Biomedical subjects

Daniel Ladant

Publications and source records attributed to Daniel Ladant.

At least 19 recordsLinked to original sources

PubMed2006

Cell entry and cAMP imaging of anthrax edema toxin.

The entry and enzymatic activity of the anthrax edema factor (EF) in different cell types was studied by monitoring EF-induced changes in intracellular cAMP with biochemical and microscopic methods. cAMP was imaged in live cells, transfected with a fluorescence resonance energy transfer biosensor based on the protein kinase A regulatory and catalytic subunits fused to CFP and YFP, respectively. The cAMP biosensor was located either in the cytosol or was membrane-bound owing to the addition of a tag determining its myristoylation/palmitoylation. Real-time imaging of cells expressing the cAMP biosensors provided the time course of EF catalytic activity and an indication of its subcellular localization. Bafilomycin A1, an inhibitor of the vacuolar ATPase proton pump, completely prevented EF activity, even when added long after the toxin. The time course of appearance of the adenylate cyclase activity and of bafilomycin A1 action suggests that EF enters the cytosol from late endosomes. EF remains associated to these compartments and its activity shows a perinuclear localization generating intracellular cAMP concentration gradients from the cell centre to the periphery.

Adenylyl Cyclases↗

PubMed2006

Structural and functional characterization of an essential RTX subdomain of Bordetella pertussis adenylate cyclase toxin.

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells by a unique mechanism that consists in a calcium-dependent, direct translocation of the CyaA catalytic domain across the plasma membrane of the target cells. CyaA possesses a series of a glycine- and aspartate-rich nonapeptide repeats (residues 1006-1613) of the prototype GGXG(N/D)DX(L/I/F)X (where X represents any amino acid) that are characteristic of the RTX (repeat in toxin) family of bacterial cytolysins. These repeats are arranged in a tandem fashion and may fold into a characteristic parallel beta-helix or beta-roll motif that constitutes a novel type of calcium binding structure, as revealed by the three-dimensional structure of the Pseudomonas aeruginosa alkaline protease. Here we have characterized the structure-function relationships of various fragments from the CyaA RTX subdomain. Our results indicate that the RTX functional unit includes both the tandem repeated nonapeptide motifs and the adjacent polypeptide segments, which are essential for the folding and calcium responsiveness of the RTX module. Upon calcium binding to the RTX repeats, a conformational rearrangement of the adjacent non-RTX sequences may act as a critical molecular switch to trigger the CyaA entry into target cells.

Adenylate Cyclase Toxin↗

PubMed2006

Delivery of the HIV-1 Tat protein to dendritic cells by the CyaA vector induces specific Th1 responses and high affinity neutralizing antibodies in non human primates.

The human immunodeficiency virus type 1 (HIV-1) Tat is a key protein playing a major role in the infectivity of the virus. Thus, HIV-Tat based vaccines have been proposed as an attractive option to treat AIDS. Recently, we have shown that the recombinant detoxified adenylate cyclase (CyaA) from Bordetella pertussis carrying HIV-Tat (CyaA-E5-Tat), targets dendritic cells (DCs) and induces specific Th1 polarized and neutralizing antibody responses in mice. To further explore the potentialities of this prototype vaccine for human use, we analyzed the CyaA-E5-Tat induced antibody responses in non-human primates and established the biological characteristics of these antibodies. African Green Monkeys (AGM) were immunized with CyaA-E5-Tat in the presence or in the absence of alum adjuvant. First, we showed that the anti-CyaA antibodies induced by such immunization does not interfere with the binding of CyaA-E5-Tat to its receptor at the DC surface, the alphaMbeta2 integrin. Monkeys immunized with CyaA-E5-Tat, with or without alum, produced anti-Tat antibodies that mainly recognized the N-terminal domain of the Tat protein. Importantly, all sera obtained after three immunizations displayed the capacity to bind to Tat and neutralize its transactivating function in vitro. Finally, in the absence of alum, CyaA-E5-Tat, induced Th1 Tat specific T cell responses. These findings reveal that CyaA-E5-Tat is efficiently delivered in non-human primates and had a significant impact on the generation of neutralizing anti-Tat antibodies. These observations are, thus, encouraging for the use of the CyaA vector in human and also suggest that CyaA-E5-Tat might be a useful tool to decipher the biological characteristic of such antibodies.

AIDS Vaccines↗

PubMed2006

An increase in antimycobacterial Th1-cell responses by prime-boost protocols of immunization does not enhance protection against tuberculosis.

Bordetella pertussis adenylate cyclase (CyaA) toxoid is a powerful nonreplicative immunization vector targeting dendritic cells, which has already been used successfully in prophylactic and therapeutic vaccination in various preclinical animal models. Here, we investigated the potential of CyaA, harboring strong mycobacterial immunogens, i.e., the immunodominant regions of antigen 85A or the complete sequence of the 6-kDa early secreted antigenic target (ESAT-6) protein, to induce antimycobacterial immunity. By generating T-cell hybridomas or by using T cells from mice infected with mycobacteria, we first demonstrated that the in vitro delivery of 85A or ESAT-6 to antigen-presenting cells by CyaA leads to processing and presentation, by major histocompatibility complex class II molecules, of the same epitopes as those displayed upon mycobacterial infection. Importantly, compared to the recombinant protein alone, the presentation of ESAT-6 in vitro was 100 times more efficient upon its delivery to antigen-presenting cells in fusion to CyaA. Immunization with CyaA-85A or CyaA-ESAT-6 in the absence of any adjuvant induced strong antigen-specific lymphoproliferative, interleukin-2 (IL-2) and gamma interferon (IFN-gamma) cytokine responses, in the absence of any IL-4 or IL-5 production. When used as boosters after priming with a BCG expressing ESAT-6, the CyaA-85A and CyaA-ESAT-6 proteins were able to strikingly increase the sensitivity and intensity of proliferative and Th1-polarized responses and notably the frequency of antigen-specific IFN-gamma-producing CD4+ T cells. However, immunization with these CyaA constructs as subunit vaccines alone or as boosters did not allow induction or improvement of protection against Mycobacterium tuberculosis infection. These results question the broadly admitted correlation between the frequency of IFN-gamma-producing CD4+ T cells and the level of protection against tuberculosis.

Adenylate Cyclase Toxin↗

PubMed2005

Eradication of established tumors by vaccination with recombinant Bordetella pertussis adenylate cyclase carrying the human papillomavirus 16 E7 oncoprotein.

High-risk human papillomaviruses (HPV) such as HPV16 are associated with the development of cervical cancer. The HPV16-E6 and HPV16-E7 oncoproteins are expressed throughout the replicative cycle of the virus and are necessary for the onset and maintenance of malignant transformation. Both these tumor-specific antigens are considered as potential targets for specific CTL-mediated immunotherapy. The adenylate cyclase (CyaA) of Bordetella pertussis is able to target dendritic cells through specific interaction with the alpha(M)beta(2) integrin. It has been previously shown that this bacterial protein could be used to deliver CD4(+) and CD8(+) T cell epitopes to the MHC class II and class I presentation pathways to trigger specific Th and CTL responses in vivo, providing protection against subsequent viral or tumoral challenge. Here, we constructed recombinant CyaA containing either the full sequence or various subfragments from the HPV16-E7 protein. We show that, when injected to C57BL/6 mice in absence of any adjuvant, these HPV16-recombinant CyaAs are able to induce specific Th1 and CTL responses. Furthermore, when injected into mice grafted with HPV16-E7-expressing tumor cells (TC-1), one of these recombinant proteins was able to trigger complete tumor regression in 100% of the animals tested. This therapeutic efficacy compared favorably to that of strongly adjuvanted peptide and was marginally affected by prior immunity to CyaA protein. This study represents the first in vivo demonstration of the antitumoral therapeutic activity of recombinant CyaA proteins carrying human tumor-associated antigens and paves the way for the testing of this vector in clinical trials.

Adenylyl Cyclases↗

PubMed2005

Interaction network among Escherichia coli membrane proteins involved in cell division as revealed by bacterial two-hybrid analysis.

Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.

Adenylyl Cyclases↗

PubMed2005

Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis.

HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8(+) and CD4(+) T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8(+) T cells were generated after vaccination with CyaA-E5-Tat in a CD4(+) T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.

AIDS Vaccines↗

PubMed2004

Bordetella pertussis adenylate cyclase delivers chemically coupled CD8+ T-cell epitopes to dendritic cells and elicits CTL in vivo.

The adenylate cyclase (CyaA) produced by Bordetella pertussis is able to deliver CD8+ and CD4+ T-cell epitopes genetically grafted within the catalytic domain of the molecule into antigen presenting cells in vivo. We develop now a new approach in which peptides containing CD8+ epitopes are chemically linked to CyaA. We show that CTL responses were induced in mice immunized with CyaA bearing these CD8+ epitopes. Moreover, we demonstrate that the OVA257-264 CD8+ epitope chemically grafted to CyaA is presented to CD8+ T cells by a mechanism requiring (1) proteasome processing, (2) TAP and (3) neosynthesis of MHC class I molecules. Thus, this novel strategy represents a very versatile system as a single CyaA carrier protein could be easily and rapidly coupled to any desired synthetic peptide.

Adenylate Cyclase Toxin↗

PubMed2004

Essential role of methionine residues in calmodulin binding to Bordetella pertussis adenylate cyclase, as probed by selective oxidation and repair by the peptide methionine sulfoxide reductases.

Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels. In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages. Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC. We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC. Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein. We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC. The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization.

Adenylyl Cyclases↗

PubMed2004

Relief of catabolite repression in a cAMP-independent catabolite gene activator mutant of Escherichia coli.

We isolated and characterized a new catabolite gene activator mutant (crp*) of Escherichia coli that confers cAMP-independent expression and total relief of catabolite repression of beta-galactosidase and tryptophanase synthesis. The two mutations responsible for this phenotype change the amino acids at codon 72 from Glu to Ala and at codon 144 from Ala to Thr in the corresponding CAP* protein.

Cyclic AMP↗

PubMed2003

Differential mechanisms for calcium-dependent protein/membrane association as evidenced from SPR-binding studies on supported biomimetic membranes.

In this work, two different types of supported biomimetic membranes were designed to study the membrane binding properties of two different proteins that both interact with cellular membranes in a calcium-dependent manner. The first one, neurocalcin, is a member of a subfamilly of EF-hand calcium-binding proteins that exhibit a calcium-myristoyl switch. The second protein is a bacterial toxin, the adenylate cyclase produced by Bordetella pertussis, the causative agent of whooping cough. The biomimetic membranes constructed in this study were either hybrid bilayer membranes or polymer-tethered membranes. Hemimembrane formation was obtained in two steps: a monolayer of 1-octadecanethiol or octadecyltrichlorosilane was self-assembled on top of the gold or glass surface, respectively, and then the egg-phosphatidyl choline (PC) vesicle fused on the hydrophobic alkyl layer. Polymer-tethered membranes on solid support were obtained using N-hydroxysuccinimide (NHS)-terminated-poly(ethyleneglycol) (PEG)-phospholipids as anchoring molecules. Egg-PC/1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-poly(ethyleneglycol)-N-hydroxy-succinimide (DSPE-PEG-NHS) mixture liposomes were injected on the top of an amine grafted surface (cysteamine-coated gold or silanized glass); vesicles were linked to the surface and disrupted, leading to the formation of a bilayer. The biomimetic membrane constructions were followed by surface plasmon spectroscopy, while membrane fluidity and continuity were observed by fluorescence microscopy. Protein/membrane binding properties were determined by resonance surface plasmon measurements. The tethered bilayer, designed here, is very versatile as it can be adapted easily to different types of support. The results demonstrate the potentialities of such polymer-tethered artificial membranes for the study of proteins that insert into biological membranes such as toxins and/or integral membrane proteins.

Adenylate Cyclase Toxin↗

PubMed2003

In vitro selection for enzymatic activity: a model study using adenylate cyclase.

An in vitro enzyme selection that can, in principle, be generalised to most chemical reactions, is described. It makes use of filamentous phage display and of a tailor-made antibody fragment directed against the reaction product. The conversion of ATP into 3',5'-cyclic AMP catalysed by Bordetella pertussis adenylate cyclase is taken as an example.

Adenylyl Cyclases↗

PubMed2003

Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain.

Adenylate cyclase toxin (CyaA) is one of the major virulence factors produced by Bordetella pertussis, the whooping cough agent. CyaA belongs to the repeat in toxin protein family and requires a post-translational fatty acylation to form cation-selective channels in target cell membranes and to penetrate into cytosol. We have demonstrated recently that CyaA uses the alphaMbeta2 integrin (CD11b/CD18) as a specific cellular receptor. Here we show that the acylation of CyaA is required for a productive and tight interaction of the toxin with cells expressing CD11b. In addition, we demonstrate that the catalytic domain is not required for binding of CyaA to CD11b and that the main integrin interacting domain of CyaA is located in its glycine/aspartate-rich repeat region. These data decipher, for the first time, the interaction of CyaA with CD11b-positive cells and open new prospects for understanding the interaction of Bordetella pertussis with innate and adaptive immune systems.

Adenylyl Cyclases↗

PubMed2003

Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes.

The adenylate cyclase (CyaA) of Bordetella pertussis is able to deliver CD8(+) T cell epitopes into the cytosol of CD11b(+) dendritic cells (DC) following its specific interaction with the alpha(M)beta(2) integrin (CD11b/CD18). This delivery results in intracellular processing and presentation by MHC class I molecules of the CD8(+) T cell epitopes inserted into CyaA. Indeed, we previously showed that CyaA toxins carrying a single cytotoxic T lymphocyte (CTL) epitope can induce efficient protective and therapeutic antitumor immunity in mice. With a view to elaborating cancer immunotherapy in humans using CyaA, we constructed two recombinant CyaA carrying HLA*0201-restricted melanoma epitopes. Here we show that these recombinant CyaA induce strong anti-melanoma CTL responses in HLA*0201 transgenic mice, even after a single i.v. immunization without adjuvant. These responses are long lasting, since they were also detected 5 months after the last injection. Finally, human DC treated with the recombinant CyaA were shown to process and present efficiently the melanoma epitopes to human CTL clones. Altogether, our results demonstrate that tumoral epitopes inserted into CyaA are efficiently processed and presented in association with human MHC molecules. These observations suggest that CyaA is capable of activating antitumoral CTL in humans and highlight the potential of CyaA for use in cancer immunotherapy.

Adenylate Cyclase Toxin↗

PubMed2003

Human immunodeficiency virus (HIV) type 1 transframe protein can restore activity to a dimerization-deficient HIV protease variant.

The protease (PR) from human immunodeficiency virus (HIV) is essential for viral replication: this aspartyl protease, active only as a dimer, is responsible for cleavage of the viral polyprotein precursors (Gag and Gag-Pol), to release the functional mature proteins. In this work, we have studied the structure-function relationships of the HIV PR by combining a genetic test to detect proteolytic activity in Escherichia coli and a bacterial two-hybrid assay to analyze PR dimerization. We showed that a drug-resistant PR variant isolated from a patient receiving highly active antiretroviral therapy is impaired in its dimerization capability and, as a consequence, is proteolytically inactive. We further showed that the polypeptide regions adjacent to the PR coding sequence in the Gag-Pol polyprotein precursor, and in particular, the transframe polypeptide (TF), located at the N terminus of PR, can facilitate the dimerization of this variant PR and restore its enzymatic activity. We propose that the TF protein could help to compensate for folding and/or dimerization defects in PR arising from certain mutations within the PR coding sequence and might therefore function to buffer genetic variations in PR.

Amino Acid Sequence↗

PubMed2003

Efficient display of two enzymes on filamentous phage using an improved signal sequence.

Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50- fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.

Adenylyl Cyclases↗

PubMed2002

In vivo receptor-mediated delivery of a recombinant invasive bacterial toxoid to CD11c + CD8 alpha -CD11bhigh dendritic cells.

The precise contribution of mouse dendritic cells (DC) CD8 alpha +CD11blow and CD8 alpha -CD11bhigh subsets to CTL priming is not fully defined. Here we show that CyaA, the adenylate cyclase toxin of Bordetella pertussis, an invasive bacterial toxin that binds cells through CD11b/CD18 can be exploited for the targeted delivery of an exogenous peptide to the CD8 alpha -CD11bhigh subset in vivo. Antigen (Ag) genetically inserted in the N-terminal domain of mutant CyaA devoid of catalytic activity, are targeted to CD8 alpha -CD11bhigh DC by the CD11b/CD18-dependent binding of CyaA to the cell surface. Ag is then presented by MHC class I molecules of CD8 alpha -CD11bhigh DC after a TAP-dependent, cytosolic processing. As a result, CTL are primed after a single injection, bypassing requirement for adjuvant, CD4+ T cell help and CD40 signaling. Beside the interest of the CyaA vector for vaccine development, these results show that Ag presentation focused on CD8 alpha -CD11bhigh DC in vivo is sufficient for eliciting a vigorous CTL response and that CD11b/CD18 could be a suitable surface molecule for targeting Ag to DC.

Adenylate Cyclase Toxin↗

PubMed2002

Analysis of mammalian peroxin interactions using a non-transcription-based bacterial two-hybrid assay.

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pbeta; and (iii) the C(3)HC(4) RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions.

Animals↗