Biomedical subjects

Françoise Jacob-Dubuisson

Publications and source records attributed to Françoise Jacob-Dubuisson.

12 recordsLinked to original sources

PubMed2006

Structural analysis of Bordetella pertussis BugE solute receptor in a bound conformation.

The Bug proteins form a large family of periplasmic solute-binding receptors present in a number of bacterial species. Here, the crystal structure of Bordetella pertussis BugE, a member of the Bug family coded by the gene BP0250, is reported. It adopts the Venus flytrap architecture of periplasmic binding proteins, with two domains separated by a deep cleft. BugE has a bound ligand, identified as a glutamate. The structure of B. pertussis BugD, which is an aspartic acid transporter, has recently been reported. These structures reveal high conservation of the Bug architecture, despite limited sequence identity. They share a common carboxylate-binding motif defined by two strand-beta-turn-alpha-helix motifs, also involving two water molecules to bridge the carboxylate O atoms to the protein. The two water molecules are hydrogen bonded to a common main-chain carbonyl group. Although the features of the carboxylate-binding motif are totally conserved, the ligand in BugE is bound by its side-chain carboxylate group rather than by its alpha-carboxylate as in BugD. This specific ligand-binding motif is highly conserved in Bug proteins and the BugE structure suggests that the cavity of the Bug proteins might also accommodate carboxylated solutes other than amino acids. The vast expansion of the Bug family in several bacterial genera is likely to be explained by the possible diversity of ligands. No charged residues are involved in glutamate binding by BugE, unlike what has been described for all glutamate receptors reported so far.

Amino Acid Motifs↗

PubMed2006

Crystallization and preliminary X-ray diffraction analysis of two extracytoplasmic solute receptors of the DctP family from Bordetella pertussis.

DctP6 and DctP7 are two Bordetella pertussis proteins which belong to the extracytoplasmic solute receptors (ESR) superfamily. ESRs are involved in the transport of substrates from the periplasm to the cytosol of Gram-negative bacteria. DctP6 and DctP7 have been crystallized and diffraction data were collected using a synchrotron-radiation source. DctP6 crystallized in space group P4(1)2(1)2, with unit-cell parameters a = 108.39, b = 108.39, c = 63.09 A, while selenomethionyl-derivatized DctP7 crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 64.87, b = 149.83, c = 170.65 A. The three-dimensional structure of DctP7 will be determined by single-wavelength anomalous diffraction, while the DctP6 structure will be solved by molecular-replacement methods.

Amino Acid Sequence↗

PubMed2006

Secretion signal of the filamentous haemagglutinin, a model two-partner secretion substrate.

The sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram-negative bacteria, the widespread two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-related proteins. The secreted TpsA proteins carry a characteristic 250-residue-long N-terminal 'TPS domain' essential for secretion, while their TpsB transporters are pore-forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. However, the nature of the secretion signal has not been elucidated yet. The whooping cough agent Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter FhaC. In this work, we show specific interactions between an N-terminal fragment of FHA containing the TPS domain and FhaC by using two different techniques, an overlay assay and a pull-down of the complex. FhaC recognizes only non-native conformations of the TPS domain, corroborating the model that in vivo, periplasmic FHA is not yet folded. By generating single amino acid substitutions, we have identified interaction determinants forming the secretion signal. They are found unexpectedly far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA-TpsB interaction. The N-terminal domain of FhaC is involved in the FHA-FhaC interaction, in agreement with its proposed function and periplasmic localization.

Adhesins, Bacterial↗

PubMed2005

Crystal structure of Bordetella pertussis BugD solute receptor unveils the basis of ligand binding in a new family of periplasmic binding proteins.

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.

Amino Acid Sequence↗

PubMed2005

Channel properties of TpsB transporter FhaC point to two functional domains with a C-terminal protein-conducting pore.

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a beta-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.

Bacterial Outer Membrane Proteins↗

PubMed2005

The periplasmic binding protein of a tripartite tricarboxylate transporter is involved in signal transduction.

A new type of solute importer has been identified recently in various bacterial genera and called the tripartite tricarboxylate transporter (TTT). TTTs consist of two cytoplasmic membrane proteins and a periplasmic solute-binding protein. In the whooping cough agent Bordetella pertussis, a TTT system that has been called BctCBA mediates the uptake of citrate, with BctA and BctB being the membrane components and BctC, the periplasmic protein. Here, we describe that the expression of the bctCBA operon is induced by the presence of citrate in the milieu. The signalling cascade involves both BctC and the signal transduction two-component system BctDE, encoded by an operon adjacent to bctCBA. Furthermore, two-hybrid analyses and affinity chromatography experiments indicated that citrate-liganded BctC interacts with the periplasmic domain of the sensor protein, BctE. Thus, BctC is part of the signalling cascade leading to upregulation of the transporter operon in the presence of its solute, a new function for periplasmic binding proteins of TT transporters.

Bacterial Proteins↗

PubMed2004

Protein secretion through autotransporter and two-partner pathways.

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.

Amino Acid Sequence↗

PubMed2004

The crystal structure of filamentous hemagglutinin secretion domain and its implications for the two-partner secretion pathway.

Filamentous hemagglutinin (FHA), the major 230-kDa adhesin of the whooping cough agent Bordetella pertussis, is one of the most efficiently secreted proteins in Gram-negative bacteria. FHA is secreted by means of the two-partner secretion (TPS) pathway. Several important human, animal, and plant pathogens also secrete adhesins and other virulence factors by using this mode of secretion. A TPS system is composed of two separate proteins, with TpsA the secreted protein and TpsB its associated specific outermembrane transporter. All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. We report here the 1.7- A structure of a functionally secreted 30-kDa N-terminal fragment of FHA. It reveals that the TPS domain folds into a beta-helix, with three extrahelical motifs, a beta-hairpin, a four-stranded beta-sheet, and an N-terminal capping, mostly formed by the nonconserved regions of the TPS domain. The structure thus explains why the TPS domain is able to initiate folding of the beta-helical motifs that form the central domain of the adhesin, because it is itself a beta-helical scaffold. It also contains less conserved extrahelical regions most likely involved in specific properties, such as the recognition of the outer-membrane transporter. This structure is representative of the TPS domains found so far in >100 secreted proteins from pathogenic bacteria. It also provides a mechanistic insight into how protein folding may be linked to secretion in the TPS pathway.

Adhesins, Bacterial↗

PubMed2004

Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide.

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.

Adenosine Triphosphatases↗

PubMed2003

Surface anchoring of bacterial subtilisin important for maturation function.

Many extracytoplasmic proteins undergo proteolytic processing during secretion, which is essential to their maturation. These post-translational modifications are carried out by specific enzymes whose subcellular localization is important for function. We have described a maturation subtilisin in Gram-negative Bordetella pertussis, the autotransporter SphB1. SphB1 catalyses the maturation of the precursor of the adhesin filamentous haemagglutinin (FHA) at the bacterial surface, in addition to the processing of its own precursor. Here, we show that the outer membrane anchor of SphB1 is crucial to its function, as evidenced by the lack of FHA maturation in a strain releasing a variant of SphB1 into the milieu. In contrast, surface association is not required for automaturation of SphB1. The surface retention of mature SphB1 is mediated by lipidation of the protein. The tethered protease appears to be stabilized by unusual Gly- and Pro-rich motifs at the N-terminus of the protein. This represents a new mode of localization for a protease involved in protein secretion.

Adhesins, Bacterial↗

PubMed2003

Overrepresentation of a gene family encoding extracytoplasmic solute receptors in Bordetella.

A family of genes that are likely to encode extracytoplasmic solute receptors is strongly overrepresented in several beta-proteobacteria, including Bordetella pertussis. This gene family, of which members have been called bug genes, contains some examples that are contained within polycistronic operons coding for tripartite uptake transporters of the TTT family, while the vast majority are "orphan" genes. Proteomic and functional analyses demonstrated that several of these genes are expressed in B. pertussis, and one is involved in citrate uptake. The bug genes probably form an ancient family that has been subjected to a large expansion in a restricted phylogenic group.

Bacterial Proteins↗

PubMed2000

Molecular characterization of Bordetella bronchiseptica filamentous haemagglutinin and its secretion machinery.

Two closely related pathogens, Bordetella pertussis and Bordetella bronchiseptica, share a number of virulence factors. Filamentous haemagglutinin (FHA) is widely regarded as the dominant adhesin of B. pertussis, and its multiple binding activities have been well characterized. This large protein is produced and secreted at high levels by B. pertussis and significantly lower levels by B. bronchiseptica strains. FHA secretion is mediated by a single outer-membrane accessory protein, FhaC. The genes encoding FHA and FhaC in B. bronchiseptica were characterized by sequencing and functional analyses and are highly similar to those of B. pertussis. The most distinctive feature of B. bronchiseptica FHA is additional repeats in the N-terminal portion of the predicted protein. Interestingly, a point mutation in the fhaB promoter region of the B. bronchiseptica GP1 isolate, relative to other isolates, was found to be detrimental to promoter activity and to FHA production. FhaC and the N-terminal secretion domain of FHA of B. bronchiseptica were fully functional for secretion in B. pertussis. Thus, the different levels of FHA secretion by these Bordetella species might reflect differences in physiology, composition and structure of cell envelope, or differential protein degradation. Characterization of FHA expression and function may provide clues as to the basis of host species tropism, tissue localization and receptor recognition.

Adhesins, Bacterial↗