The Bvg accessory factor (Baf) enhances pertussis toxin expression in Escherichia coli and is essential for Bordetella pertussis viability

Pertussis toxin expression in the Gram-negative respiratory pathogen, Bordetella pertussis, is regulated by the BvgAS two-component system. Previous studies suggested that an additional gene encoding a Bvg accessory factor (Baf) was required, along with BvgAS, for expression of a ptx-lacZ fusion in Escherichia coli grown in rich medium. However, other studies showed that BvgAS is sufficient for ptx-lacZ expression in minimal medium. Here we show that Baf acts with BvgAS to further increase ptx-lacZ expression in E. coli grown in minimal media and this is concomitant with a two-fold increase in BvgA protein levels. Gene replacement experiments show that baf is essential for viability of B. pertussis, suggesting that Baf affects the expression of other genes in addition to ptx.     

Role of BvgA phosphorylation and DNA binding affinity in control of Bvg-mediated phenotypic phase transition in Bordetella pertussis

To investigate the mechanism by which the Bordetella BvgAS phosphorelay controls expression of at least three distinct phenotypic phases, we isolated and characterized two B. pertussis mutants that were able to express Bvg- and Bvg(i) phase phenotypes but not Bvg+ phase phenotypes. In both cases, the mutant phenotype was due to a single nucleotide change in bvgA resulting in a single amino acid substitution in BvgA. In vitro phosphorylation assays showed that BvgA containing the T194M substitution was significantly impaired in its ability to use either BvgS or acetyl phosphate as a substrate for phosphorylation. Binding studies indicated that this mutant protein was able to bind an oligonucleotide containing a high-affinity BvgA binding site in a manner similar to wild-type BvgA, but was defective for binding the fhaB promoter in the absence of RNA polymerase (RNAP). By contrast, BvgA containing the R152H substitution had wild-type phosphorylation properties but was severely defective in its ability to bind either the high-affinity BvgA binding site-containing oligonucleotide or the fhaB promoter by itself. Both mutant BvgA proteins were able to bind the fhaB promoter in the presence of RNAP however, demonstrating the profound effect that RNAP has on stabilizing the ternary complexes between promoter DNA, BvgA and RNAP. Our results are consistent with the hypothesis that BvgAS controls expression of multiple phenotypic phases by adjusting the intracellular concentration of BvgA-P and they demonstrate the additive nature of BvgA binding site affinity and protein-protein interactions at different Bvg-regulated promoters.     

The Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica

Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg+ phase, a nonvirulent Bvg- phase, and an intermediate Bvgi phase. Genes expressed in the Bvg+ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvgi phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvgi phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvgi phase in B. bronchiseptica.     

Iron stress increases Bordetella pertussis mucin-binding capacity and attachment to respiratory epithelial cells

Whooping cough is a reemerging infectious disease of the respiratory tract caused by Bordetella pertussis. The incomplete understanding of the molecular mechanisms of host colonization hampers the efforts to control this disease. Among the environmental factors that commonly determine the bacterial phenotype, the concentration of essential nutrients is of particular importance. Iron, a crucial and scarce nutrient in the natural environment of B. pertussis, has been found to induce substantial phenotypic changes in this pathogen. However, the relevance of this phenotype for the interaction with host cells was never investigated. Using an in vitro model for bacterial attachment, it was shown that the attachment capacity of B. pertussis to epithelial respiratory cells is enhanced under iron stress conditions. Attachment is mediated by iron-induced surface-exposed proteins with sialic acid-binding capacity. The results further suggest that some of these iron-induced surface-associated proteins are immunogenic and may represent attractive vaccine candidates.     

Molecular characterization of the BvgA response regulator of Bordetella holmesii

The BvgAS system controls the expression of most virulence factors in Bordetella pertussis. Recently, we identified an orthologous system in the related human pathogen Bordetella holmesii. However, while we found that the orthologous histidine kinases BvgS could be functionally exchanged between the two species, the B. holmesii response regulator BvgA(BH) could not substitute for its B. pertussis counterpart in vivo and, accordingly, was not able to bind to B. pertussis virulence promoters in vitro. Here we show that a hybrid response regulator consisting of the B. pertussis derived DNA-binding output domain of BvgA(BP) combined with the B. holmesii receiver domain binds to BvgA(BP) regulated virulence promoters of B. pertussis in vitro and is functional in B. pertussis in vivo. This shows that the inability of BvgA(BH) to complement BvgA(BP) in B. pertussis is due to the small number of sequence variations present in its output domain. However, by mutation analysis we show that four amino acid exchanges present in the helix-turn-helix motif of BvgA(BH) as compared to BvgA(BP) are not the only reason for its inability to substitute for BvgA(BP) but additional mutations present in the output domain must play a role.     

Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis.

Expression of virulence factors by Bordetella pertussis is altered by environmental signals (antigenic modulation) and is dependent on an activator encoded by a gene called vir. We have used TnphoA (Tn5 IS50L::phoA) gene fusions to define two sets of genes whose expression is either activated (vag loci) or repressed (vrg loci) by modulation signals. Both groups of genes appear to be regulated by the vir gene product in that, in the absence of modulators, null mutations in vir lead to the repression of vag gene fusions and derepression of vrg gene fusions. Mutants of B. pertussis were isolated that constitutively express virulence factors in the presence of the modulator MgSO4, nicotinic acid, or low incubation temperature. We designate the gene that carries such mutations mod (modulation) and have characterized one (mod-1) of these mod constitutive mutations. A method was developed for the insertional inactivation of the vir gene by using the integration of a suicide replicon. Inactivation of the vir gene in the mod-1 mutant, followed by transcomplementation with the cloned wild-type vir gene, gives the Mod-1 constitutive phenotype, showing that the mod-1 mutation defines a gene distinct from vir. The gene carrying the mod-1 mutation is linked to vir and was cloned on a recombinant cosmid (pLAF-C1) which transcomplements the vir-1::Tn5 mutation in B. pertussis 347. Introduction of pLAF-C1 into vir mutant and vir+ B. pertussis strains also gives the Mod-1 constitutive phenotype, indicating that mod-1 is a dominant allele. These data suggest that the mod gene product could have sensory functions for the environmental signals that affect the expression of vir-regulated genes of B. pertussis. The mod constitutive strains and plasmids described here also have applications in pertussis vaccine development.     

Alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is not repressed by the BvgAS virulence control system

A previous study found that alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is Bvg repressed. In contrast, we report that alcaligin production by RB50 does not require Bvg phenotypic phase modulation and that isogenic Bvg(Con) and Bvg(-) phase-locked mutants both produce alcaligin in response to iron starvation.     

Characterization of intermediate phenotypes induced by chemically undefined laboratory media in virulent Bordetella bronchiseptica strains

The expression of many virulence factors of Bordetella bronchiseptica is regulated by the bvgAS locus and reduced in response to environmental signals called modulators. Virulent strains can alternate between virulent (Bvg(+)), intermediate (Bvg(i)), and modulated (Bvg(+)mod) phenotypes. Potential vaccine antigens can be expressed by Bvg1 strains grown only in the absence of modulators. In the present study we evaluated filamentous hemagglutinin (FHA) and outer membrane protein (OMP) expression in Bvg(+) B. bronchiseptica strains grown in chemically undefined media: nutrient agar (NA), tryptic soy agar (TSA), tryptose phosphate broth (TPB), and brain-heart infusion (BHI). Our results suggest that TSA and TPB usually induce semimodulation, since Bvg(+) strains cultured in these media retained the expression of FHA and virulence-associated OMPs in the 30 kDa region, but failed to express other virulence markers such as OMPs in the regions of 90 and 200 kDa, though they expressed flagellin (avirulence marker). On the other hand, NA and BHI usually induce modulation. Thus the assayed chemically undefined media should not be used in vaccine production. Semimodulation induced by TSA and TPB can be accurately detected by SDS-PAGE Sarkosyl-insoluble OMP-enriched profiles. The reduction or absence of OMPs in the regions of 90 and 200 kDa is the most sensitive marker, and in some cases the presence of flagellin in intermediate profiles is another trait of the Bvg(i) phenotypes. Therefore these markers could be useful for selecting media for vaccine production. We also characterized the phenotype of Bvg(+) strains grown in Stainer-Scholte broth, an expensive medium, with and without glutathione, and we have detected no differences; this is the first attempt to reduce the cost of a Bordetella growth medium for veterinary vaccine production.     

Fc gamma RIIB regulates nasal and oral tolerance: a role for dendritic cells

Mucosal tolerance prevents the body from eliciting productive immune responses against harmless Ags that enter the body via the mucosae, and is mediated by the induction of regulatory T cells that differentiate in the mucosa-draining lymph nodes (LN) under defined conditions of Ag presentation. In this study, we show that mice deficient in FcgammaRIIB failed to develop mucosal tolerance to OVA, and demonstrate in vitro and in vivo a critical role for this receptor in modulating the Ag-presenting capacity of dendritic cells (DC). In vitro it was shown that absence of FcgammaRIIB under tolerogenic conditions led to increased IgG-induced release of inflammatory cytokines such as MCP-1, TNF-alpha, and IL-6 by bone marrow-derived DC, and increased their expression of costimulatory molecules, resulting in an altered immunogenic T cell response associated with increased IL-2 and IFN-gamma secretion. In vivo we could show enhanced LN-DC activation and increased numbers of Ag-specific IFN-gamma-producing T cells when FcgammaRIIB(-/-) mice were treated with OVA via the nasal mucosa, inferring that DC modulation by FcgammaRIIB directed the phenotype of the T cell response. Adoptive transfer of CD4(+) T cells from the spleen of FcgammaRIIB(-/-) mice to naive acceptor mice demonstrated that OVA-responding T cells failed to differentiate into regulatory T cells, explaining the lack of tolerance in these mice. Our findings demonstrate that signaling via FcgammaRIIB on DC, initiated by local IgG in the mucosa-draining LN, down-regulates DC activation induced by nasally applied Ag, resulting in those defined conditions of Ag presentation that lead to Tr induction and tolerance.     

Demonstration of differential virulence gene promoter activation in vivo in Bordetella pertussis using RIVET

Bordetella pertussis, the etiologic agent of whooping cough, causes disease by employing an array of virulence factors controlled by the BvgA-BvgS two-component signal transduction system. Regulation by this system has been extensively characterized in vitro, where bvg-activated genes are repressed in a process known as phenotypic modulation. Differential regulation of these genes by the response regulator BvgA results in promoters that are activated early, middle, or late after being released from modulation. However, the in vivo environmental signal and regulation pattern has not been described. In order to investigate BvgAS-mediated regulation of B. pertussis virulence factors in vivo using the mouse aerosol challenge model, we have adapted the recombinase-based in vivo technology (RIVET) system for use in B. pertussis. We have demonstrated that these strains show resolution during in vitro growth under non-modulating conditions. In addition, we have demonstrated that modulating strains by growth on media containing MgSO4 does not affect virulence in the mouse aerosol challenge model. We have therefore used the RIVET system to reveal the time-course of gene expression in vivo for selected B. pertussis virulence factors (cya, fha, prn and ptx). Our data indicate that this method can be effectively used to monitor and compare in vivo and in vitro gene expression in B. pertussis, and that temporal regulation patterns previously observed in vitro are mirrored in vivo.     

Bordetella pertussis autotransporter Vag8 binds human C1 esterase inhibitor and confers serum resistance

Bordetella pertussis employs numerous strategies to evade the immune system, including the ability to resist killing via complement. Previously we have shown that B. pertussis binds a complement regulatory protein, C1 esterase inhibitor (C1inh) to its surface in a Bvg-regulated manner (i.e. during its virulence phase), but the B. pertussis factor was not identified. Here we set out to identify the B. pertussis C1inh-binding factor. Using a serum overlay assay, we found that this factor migrates at approximately 100 kDa on an SDS-PAGE gel. To identify this factor, we isolated proteins of approximately 100 kDa from wild type strain BP338 and from BP347, an isogenic Bvg mutant that does not bind C1inh. Using mass spectrometry and bioinformatics, we identified the autotransporter protein Vag8 as the putative C1inh binding protein. To prove that Vag8 binds C1inh, vag8 was disrupted in two different B. pertussis strains, namely BP338 and 18-323, and the mutants were tested for their ability to bind C1inh in a surface-binding assay. Neither mutant strain was capable of binding C1inh, whereas a complemented strain successfully bound C1inh. In addition, the passenger domain of Vag8 was expressed and purified as a histidine-tagged fusion protein and tested for C1inh-binding in an ELISA assay. Whereas the purified Vag8 passenger bound C1inh, the passenger domain of BrkA (a related autotransporter protein) failed to do so. Finally, serum assays were conducted to compare wild type and vag8 mutants. We determined that vag8 mutants from both strains were more susceptible to killing compared to their isogenic wild type counterparts. In conclusion, we have discovered a novel role for the previously uncharacterized protein Vag8 in the immune evasion of B. pertussis. Vag8 binds C1inh to the surface of the bacterium and confers serum resistance.     

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

AIMS: The present study shows that Congo red binding and urease activity assays are useful for selection of virulent (Bvg+) Bordetella bronchiseptica cultures. METHODS AND RESULTS: Congo red binding and urease activity of Bvg+ B. bronchiseptica cultures in different liquid media were compared with the expression of virulence markers such as filamentous haemagglutinin and some outer membrane proteins (OMP). The correlation with the reference virulence markers allowed the establishment of cut-off values for the proposed markers to assure the virulent phenotype (> or = 26 nmol ml-1 of CR and < or = 2.6 U). Using both assays, modulated cultures with avirulent phenotype (Stainer-Scholte broth, with MgSO4 20 mmol l-1 and brain heart infusion broth) and semi-modulated cultures with intermediate phenotypes (tryptose phosphate broth and 83% Stainer-Scholte with MgSO4 5 mmol l-1 cultures) could be distinguished. CONCLUSION: CR binding assay and urease activity are specific and sensitive enough to detect intermediate phenotypes that could only be detected by subtle changes in OMP profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of effective veterinary vaccines is hampered by reversible B. bronchiseptica antigenic modulation. The proposed assays are technically suitable for selection of virulent cultures to optimize vaccine production.