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E Duflot

Publications and source records attributed to E Duflot.

15 recordsLinked to original sources
PubMed1999

Functional analysis of the carboxy-terminal domain of Bacillus anthracis protective antigen.

Protective antigen (PA) is the common receptor-binding component of the two anthrax toxins. We investigated the involvement of the PA carboxy-terminal domain in the interaction of the protein with cells. A deletion resulting in removal of the entire carboxy-terminal domain of PA (PA608) or part of an exposed loop of 19 amino acids (703 to 722) present within this domain was introduced into the pag gene. PA608 did not induce the lethal-factor (LF)-mediated cytotoxic effect on macrophages because it did not bind to the receptor. In contrast, PA711- and PA705-harboring lethal toxins (9- and 16-amino-acid deletions in the loop, starting after positions 711 and 705, respectively) were 10 times less cytotoxic than wild-type PA. After cleavage by trypsin, the mutant PA proteins formed heptamers and bound LF. The capacity of PA711 and PA705 to interact with cells was 1/10 that of wild-type PA. In conclusion, truncation of the carboxy-terminal domain or deletions in the exposed loop resulted in PA that was less cytotoxic or nontoxic because the mutated proteins did not efficiently bind to the receptor.

Animals↗
PubMed1997

Anthrax lethal toxin-induced mitogenic response of human T-cells.

Bacillus anthracis lethal toxin (PALF) stimulated the proliferation of human peripheral blood T-cells in vitro. Activation of T-lymphocytes by PALF required the presence of monocytes and did not result from a collaborative effect between T-cells and B-cells. PALF acted directly on monocytes and independently of T-cells. The monocytes contributed to the proliferation of T-cells by secretion of mediator(s). The mitogenic activity of the lethal toxin was dependent on its metalloprotease activity.

Antigens, Bacterial↗
PubMed1997

A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines.

ADP Ribose Transferases↗
PubMed1995

Characterization of the Bacillus anthracis S-layer: cloning and sequencing of the structural gene.

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined. The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide. The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues. The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp alpha protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.

Amino Acid Sequence↗
PubMed1993

Construction of Bacillus anthracis mutant strains producing a single toxin component.

The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo.

Animals↗
PubMed1991

Structural and functional relationships between Pasteurella multocida and enterobacterial adenylate cyclases.

The Pasteurella multocida adenylate cyclase gene has been cloned and expressed in Escherichia coli. The primary structure of the protein (838 amino acids) deduced from the corresponding nucleotide sequence was compared with that of E. coli. The two enzymes have similar molecular sizes and, based on sequence conservation at the protein level, are likely to be organized in two functional domains: the amino-terminal catalytic domain and the carboxy-terminal regulatory domain. It was shown that P. multocida adenylate cyclase synthesizes increased levels of cyclic AMP in E. coli strains deficient in the catabolite gene activator protein compared with wild-type strains. This increase does not occur in strains deficient in both the catabolite gene activator protein and enzyme III-glucose, indicating that a protein similar to E. coli enzyme III-glucose is involved in the regulation of P. multocida adenylate cyclase. It also indicates that the underlying process leading to enterobacterial adenylate cyclase activation has been conserved through evolution.

Adenylyl Cyclases↗
PubMed1989

Staphylococcal alpha-toxin: a study of membrane penetration and pore formation.

Cell lysis by staphylococcal alpha-toxin, a potent virulence factor of most pathogenic strains of Staphylococcus aureus, follows a three-step sequence: binding of toxin to the membrane, leaking of ions caused by membrane injury, and rupturing of the membrane caused by osmotic swelling. The membrane injury step is composed of two separate events, membrane penetration and membrane perturbation. The membrane penetration event involves conversion of the soluble toxin monomer into an amphipathic molecule, which inserts into the lipid bilayer of the membrane. The membrane perturbation event involves association of the toxin monomers, in the plane of the membrane, to form hexameric transmembrane pores. In this study, we demonstrate that, in an asolectin liposome system, controlling the pH of the external buffer permits these two events to be temporally resolved. Using Controlled-Pore Glass bead-purified alpha-toxin, four events are measured as a function of pH: (a) release of potassium from prelabeled asolectin vesicles, (b) conversion of the toxin to a globally hydrophobic molecule, (c) binding of detergent by the toxin, and (d) labeling of the toxin with photoactivable, radiolabeled, hydrophobic probes. Two of these events, potassium release and conversion to a net hydrophobic state, are paired in that, for the event to occur, each requires a pH of 4.6 or less. In contrast, photolabeling with the membrane probes PC I and PC II (where PC represents phosphatidylcholine) is easily detectable at pH values as high as 5.0 and 6.0. These results demonstrate that, as the pH is lowered, two distinct changes in the physical properties of alpha-toxin occur. The first, which occurs under mild acidic conditions, converts the toxin from a water-soluble molecule into an amphipathic molecule. The second, requiring relatively more acidic conditions, converts the amphipathic toxin molecule into a globally hydrophobic molecule. Correlated with these physical changes in the alpha-toxin molecule is the acquisition of two new biological properties. The conversion of alpha-toxin into an amphipathic conformation correlates with the acquisition of the biological property of the reversible penetration into the bilayer of the asolectin liposome membrane, as evidenced by labeling with the photoactivable probes. At lower pH, the conversion of the toxin into a globally hydrophobic molecule correlates with the biological property of causing damage to the cell membrane, as measured by the release of internal potassium ions, presumably by the formation of transmembrane hexamer pores.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial Toxins↗
PubMed1988

Structural homology between virulence-associated bacterial adenylate cyclases.

The primary structure of the calmodulin-sensitive adenylate cyclase toxin from Bacillus anthracis has been determined from the corresponding nucleotide sequence and compared to that of the homologous toxin secreted by Bordetella pertussis. The cya gene of Bacillus anthracis encodes an 800 amino acid (aa) protein beginning with an N-terminal signal peptide. The central part of the B. anthracis adenylate cyclase includes a region of striking homology with the N-terminal part of the B. pertussis enzyme. In this region a particularly well conserved 24-aa peptide and two other less homologous peptides have been identified. These data corroborate the immunological relatedness of the two enzymes and suggest that the two prokaryotic calmodulin-sensitive adenylate cyclases originate from a common ancestor.

Adenylyl Cyclases↗
PubMed1986

Tetanus toxin is labeled with photoactivatable phospholipids at low pH.

The mechanism of cell penetration by tetanus toxin is unknown; it has been suggested that the toxin may penetrate into the lipid bilayer from a low-pH vesicular compartment. In this work, the interaction of tetanus toxin with liposomal model membranes has been studied by following its photoinduced cross-linking with either a nitrene or a carbene photolytically generated from corresponding light-sensitive phosphatidylcholine analogues. The toxin was labeled only at pHs lower than 5.5. The low pH acquired hydrophobicity of tetanus toxin appears to be confined to its light chain and to the 45-kDa NH2-terminal fragment of the heavy chain. Negatively charged lipids promote the interaction of this toxin with the hydrocarbon chain of phospholipids. The relevance of the present findings to the possible mechanism of nerve cell penetration by tetanus toxin is discussed.

Carbon Radioisotopes↗
PubMed1984

Low pH induces a hydrophobic domain in the tetanus toxin molecule.

Binding of the non-ionic detergent [3H]Triton X-100 by tetanus toxin, by its fragment C and by its alpha chain has been studied. At pH 4.00 or above, tetanus toxin does not bind Triton X-100. At pH lower than 4.00, binding of detergent to the toxin occurs. At pH 3.00, a maximum of 100 mol bound/mol of protein is reached only when the detergent concentration exceeds its critical micelle concentration. No measurable amount of Triton X-100 is bound by the toxin C fragment at pH 3.00. Most of the tetanus toxin alpha chain precipitates out in Triton X-100 at pH 3.00. Leakage of K+ from single-walled asolectin vesicles loaded with potassium was observed with tetanus toxin at pH lower than 4.00. When ganglioside GDlb was present on the asolectin vesicles, release of K+ was obtained with tetanus toxin between pH 4.00 to 5.00. We suggest that, as for diphtheria toxin, entry of tetanus toxin into an acidic compartment of target cells might be required for the expression of its biological activity.

Gangliosides↗
PubMed1982

Characteristics of guinea-pig immune sera elicited by a synthetic diphtheria toxin oligopeptide.

Several oligopeptides of different lengths contained within the Cys 186-Cys 201 first disulfide loop of the diphtheria toxin molecule have been synthesized by a solid-phase method. 125I-labeled rabbit antibodies raised against diphtheria toxin reacted specifically with oligopeptides linked to m-nitrobenzhydrylamine resin when the amino acid chain length was equal to or greater than 10 residues. The synthetic tetradecapeptide (STDP) corresponding to the sequence Gly 188-Cys 201 was used to immunize guinea-pigs. The immune sera obtained reacted with the whole diphtheria toxin molecule as judged by an antigen-linked immunosorbent assay. Anti-STDP sera exhibited a clear, albeit limited, neutralizing effect against the lethal action of diphtheria toxin on cultivated Vero cells. The anti-STDP sera were also able to partially block the ADP-ribosylation of elongation factor 2 mediated by whole diphtheria toxin. In contrast, anti-STDP sera were almost inactive on the enzymic activities of either toxin fragment A or crm 45, a mutant protein which lacks the 15,000 mol. wt C-terminal sequence of the toxin molecule. On the basis of the results obtained, a possible localization of the Cys 188-Cys 201 loop region on the toxin molecule is proposed.

Animals↗
PubMed1982

Tetanus toxin fragment forms channels in lipid vesicles at low pH.

Single-walled asolectin vesicles loaded with K+ at pH 7.00 released their K+ content upon incubation with tetanus toxin fragment B but only when the incubation was at pH below 5.00. Whole tetanus toxin exhibited only a weak releasing activity. Toxin light alpha chain and the carboxyl-terminal 48,000-dalton moiety of the heavy chain (fragment IIC) were unable to provoke K+ release from vesicles at any pH. K+ release from lipid vesicles could also be detected with tetanus toxin heavy beta chain at low pH. Furthermore, using a detergent binding assay ([3H]Triton X-100), we have also shown that an hydrophobic domain, localized in the 50,000-dalton terminal polypeptide of tetanus toxin heavy chain, is detectable at pH 3.60 but not at pH 5.00. These results lead us to conclude that the ability of tetanus toxin fragment B to release K+ from asolectin vesicles at low pH is due to the 50,000-dalton amino-terminal polypeptide of the heavy chain present in toxin fragment B. We propose that this phenomenon is caused by channel formation across the vesicle membrane as has been observed for the 23,000-dalton amino-terminal moiety of diphtheria toxin fragment B.

Amino Acid Sequence↗
PubMed1981

Studies on the role of a nucleoside-phosphate-binding site of diphtheria toxin in the binding of toxin to Vero cells or liposomes.

Using Fab antibody fragments directed against protein crm 45 (non-toxic cross-reacting material serologically-related to diphtheria toxin mutant protein, the product of a non-sens mutation in the diphtheria toxin gene) or the B 45 domain of this molecule (the remaining portion of the toxin B fragment present in protein crm 45)., we have shown that the diphtheria toxin polyphosphate-binding site (P site) [Lory and Collier (1980) Proc. Natl Acad. Sci. USA, 1, 267-271], evidenced by the ability of the toxin to bind ATP, is affected upon incubation of this protein with the Fab fragment of anti-protein (crm 45) IgG but not with the Fab fragment of anti-polypeptide (B 45) IgG. After incubation with an excess of the Fab fragment of anti-polypeptide (B 45) IgG, 125I-labeled toxin does not bind to toxin-sensitive Vero cells. We therefore suggest that the P site is not by itself the binding domain of diphtheria toxin to the cell membrane receptor and that the region of the toxin molecule devoted to this particular activity overlapped probably on the B 45 polypeptide. In contrast with the results obtained by Alving et al. (Proc. Natl Acad. Sci. USA, 1980, 4, 1986-1990) with or without a functional P site, diphtheria toxin is able to bind liposomes. However interaction with phospholipid vesicles with toxin is blocked upon incubation of the protein with the Fab fragment of anti-polypeptide (B 45) IgG. Our data indicate that the P site of toxin does not participate in the ability of this molecule to bind liposomes.

Adenosine Triphosphate↗
PubMed1979

Alternation of low and high affinities of secreted and cell-bound antibodies during the anamnestic response of rabbits to Salmonella senftenberg microorganisms.

We have investigated at the cellular level the variations in affinities of secreted antibodies and of cell receptors during the anamnestic response of rabbits, by using an immunization schedule made up of administrations, over successive intervals of 40 days, of Salmonella senftenberg vaccine. Affinities of free and membrane-bound antibodies biosynthesized at a given time were measured by inhibition of haemolytic plaque-forming cells (PFC) and rosette-forming cells (RFC) with spleen, lymph node, and peripheral blood cells. The immunodominant monosaccharide, methyl-alpha-D-glucopyranoside (alpha-Me-Glc), was used as inhibitor, and the target cells were coated with the O-specific polysaccharide of the immunogenic microorganisms. We observed that the monosaccharide concentration causing a 50% inhibition (I50) alternated between low and high values in both PFC and RFC assays. Furthermore, when sheep erythrocytes were coated with polysaccharides from other strains of Salmonella bearing the determinants for serotypes 1 or 3 the percentage of cross-reactive PFC obtained exhibited a reverse oscillating pattern of variation. These results demonstrated that affinity of secreted antibodies or of antigen-receptors of cells, directed against different determinants of the antigenic polysaccharide, alternated between low and high values after each new vaccine administration. Possible mechanisms accounting for this phenomenon are discussed.

Animals↗