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.

Diphtheria toxin induces fusion of small unilamellar vesicles at low pH.

Model membrane systems such as phospholipid vesicles have been extensively used to study the mechanism of membrane fusion at the molecular level. We report here on the capacity of diphtheria toxin to induce fusion of small unilamellar vesicles of dipalmitoylphosphatidylcholine at low pH. Fluorescence polarization and differential scanning calorimetry make it possible to demonstrate the mixing of the lipid phase. Mixing of the internal aqueous compartments of liposome was established using the terbium fluorescence technique. The analogy of structure and properties between melittin and a diphtheria toxin fragment is discussed.

Fusion of Newcastle disease virus with liposomes: role of the lipid composition of liposomes.

We demonstrate here that fusion occurs between the membrane of the Newcastle disease virus (NDV) and liposomes. Fluorescence dequenching studies (using Rhodamine-bearing viral envelopes) revealed the mixing of the lipids constituting the viral and liposomal membrane. The digestion of internal viral proteins by trypsin-containing liposomes indicated the mixing of the internal aqueous compartments. This last assay is independent of exchange of lipids between liposomal and viral membrane in the absence of fusion. Investigation of the effects of liposomal composition indicated that the presence of phosphatidylethanolamine and gangliosides are essential to optimize fusion. The fact that the Newcastle disease virus membrane can fuse with liposome also confirms that fusion must be determined by the viral proteins and could be mostly independent of the nature or presence of the host proteins.

The complete amino acid sequence of diphtheria toxin fragment B. Correlation with its lipid-binding properties.

The complete amino acid sequence of fragment B from diphtheria toxin has been determined. The polypeptide chain was split with cyanogen bromide, o-iodosobenzoic acid, clostripain and trypsin; all amino acid sequence analyses were made by automated Edman degradation. Fragment B, which corresponds to the carboxy terminus of the toxin molecule, contains 342 amino acids and has an Mr of 37240. The proposed amino acid sequence fully confirms the structure recently deduced from the nucleotide sequence of the structural gene. The complete sequence is analyzed in relationship with the role of fragment B in the transfer of diphtheria toxin fragment A from the extracellular medium into the cell cytoplasm.

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.

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH.

Like diphtheria toxin and the N-terminal (Mr 23 000) region of fragment B, CB1 (Mr 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (Mr 23 000 and 13 000) share a common segment (Mr 6300) containing the predicted amphipathic, alpha-helical, transverse lipid-associating domain (Mr 2750) of fragment B [J. Cell Biol. (1980) 87, 837-840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172-176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C-terminal region of CB1 is proposed.

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.

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.

Secondary structure changes of diphtheria toxin interacting with asolectin liposomes: an infrared spectroscopy study.

Until now, the study of the secondary structure of diphtheria toxin (DT) in the presence of phospholipid vesicles as a function of the pH has been prevented by the optical turbidity of the solution. In the present paper, this problem has been overcome by the use of IR attenuated total reflection (IR-ATR) spectroscopy. Incubation of DT with asolectin liposomes at pH 7.3 results in the binding of DT on to the liposomes and in a 10% increase in its alpha-helix content. At pH 4, in the presence of asolectin liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds, which did not exist before lowering of the pH. This new type of beta-sheet (low frequency beta-sheet) involves 25% of the amino acid residues of the protein.

Detection of lytic antimycobacterial antibodies by immune lysis of liposomes sensitized to tuberculin.

Two procedures were used in order to incorporate purified protein derivative tuberculin (PPD) from M. tuberculosis, strain H37Rv, into calcein-containing liposomes: formation of multilamellar vesicles (MLV) in a PPD solution or exposure of preformed MLV to this solution. Immune lysis of these PPD-sensitized MLV was studied in the presence of a hyperimmune anti-M. tuberculosis sheep serum using a specific pathogen-free rabbit serum as a source of complement. A 50% release of encapsulated calcein was observed spectrofluorometrically after 30 min and remained unchanged up to 2 h. The release of calcein in the absence of complement or of anti-H37Rv serum or by liposomes which did not contain PPD never exceeded 1-2%. Liposomes formed in PPD solution were more sensitive to anti-H37Rv serum than preformed liposomes exposed to PPD. Trials with human sera from ten tuberculous patients revealed the presence of specific lytic immunoglobulins. In the presence of sera from skin test negative, non-tuberculous subjects, calcein release was significantly lower. This opens the way to a new method for the study of the humoral immunity in tuberculosis.

Aggregation and fusion of lipid vesicles induced by diphtheria toxin at low pH: possible involvement of the P site and the NAD+ binding site.

Model membranes have been used to study the interaction between diphtheria toxin and lipids. We report here on the ability of this toxin to induce, at low pH, fusion and aggregation of asolectin lipid vesicles. Resonance energy transfer experiments using lipid fluorescent probes make it possible to discriminate between these two processes.

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.

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.

Membrane translocation and channel-forming activities of diphtheria toxin are blocked by replacing isoleucine 364 with lysine.

A mutant of diphtheria toxin in which Ile-364 was replaced by Lys was at least 500-fold less toxic to Vero cells than the parental toxin. Its ability to undergo low-pH-triggered translocation across the plasma membrane was greatly diminished, as was its ability to form ion-conductive channels. In addition, the mutant toxin was inactive in the pH-dependent killing of Escherichia coli.

Computer modelling of the transmembrane channel formed by a CNBr peptide of diphtheria toxin B fragment.

Diphtheria toxin (DT) forms transmembrane, voltage-dependent channels in a planar lipid bilayer. Channels with similar characteristics were obtained with CB1, a cyanogen bromide peptide of diphtheria toxin B fragment (DTB) (res 340-459). Tryptophan 398 is in interaction with the hydrophobic core of the lipid bilayer. Using the Eisenberg method in association with the Shiffer-Edmunson wheel representation, we have identified two amphipathic alpha-helices within CB1 (res 346-364 and 389-406) that could be involved in the interaction with lipids. Bearing this information in mind, we are providing a model for the structure of the CB1 channel.

Secondary structure and dosage of soluble and membrane proteins by attenuated total reflection Fourier-transform infrared spectroscopy on hydrated films.

Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the alpha-helix and beta-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.

pH dependent insertion of a diphtheria toxin B fragment peptide into the lipid membrane: a conformational analysis.

A peptide of diphtheria toxin B fragment (residues 147-266) has been shown to induce pore formation in lipid bilayer membranes at low pH. Such an effect was obtained at a much lower extent or not at all at pH = 7. The region localized between residues 225 and 246 is highly hydrophobic (27.3% polarity) and characterized by a high concentration of proline residues. Since proline cis-trans isomerization is highly sensitive to the pH of the medium, we investigated the capability of the cis and trans isomers to penetrate into the lipid matrix. Obviously, the cis-trans isomerization of proline 242 and 245, assumed to be imposed by a low pH, uncovers the hydrophobic region and induces its insertion into a lipid layer of dipalmitoylphosphatidylcholine. The lipid matrix destabilization resulting from this process could promote the penetration into the lipid bilayer of an amphipatic structure (153-178) similar to the transverse lipid associating domains of membrane proteins.