of their interaction with the membrane. This last peptide was shown to most probably adopt a surface orientation. Both alpha-helices and beta-sheets were found in the structure of the protected peptides.

Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid-binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.

Translocation of Bacillus anthracis lethal and oedema factors across endosome membranes.

The two exotoxins of Bacillus anthracis, the causative agent of anthrax, are the oedema toxin (PA-EF) and the lethal toxin (PA-LF). They exert their catalytic activities within the cytosol. The internalization process requires receptor-mediated endocytosis and passage through acidic vesicles. We investigated the translocation of EF and LF enzymatic moieties across the target cell membrane. By selective permeabilization of the plasma membrane with Clostridium perfringens delta-toxin, we observed free full-size lethal factor (LF) within the cytosol, resulting from specific translocation from early endosomes. In contrast, oedema factor (EF) remained associated with the membranes of vesicles.

Structure and interaction of VacA of Helicobacter pylori with a lipid membrane.

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes.

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-A pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37 degrees C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.

Secondary structure of sea anemone cytolysins in soluble and membrane bound form by infrared spectroscopy.

Attenuated total reflection (ATR) Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of two pore-forming cytolysins from the sea anemone Stichodactyla helianthus and their interaction with lipid membranes. Frequency component analysis of the amide I' band indicated that these peptides are composed predominantly of beta structure, comprising 44-50% beta-sheet, 18-20% beta-turn, 12-15% alpha-helix, and 19-22% random coil. Upon interaction with lipid membranes a slight increase in the alpha-helical and beta-sheet structures was observed with a concomitant decrease of the unordered structure. Polarisation experiments indicated that both toxins had some disordering effect on the lipid layers. The dichroic ratio of the alpha-helical component of the membrane-bound toxin was 3.0-3.3, indicating that this element was oriented with an angle of 38 degrees-42 degrees with respect to the normal to the plane of the crystal surface, thus resulting almost parallel to the mean direction of the lipid chains.

Structure and topology of diphtheria toxin R domain in lipid membranes.

The interaction of the receptor-binding domain (R domain) of diphtheria toxin with a pure lipid membrane has been characterized by several approaches. Using a photoactivatable lipid, the R domain has been shown to deeply insert in the lipid membrane. Three regions of the R domain (residues 380-421, 422-441, and 442 to about 483) are protected by their interaction with the membrane from externally added proteases. At least one of these regions is deeply interacting with the lipid membrane, as evidenced by the location of Cys 461 and 471 determined by fluorescence experiments. Binding of the R domain to the lipid membrane is characterized by the appearance of an alpha-helical component whose orientation is compatible with a transmembrane orientation.

Use of a photoactivatable lipid to probe the topology of PA63 of Bacillus anthracis in lipid membranes.

The protective antigen of Bacillus anthracis is a key protein that promotes the translocation of the enzymatic moieties of the two toxins of B. anthracis into the cell cytoplasm. The membrane topology of the active form of the protective antigen (PA63) was investigated by proteolysis of PA63 inserted into liposomes containing a photoactivatable, radioactive lipid, and characterization of the N-terminal moiety of the deeply-inserted (and therefore radiolabeled) peptides. A single sequence starting at residue Ala258 was identified. Fourier-transform infrared spectroscopy showed that the protected peptide was mainly adopting a beta-sheet structure whose orientation was compatible with a transmembrane organization.

Structure and interaction of PA63 and EF (edema toxin) of Bacillus anthracis with lipid membrane.

The secondary structures of the two components of the Bacillus anthracis edema toxin, protective antigen (PA63) and edema factor (EF), as well as the two EF mutants: CYA30 (containing the N-terminal PA63-binding domain) and CYA62 (containing the C-terminal catalytic domain) were investigated as a function of pH in the absence and in the presence of phospholipid vesicles using attenuated total reflection Fourier transform infrared spectroscopy. Secondary structures were independent of pH, whereas, in all cases, structural modifications were observed upon lipid binding. The ability of PA63 and EF to undergo hydrogen/deuterium exchange was evaluated. The binding of these proteins and the mutants to the lipid membrane was also characterized and it was demonstrated that the association of PA63 to the lipid bilayer was pH-dependent, while the binding of EF to the lipid membrane took place at both neutral and acidic pH. Interestingly, the two EF mutants are showing different lipid binding properties in response to pH: CYA30 has a strong pH-dependence whereas CYA62, as EF, binds to the lipid vesicles at all pHs. For the two proteins characterized by a pH-dependent lipid binding, the reversibility of binding upon neutralization was tested and binding of PA63 to the membrane was found to be irreversible whereas that of CYA30 was reversible.

Secondary structures comparison of aquaporin-1 and bacteriorhodopsin: a Fourier transform infrared spectroscopy study of two-dimensional membrane crystals.

Aquaporins are integral membrane proteins found in diverse animal and plant tissues that mediate the permeability of plasma membranes to water molecules. Projection maps of two-dimensional crystals of aquaporin-1 (AQP1) reconstituted in lipid membranes suggested the presence of six to eight transmembrane helices in the protein. However, data from other sequence and spectroscopic analyses indicate that this protein may adopt a porin-like beta-barrel fold. In this paper, we use Fourier transform infrared spectroscopy to characterize the secondary structure of highly purified native and proteolyzed AQP1 reconstituted in membrane crystalline arrays and compare it to bacteriorhodopsin. For this analysis the fractional secondary structure contents have been determined by using several different algorithms. In addition, a neural network-based evaluation of the Fourier transform infrared spectra in terms of numbers of secondary structure segments and their interconnections [sij] has been performed. The following conclusions were reached: 1) AQP1 is a highly helical protein (42-48% alpha-helix) with little or no beta-sheet content. 2) The alpha-helices have a transmembrane orientation, but are more tilted (21 degrees or 27 degrees, depending on the considered refractive index) than the bacteriorhodopsin helices. 3) The helices in AQP1 undergo limited hydrogen/deuterium exchange and thus are not readily accessible to solvent. Our data support the AQP1 structural model derived from sequence prediction and epitope insertion experiments: AQP1 is a protein with at least six closely associated alpha-helices that span the lipid membrane.

Purification of IpaC, a protein involved in entry of Shigella flexneri into epithelial cells and characterization of its interaction with lipid membranes.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the secreted IpaA-D proteins. A complex containing IpaC and IpaB is able to promote uptake of inert particles by epithelial cells. This suggested that Ipa proteins, either individually or as a complex, might interact with the cell membrane. We have purified IpaC and demonstrated its interaction with lipid vesicles. This interaction is modulated by the pH, which might be relevant to the dual role of Ipa proteins, in induction of membrane ruffles upon entry and lysis of the endosome membrane thereafter.

Interaction with a lipid membrane: a key step in bacterial toxins virulence.

Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane.

Conformational changes in aerolysin during the transition from the water-soluble protoxin to the membrane channel.

Proteolytic activation, oligomerization, and membrane insertion are three steps that precede channel formation by the bacterial toxin aerolysin. Using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and hydrogen-deuterium exchange, the structural changes associated with each step were analyzed. Our results show that activation induces a significant change in secondary structure, characterized by a decrease in random structure and an increase in beta-sheet content. We show that release of the propeptide is essential for this conformational change to occur and that changes are not restricted to the vicinity of the cleavage site but appear to propagate along the molecule. In contrast, subsequent oligomerization of the mature toxin does not involve any change in overall secondary structure but does involve a modification of the tertiary interactions. Finally, insertion of the heptameric complex into dimyristoylphosphatidylcholine vesicles also occurs without major modification of the secondary structure. Studies on the orientations of the secondary structures of the heptamer in the lipid bilayer have also been performed.

Secondary structure of anthrax lethal toxin proteins and their interaction with large unilamellar vesicles: a fourier-transform infrared spectroscopy approach.

Attenuated total reflection Fourier transform infrared spectroscopy has been used to study the secondary structure of anthrax lethal toxin proteins: protective antigen (PA) and lethal factor (LF), as a function of pH in the absence and in the presence of phospholipid vesicles. We first characterized the binding of LF and PA to the lipid membrane and demonstrated the strong pH dependence of the association of PA and LF to the lipid bilayer as well as the effect of pH neutralization on this binding. Binding of LF to the lipid membrane can be, at least partially, reversed when the pH is brought to neutral whereas in the same conditions PA binding is irreversible. Characterization of the conformational changes undergone by PA and LF upon pH lowering, lipid binding, and, in the case of LF, reversal of binding was carried out (i) by determining the secondary structure of the proteins and (ii) by evaluating their ability to undergo an hydrogen/deuterium exchange.

Topology of diphtheria toxin in lipid vesicle membranes: a proteolysis study.

The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non-specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu-C), led us to propose a topological model of the N-terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two alpha-helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third alpha-helix could adopt a transmembrane topology only in a small proportion of DT molecules.

Secondary structure and membrane interaction of PR-39, a Pro+Arg-rich antibacterial peptide.

PR-39 is a 4719-Da peptide isolated from pig intestine and belonging to the recently discovered family of Pro+Arg-rich antibacterial peptides. PR-39 does not lyse Escherichia coli, instead the lethal action is probably linked to the termination of DNA and protein synthesis [Boman, H. G., Agerberth, B. & Boman, A. (1993) Infect. Immun. 61, 2978-2984]. Circular dichroism and Fourier-transform infrared spectroscopy have been used to investigate the secondary structure of PR-39 in the absence or presence of lipids. According to the circular dichroic data, this structure is not altered upon incubation of PR-39 with negatively charged vesicles, although the infrared spectra suggest that the hydrogen bond pattern is modified upon the peptide-lipid interaction. This is detected by a shift in the maximum wavelength of absorption of PR-39 from 1636 cm-1 in the absence of lipids to 1645 cm-1 in the presence of lipids. We have further addressed the question of the possible mechanism of interaction of PR-39 with model membranes (liposomes and planar lipid bilayers) whose lipid compositions mimick that of the E. coli inner membrane. PR-39 induced a calcein release from large unilamellar vesicles, which is dependent upon the peptide concentration and upon the presence of negatively charged lipid (glycerophosphoglycerol) in the membrane. The binding study of PR-39 to dioleoylglycerophosphoglycerol vesicles suggests that nearly 100% of the added peptide is membrane-bound. Addition of PR-39 to a planar lipid bilayer induced a linear increase in the current but no channel formation was observed since no discrete steps of conductance occurred.

Sequence and structure of the membrane-associated peptide of glycophorin A.

Glycophorin A (GPA) has been reconstituted into dimyristoylphosphatidylcholine vesicles and digested with proteinase K to identify the membrane domain and to characterize its structure and orientation. After digestion of the inner and outer domain of GPA by protease action restricted to the aqueous phase, a protected peptide migrates on an electrophoresis gel as a 7.5-kDa dimer (His66-Ile95). The secondary structure and orientation in a lipid bilayer of the 7.5-kDa dimer have been studied by Fourier transform infrared spectroscopy. Our proteolytic and spectroscopic data are in agreement with a topological model in which the His66-Glu72 peptide adopts a beta-sheet conformation and is oriented parallel to the lipid-water interface and the Ile73-Ile95 domain is helical and oriented parallel to the lipid acyl chains, in a transmembrane configuration. Digestion of the domain protruding to the outside of the liposome generates "head-head" and "head-tail" dimers of 16 and 38 kDa, respectively. This observation is discussed in terms of the specificity of the dimer formation process.