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D Ladant

Publications and source records attributed to D Ladant.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of mutant Bordetella pertussis adenylate cyclase toxins with reduced affinity for calmodulin. Implications for the mechanism of toxin entry into target cells.

Bordetella pertussis secretes a calmodulin-stimulated adenylate cyclase toxin (CyaA) that is one of the major virulence factors of this organism. The toxin is able to enter various types of eukaryotic cells where, upon activation by calmodulin, it catalyzes the production of non-physiological amounts of cyclic AMP. The mechanism of toxin entry into target cells is unknown, although it has been shown that it does not involve receptor-mediated endocytosis. The adenylate cyclase toxin exhibits a very high affinity for calmodulin, and it has been proposed that the energy of calmodulin-binding to CyaA might be required for the entry of the toxin into the target cells [Oldenburg, D.J., Gross, M. K., Wong, C. S. & Storm, D. R. (1992) Biochemistry 31, 8884-8891]. In the present study, we have reexamined this issue by analyzing the cytotoxicity of various modified CyaA toxins that have altered calmodulin affinity. We show that despite their low affinity for calmodulin (at least 1000-times less than that of the wild type CyaA), these toxins were able to efficiently deliver their catalytic domain into the cytoplasm of the target cells, erythrocytes. These results demonstrate that high-affinity calmodulin binding is not required for the entry of B. pertussis adenylate cyclase into eukaryotic cells. However, the high-affinity of CyaA for calmodulin is crucial for an efficient synthesis of cAMP within the target cells.

Adenylyl Cyclases↗
PubMed1996

In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying viral CD8+ T cell epitopes.

Exogenous Ags enter the endosomal pathway and are presented to CD4+ T cells in association with class II molecules whereas endogenously synthesized Ags, such as viral proteins, are presented to CD8+ T cells in association with MHC class I molecules. Therefore, most CTL activation strategies use live vectors although an alternative possibility could be to deliver the epitope into the cytosol by targeting it to an invasive nonreplicative vector. The adenylate cyclase toxin of Bordetella pertussis is able to invade a large number of eukaryotic cells and to deliver its catalytic domain to the cytosol of the cells. In the present study, we have tested the in vivo immunogenicity of recombinant adenylate cyclase toxins expressing CTL epitopes either from the nucleoprotein of lymphocytic choriomeningitis virus or from the V3 region of HIV-1 gp120. BALB/c mice immunized with these toxins developed high specific CTL responses that were shown to be mediated by class I-restricted CD8+ CTL. The induction of CTL responses by recombinant toxins did not require CD4+ T cells and the cytotoxic activity persisted 2 mo after immunization. The activation of CTL responses by the recombinant adenylate cyclase toxin required the full-length invasive activity of the toxin but did not depend upon its catalytic adenylate cyclase activity as demonstrated with a genetically inactivated recombinant toxin expressing the lymphocytic choriomeningitis virus epitope. This genetically detoxified invasive toxin represents, therefore, an attractive new vector for CTL activation.

Adenylyl Cyclases↗
PubMed1995

Interaction of calcium with Bordetella pertussis adenylate cyclase toxin. Characterization of multiple calcium-binding sites and calcium-induced conformational changes.

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity. Binding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (KD = 0.5-0.8 mM) Ca2+ binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca(2+)-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.

Adenylate Cyclase Toxin↗
PubMed1995

Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli.

Neurocalcins are brain-specific proteins that belong to a new subclass of the EF-hand superfamily of calcium binding proteins, defined by the photoreceptor cell-specific protein, recoverin. Recoverin, which regulates the desensitization of photo-excited rhodopsin, is myristoylated and exhibits a calcium-myristoyl switch. Like recoverin, neurocalcins have a signal for N-myristoylation and possess four EF-hands, although the first one lacks some residues critical for calcium binding. In this work, I have examined the calcium and membrane binding properties of recombinant myristoylated and unmyristoylated neurocalcin delta. I show that neurocalcin, like recoverin, binds to biological membranes in a calcium- and myristoyl-dependent manner. Both myristoylated and unmyristoylated proteins bind three calcium ions. However, the unmyristoylated form exhibits a higher affinity for calcium than the myristoylated protein but shows a lower cooperativity in binding calcium. These data support the model for the calcium-myristoyl switch mechanism proposed for recoverin (Zozulya, S., and Stryer, L. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11569-11573; Dizhoor, A. M., Chen, C. K., Olshevskaya, E., Sinelnikova, V. V., and Hurley, J. B. (1993) Science 259, 829-832). Using point mutations, I have investigated the relative importance of each of the three functional EF hands (EF2, EF3, and EF4) in the calcium and membrane binding properties of neurocalcin. Calcium and membrane binding properties of the mutant-myristoylated proteins suggest that binding of calcium to EF2 is critical in triggering the binding of the protein to membranes.

Amino Acid Sequence↗
PubMed1995

Cell-invasive activity of epitope-tagged adenylate cyclase of Bordetella pertussis allows in vitro presentation of a foreign epitope to CD8+ cytotoxic T cells.

The adenylate cyclase (AC) toxin (CyaA) of Bordetella pertussis has an invasive catalytic domain (AC domain) which penetrates the cytoplasmic membrane of a variety of eukaryotic cells and intoxicates them by unregulated synthesis of cyclic AMP. Previous work led to identification of five permissive sites in the AC domain at which heterologous peptides are accommodated without affecting its enzymatic properties. We have constructed a set of CyaA toxins tagged at these permissive sites by insertion of a CD8+ T-cell epitope, RPQASGVYMGNLTAQ, from the nucleoprotein of lymphocytic choriomeningitis virus. Introduction of the epitope at any of the five sites did not affect the capacity of the toxin to deliver its AC domain into target cells. Moreover, the toxin with the inserted epitope was shown to sensitize target cells for lysis by epitope-specific CD8+ cytotoxic T lymphocytes in vitro, showing that the tagged AC was processed for presentation of the lymphocytic choriomeningitis virus epitope in association with the major histocompatibility complex class I molecules. This finding indicates that by virtue of delivery of foreign epitopes into the antigen-presenting cells, purpose-designed recombinant CyaAs may be useful for induction of specific major histocompatibility complex class I-restricted cell-mediated immunity also in vivo.

Adenylyl Cyclases↗
PubMed1994

Adenylate cyclase toxin (CyaA) of Bordetella pertussis. Evidence for the formation of small ion-permeable channels and comparison with HlyA of Escherichia coli.

The interaction between the adenylate cyclase toxin (CyaA) of Bordetella pertussis and lipid was studied using the lipid bilayer assay. The addition of CyaA to the aqueous phase bathing lipid bilayer membranes composed of different lipids resulted in the increase of the membrane conductance. This increase was rather small for membranes formed of pure lipids as compared with lipid mixtures such as asolectin. The toxin formed in asolectin membranes small transient ion-permeable channels with a single-channel conductance of 27 pS in 1 M KCl, which is considerably smaller than that of the alpha-hemolysin (HlyA) of Escherichia coli (1500 pS). Experiments with different salts suggested that the CyaA-induced channels were exclusively cation-selective because of negative charges localized at the channel mouth. The single-channel conductance of channels initiated by CyaA was independent of whether the toxin was purified from B. pertussis or from recombinant E. coli. However, the channel-forming activity of the CyaA expressed in B. pertussis was substantially higher than that of the recombinant toxin. Experiments with mutant forms of CyaA suggested that both the activation of CyaA by CyaC and the hemeolytic part of the toxin, but not the repeats and the cyclase activity, are required for channel formation in lipid bilayer membranes.

Adenylate Cyclase Toxin↗
PubMed1993

Bordetella pertussis adenylate cyclase: a toxin with multiple talents.

The adenylate cyclase toxin of Bordetella pertussis is a secreted multifunctional protein, endowed with calmodulin-activated catalytic, haemolytic and cytotoxic activities. Residues and domains involved in different functions have been localized and several permissive sites, able to accommodate insertion of peptides without impairing the different functions of the toxin, have been identified. A 400-bp region in the promoter upstream region of the cyaA gene, encoding the toxin, has been defined as the target of transcriptional activation.

Adenylyl Cyclases↗
PubMed1993

Repeat sequences in the Bordetella pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the Escherichia coli alpha-haemolysin translocator.

The 1706-residue adenylate cyclase toxin (CyaA) of Bordetella pertussis is an RTX protein with extensive carboxy-proximal glycine and aspartate-rich repeats. CyaA does not have a cleavable amino-terminal signal peptide and can be secreted across both bacterial membranes of the Escherichia coli cell envelope by the alpha-haemolysin (HlyA) translocator (HlyBD/TolC). We performed deletion mapping of secretion signals recognized in CyaA by this heterologous translocator. Truncated proteins with N-terminal and internal deletions were secreted at levels up to 10 times higher than intact CyaA and similar to HlyA. A secretion signal recognized by HlyBD/TolC was found within the last 74 residues of CyaA. However, secretion of CyaA was reduced but not abolished upon deletion of the last 75 or 217 residues, indicating that at least two additional secretion signals recognized by HlyBD/TolC are within CyaA. One of them was localized to the repeat sequence between residues Asp-1587 to Ile-1631. Interestingly, a conserved 'acidic' motif (Glu/Asp)-(X)11-Asp-(X)3/5-(Glu/Asp)-(X)14-Asp was found in the C-terminal sequences of HlyA, CyaA and the two secreted CyaA derivatives. We speculate that the presence and spacing of acidic residues may be an important feature of secretion signals recognized by the haemolysin translocator.

Adenylate Cyclase Toxin↗
PubMed1992

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.

Adenylate Cyclase Toxin↗
PubMed1992

Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.

Adenylyl Cyclases↗
PubMed1990

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase.

The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.

Adenosine Triphosphate↗
PubMed1990

Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase.

The Bordetella pertussis cyaA gene encodes a virulence factor which is a bifunctional protein exhibiting calmodulin-sensitive adenylate cyclase and hemolytic activities (P. Glaser, H. Sakamoto, J. Bellahov, A. Ullmann, and A. Danchin, EMBO J. 7:3997-4004, 1988). We characterized the hemolytic and toxin activities of the 200-kilodalton (kDa) bifunctional (CyaA) protein and showed that, whether cell associated or secreted, the 200-kDa CyaA protein carries hemolytic and toxin functions. The catalytically active 45-kDa form of adenylate cyclase released by proteolytic digestion of the 200-kDa CyaA protein displayed neither hemolytic nor toxin activities. We constructed in-phase deletions in the 3' region of the cyaA gene, which presumably carries the hemolytic determinant, and showed that the resulting proteins exhibited wild-type adenylate cyclase activity and were secreted without processing into culture supernatants. The hemolytic activities of these mutant CyaA proteins were severely reduced, and their toxin activities were abolished. These results suggest that the structural integrity of the 200-kDa CyaA protein is necessary for toxin activity and that distinct structural determinants within the CyaA protein are involved in secretion, pore formation, and entry into target cells.

Adenylyl Cyclases↗
PubMed1990

Synthesis and secretion of Bordetella pertussis adenylate cyclase as a 200-kilodalton protein.

Bordetella pertussis, the etiological agent of whooping cough, synthesizes a calmodulin-sensitive adenylate cyclase that is suspected to play a major role in the virulence of this bacterium. We show that adenylate cyclase synthesized as a 200-kilodalton protein is the product of the cyaA gene and that various virulent Bordetella species secrete this high-molecular-weight polypeptide without apparent proteolytic processing. When submitted to trypsin digestion, the 200-kilodalton protein was converted to a stable 45- to 50-kilodalton species. This corresponds to the size of the enzyme previously purified from a culture supernatant. The molecular heterogeneity reported for the various identified forms of adenylate cyclase could therefore result in part from proteolytic degradation or molecular aggregation of the major 200-kilodalton form of the enzyme.

Adenylyl Cyclases↗
PubMed1989

Characterization of the calmodulin-binding and of the catalytic domains of Bordetella pertussis adenylate cyclase.

The structural organization of the low molecular mass form (43 kDa) of Bordetella pertussis adenylate cyclase was dissected taking advantage of the known sequence of the bacterial cya gene (Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A., and Danchin, A. (1988) Mol. Microbiol. 2, 19-30) and its low content of Trp and Met residues. Cleavage of the 43-kDa protein and of its complementary tryptic fragments (T25 and T18 peptides) with N-chlorosuccinimide and cyanogen bromide followed by sodium dodecyl sulfate-polyacrylamide gel analysis of digestion products allowed the following conclusions: (i) the catalytically active 43-kDa form of B. pertussis adenylate cyclase is within the first 400 residues of the protein encoded by the cya gene. T25 occupies the N-terminal domain of the protein (residues 1-235/237). Isolated T25 fragment exhibits a low but measurable enzymatic activity which indicates that it harbors the catalytic site; (ii) T18 which is the main calmodulin-binding domain, occupies the C-terminal segment of protein (residues 236/238-399) and is devoid of catalytic properties; (iii) the two complementary peptides T25 and T18 reassociated only in the presence of calmodulin, leading to significant recovery of the original activity. These results demonstrate that both fragments of the 43-kDa form of adenylate cyclase are essential for a high level of enzymatic activity.

Adenylyl Cyclases↗
PubMed1989

Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.

Adenylyl Cyclases↗
PubMed1988

Affinity-based chromatography utilizing genetically engineered proteins. Interaction of Bordetella pertussis adenylate cyclase with calmodulin.

An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution. However, when immobilized VU-8 calmodulin was tested as a calcium-dependent, affinity-based, adsorption chromatography step in the purification of the cyclase from culture media or bacterial extracts, the enzyme bound to the column in a calcium-dependent manner, and a nearly homogeneous enzyme was obtained in high yield. These results demonstrate the feasibility of using engineered calmodulins that have selective differences in activity for the rational design of rapid purification protocols for calmodulin-binding proteins as well as indicate the importance of the conserved negative charge cluster at residues 82-84 of calmodulin for activation of this cyclase.

Adenylyl Cyclases↗
PubMed1988

Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains.

The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a photoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:1 complex with CaM or azido-CaM. CaM-bound adenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically active conformation resembling that of the undigested complex. When proteolysis was carried out in the absence of CaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since this fragment cannot be cross-linked with azido-CaM we suggest that the CaM-binding site on the 25-kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.

Adenylyl Cyclases↗