Balance between Coiled-Coil Stability and Dynamics Regulates Activity of BvgS Sensor Kinase in Bordetella.

UNLABELLED: The two-component system BvgAS controls the expression of the virulence regulon of Bordetella pertussis. BvgS is a prototype of bacterial sensor kinases with extracytoplasmic Venus flytrap perception domains. Following its transmembrane segment, BvgS harbors a cytoplasmic Per-Arnt-Sim (PAS) domain and then a predicted 2-helix coiled coil that precede the dimerization-histidine-phosphotransfer domain of the kinase. BvgS homologs have a similar domain organization, or they harbor only a predicted coiled coil between the transmembrane and the dimerization-histidine-phosphotransfer domains. Here, we show that the 2-helix coiled coil of BvgS regulates the enzymatic activity in a mechanical manner. Its marginally stable hydrophobic interface enables a switch between a state of great rotational dynamics in the kinase mode and a more rigid conformation in the phosphatase mode in response to signal perception by the periplasmic domains. We further show that the activity of BvgS is controlled in the same manner if its PAS domain is replaced with the natural α-helical sequences of PAS-less homologs. Clamshell motions of the Venus flytrap domains trigger the shift of the coiled coil's dynamics. Thus, we have uncovered a general mechanism of regulation for the BvgS family of Venus flytrap-containing two-component sensor kinases. IMPORTANCE: The two-component system BvgAS of the whooping cough agent Bordetella pertussis regulates the virulence factors necessary for infection in a coordinated manner. BvgS is the prototype of a family of sensor kinase proteins found in major bacterial pathogens. When BvgS functions as a kinase, B. pertussis is virulent, and the bacterium shifts to an avirulent phase after BvgS senses chemicals that make it switch to phosphatase. Our goal is to decipher the signaling mechanisms of BvgS in order to understand virulence regulation in Bordetella, which may lead to new antimicrobial treatments targeting those two-component systems. We discovered that the activity of BvgS is regulated in a mechanical manner. A short region of the protein that precedes the enzymatic domain switches between two states in response to signal perception by other BvgS domains. This switch region is conserved among BvgS homologs, and thus, the regulation uncovered here will likely be relevant for the family.

Role of DegP for two-partner secretion in Bordetella.

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in beta-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis, represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long beta-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37 degrees C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro, DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.

Structure-based mechanism of ligand binding for periplasmic solute-binding protein of the Bug family.

Bug proteins form a large family of periplasmic solute-binding proteins well represented in beta-proteobacteria. They adopt a characteristic Venus flytrap fold with two globular domains bisected by a ligand-binding cleft. The structures of two liganded Bug proteins have revealed that the family is specific for carboxylated solutes, with a characteristic mode of binding involving two highly conserved beta strand-beta turn-alpha helix motifs originating from each domain. These two motifs form hydrogen bonds with a carboxylate group of the ligand, both directly and via conserved water molecules, and have thus been termed the carboxylate pincers. In both crystallized Bug proteins, the ligands were found enclosed between the two domains and inaccessible to solvent, suggesting an inter-domain hinge-bending motion upon ligand binding. We report here the first structures of an open, unliganded Bug protein and of the same protein with a citrate ion bound in the open cavity. One of the ligand carboxylate groups is bound to one half of the carboxylate pincers by the beta strand-beta turn-alpha helix motif from domain 1, and the citrate ion forms several additional interactions with domain 1. The ligand is accessible to solvent and has very few contacts with domain 2. In this open, liganded structure, the second part of the carboxylate pincers originating from domain 2 is not stabilized by ligand binding, and a loop replaces the beta turn. In the unliganded structure, both motifs of the carboxylate pincers are highly mobile, and neither of the two beta turns is formed. Thus, ligand recognition is performed by domain 1, with the carboxylate group serving as an initial anchoring point. Stabilization of the closed conformation requires proper interactions to be established with domain 2, and thus domain 2 discriminates between productively and non-productively bound ligands.

Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

Tetanus toxin fragment C-specific priming by intranasal infection with recombinant Bordetella pertussis.

As an alternative to parenteral administration, mucosal administration offers several advantages including the ease of administration, safety and the ability to induce mucosal immunity. As a first step towards nasal administration of important childhood vaccines, we have previously developed attenuated Bordetella pertussis strains able to protect mice against pertussis upon nasal vaccination. Since pertussis vaccines are generally combined with tetanus and diphtheria vaccines, we constructed recombinant B. pertussis strains producing the non-toxic protective tetanus toxin fragment C (TTFC). TTFC was genetically fused to the N-terminal domain of the B. pertussis filamentous haemagglutinin. The hybrid gene was introduced into B. pertussis both on a multi-copy replicative plasmid and as a single copy inserted into the chromosome of a pertussis toxin-producing strain and a toxin-deficient attenuated strain. The hybrid protein was secreted by the recombinant strains. However, the recombinant multi-copy plasmid was unstable in vivo, and immunisation could only be carried out with the strains containing the single-copy chromosomal integration. Both the toxin-producing and the toxin-deficient recombinant B. pertussis strains were able to prime mice for the production of anti-TTFC serum antibodies upon intranasal administration, suggesting the feasibility of using recombinant attenuated B. pertussis for the development of combined childhood vaccines.

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins.

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.

Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway.

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells.

Production of Neisseria meningitidis transferrin-binding protein B by recombinant Bordetella pertussis.

Neisseria meningitidis serogroup B infections are among the major causes of fulminant septicemia and meningitis, especially severe in young children, and no broad vaccine is available yet. Because of poor immunogenicity of the serogroup B capsule, many efforts are now devoted to the identification of protective protein antigens. Among those are PorA and, more recently, transferrin-binding protein B (TbpB). In this study, TbpB of N. meningitidis was genetically fused to the N-terminal domain of the Bordetella pertussis filamentous hemagglutinin (FHA), and the fha-tbpB hybrid gene was expressed in B. pertussis either as a plasmid-borne gene or as a single copy inserted into the chromosome. The hybrid protein was efficiently secreted by the recombinant strains, despite its large size, and was recognized by both anti-FHA and anti-TbpB antibodies. A single intranasal administration of recombinant virulent or pertussis-toxin-deficient, attenuated B. pertussis to mice resulted in the production of antigen-specific systemic immunoglobulin G (IgG), as well as local IgG and IgA. The anti-TbpB serum antibodies were of the IgG1, IgG2a, and IgG2b isotypes and were found to express complement-mediated bactericidal activity against N. meningitidis. These observations indicate that recombinant B. pertussis may be a promising vector for the development of a mucosal vaccine against serogroup B meningococci.

Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins.

A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria. The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase. The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.

Bordetella pertussis, molecular pathogenesis under multiple aspects.

Recent studies, including those based on genomics, have demonstrated that besides toxins and adhesins, Bordetella pertussis uses many additional virulence determinants. Most of them are part of the BvgAS regulon, although some, in particular iron-uptake systems, are independent of BvgAS. They are regulated by iron, although in one case, the production of a siderophore receptor could be linked to the BvgAS regulon.

New virulence-activated and virulence-repressed genes identified by systematic gene inactivation and generation of transcriptional fusions in Bordetella pertussis.

An in silico scan of the partially completed genome sequence of Bordetella pertussis and analyses of transcriptional fusions generated with a new integrational vector were used to identify new potential virulence genes. The genes encoding a putative siderophore receptor, adhesins, and an autotransporter protein appeared to be regulated in a manner similar to Bordetella virulence genes by the global virulence regulator BvgAS. In contrast, the gene encoding a putative intimin-like protein appeared to be repressed under conditions of virulence.

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.

Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many virulence factors of pathogenic microorganisms are presented at the cell surface. However, protein secretion across the outer membrane of Gram-negative bacteria remains poorly understood. Here we used the extremely efficient secretion of the Bordetella pertussis filamentous hemagglutinin (FHA) to decipher this process. FHA secretion requires a single specific accessory protein, FhaC, the prototype of a family of proteins necessary for the extracellular localization of various virulence proteins in Gram-negative bacteria. We show that FhaC is heat-modifiable and localized in the outer membrane. Circular dichroism spectra indicated that FhaC is rich in beta-strands, in agreement with structural predictions for this protein. We further demonstrated that FhaC forms pores in artificial membranes, as evidenced by single-channel conductance measurements through planar lipid bilayers, as well as by liposome swelling assays and patch-clamp experiments using proteoliposomes. Single-channel conductance appeared to fluctuate very fast, suggesting that the FhaC channels frequently assume a closed conformation. We thus propose that FhaC forms a specific beta-barrel channel in the outer membrane for the outward translocation of FHA.

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin.

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA- E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44-CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.

N-terminal characterization of the Bordetella pertussis filamentous haemagglutinin.

The major adhesin of Bordetella pertussis, filamentous haemagglutinin (FHA), is produced and secreted at high levels by the bacterium. Mature FHA derives from a large precursor, FhaB, that undergoes several post-translational maturations. In this work, we demonstrate by site-directed mutagenesis that the N-terminal signal peptide of FHA is composed of 71 amino acids, including a 22-residue-long 'N-terminal extension' sequence. This sequence, although highly conserved in various other secretory proteins, does not appear to play an essential part in FHA secretion, as shown by deletion mutagenesis. The entire N-terminal signal region of FhaB is removed in the course of secretion by proteolytic cleavage at a site that corresponds to a Lep signal peptidase recognition sequence. After this maturation, the N-terminal glutamine residue is modified to a pyroglutamate residue. This modification is not crucial for heparin binding, haemagglutination or secretion. Interestingly, however, the modification is absent from Escherichia coli secreted FHA derivatives. In addition, it is dependent in B. pertussis on the presence of all three cysteines contained in the signal peptide of FhaB. These observations suggest that it does not occur spontaneously but perhaps requires a specific enzymatic machinery.

Lack of functional complementation between Bordetella pertussis filamentous hemagglutinin and Proteus mirabilis HpmA hemolysin secretion machineries.

The gram-negative bacterium Bordetella pertussis has adapted specific secretion machineries for each of its major secretory proteins. In particular, the highly efficient secretion of filamentous hemagglutinin (FHA) is mediated by the accessory protein FhaC. FhaC belongs to a family of outer membrane proteins which are involved in the secretion of large adhesins or in the activation and secretion of Ca2+-independent hemolysins by several gram-negative bacteria. FHA shares with these hemolysins a 115-residue-long amino-proximal region essential for its secretion. To compare the secretory pathways of these hemolysins and FHA, we attempted functional transcomplementation between FhaC and the Proteus mirabilis hemolysin accessory protein HpmB. HpmB could not promote the secretion of FHA derivatives. Likewise, FhaC proved to be unable to mediate secretion and activation of HpmA, the cognate secretory partner of HpmB. In contrast, ShlB, the accessory protein of the closely related Serratia marcescens hemolysin, was able to activate and secrete HpmA. Two invariant asparagine residues lying in the region of homology shared by secretory proteins and shown to be essential for the secretion and activation of the hemolysins were replaced in FHA by site-directed mutagenesis. Replacements of these residues indicated that both are involved in, but only the first one is crucial to, FHA secretion. This slight discrepancy together with the lack of functional complementation demonstrates major differences between the hemolysins and FHA secretion machineries.

Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper.

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.

Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin.

The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M(r) as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.

PapD chaperone function in pilus biogenesis depends on oxidant and chaperone-like activities of DsbA.

Adhesive P pili of uropathogenic Escherichia coli were not assembled by a strain that lacks the periplasmic disulfide isomerase DsbA. This defect was mostly attributed to the immunoglobulin-like pilus chaperone PapD, which possesses an unusual intrasheet disulfide bond between the last two beta-strands of its CD4-like carboxyl-terminal domain. The DsbA-dependent formation of this disulfide bond was critical for PapD's proper folding in vivo. Interestingly, the absence of the disulfide bond did not prevent PapD from folding in vitro or from forming a complex with the pilus adhesin in vitro. We suggest that DsbA maintains nascently translocated PapD in a folding-competent conformation prior to catalyzing disulfide bond formation, acting both as an oxidant and in a chaperone-like fashion. Disulfide bond formation in pilus subunits was also mediated by DsbA even in the absence of PapD. However, the ability of pilus subunits to achieve native-like conformations in vivo depended on PapD. These results suggest that a productive folding pathway for subunits requires sequential interactions with DsbA and the PapD chaperone.