Invasion of HeLa cells by Bordetella bronchiseptica

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis , is able to invade HeLa cells. Comparison of the invasiveness of Bvg⁺ and Bvg^-B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg^- (flagellated) phase was ten-fold lower than when Bvg⁺ (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica , which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.     

Characterization of BrkA Expression in Bordetella bronchiseptica

BrkA confers resistance to killing by complement in Bordetella pertussis. Complement resistance in Bordetella bronchiseptica was examined. Four B. bronchiseptica strains possessed the brkA gene; however, only three expressed the protein. Only the strain lacking BrkA was susceptible to complement. Introduction of the B. pertussis brkA gene restored BrkA expression to this strain but did not confer resistance. brkA was mutated in the strains that naturally expressed BrkA, and loss of BrkA did not confer sensitivity to complement. As a species, B. bronchiseptica is more resistant to complement than B. pertussis, and BrkA does not mediate resistance.     

Vag8, a Bordetella pertussis bvg-Regulated Protein

Bordetella pertussis expresses a bvg-regulated 95-kDa protein, Vag8, encoded by vag-8. Southern blot analysis indicates that strains of Bordetella bronchiseptica and Bordetella parapertussis have DNA homologous to vag-8. Antiserum raised to a fusion of maltose binding protein to an N-terminal 60-kDa fragment of Vag8 recognizes the native 95-kDa protein in immunoblots of B. pertussis and B. bronchiseptica but not B. parapertussis. A 95-kDa protein-negative derivative of B. pertussis 18323 containing a deletion of vag-8 colonized mice as efficiently as the parent B. pertussis strain in a mouse aerosol model of pertussis.     

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice.     

Detection of avirulent insertional <Emphasis Type="Italic">Bordetella pertussis bvg</Emphasis><Superscript>−</Superscript> mutants in patients with pertussis and with upper respiratory tract infection and in seemingly healthy people

Significant growth in the incidence of laboratory proven pertussis in adolescents and adults worldwide, the detection of asymptomatic carriage and intracellular survival of Bordetella pertussis in vivo, and the dependence of this process on the virulence of the causative agent denote the possibility of the persistence of bacteria that belong to the genus Bordetella in the host organism. B. pertussis phenotypic phases that cause atypical and asymptomatic forms of pertussis, as well as persistent bacteria were suggested to be formed as a result of the integration of IS elements into the bvgAS operon and other virulence genes. A test system that allows one to detect IS elements integration into the bvgAS operon for in vitro bacterial cultures and bacteria isolated from humans was developed based on the RT-PCR technique. Different quantity of avirulent B. pertussis, containing IS element insertions in the virulence operon was found in patients with pertussis, acute and chronic respiratory infections of the upper and lower airways, as well as in apparently healthy people.     

Genomic analysis of the adenylate cyclase-hemolysin C-terminal region of Bordetella pertussis, Bordetella parapertussisand Bordetella bronchiseptica

Adenylate cyclase-hemolysin plays an important role in the virulence of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species. Its C-terminal region carries protective epitopes and receptor binding site for human cells. Genomic analyses of this region indicate no polymorphism in B. pertussis and B. parapertussis regions, but substantial variability in B. bronchiseptica that might be linked to the various niches of this species.     

Neither the Bvg− Phase nor the vrg6 Locus of Bordetella pertussis Is Required for Respiratory Infection in Mice

In Bordetella species, the BvgAS sensory transduction system mediates an alteration between the Bvg⁺ phase, characterized by expression of adhesins and toxins, and the Bvg⁻ phase, characterized by the expression of motility and coregulated phenotypes in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. Since there is no known environmental or animal reservoir for B. pertussis, the causative agent of whooping cough, it has been assumed that this phenotypic alteration must occur within the human host during infection. Consistent with this hypothesis was the observation that a B. pertussis mutant, SK6, containing a TnphoA insertion mutation in a Bvg-repressed gene (vrg6) was defective for tracheal and lung colonization in a mouse model of respiratory infection (D. T. Beattie, R. Shahin, and J. Mekalanos, Infect. Immun. 60:571–577, 1992). This result was inconsistent, however, with the observation that a Bvg⁺ phase-locked B. bronchiseptica mutant was indistinguishable from the wild type in its ability to establish a persistent respiratory infection in rabbits and rats (P. A. Cotter and J. F. Miller, Infect. Immun. 62:3381–3390, 1994; B. J. Akerley, P. A. Cotter, and J. F. Miller, Cell 80:611–620, 1995). To directly address the role of Bvg-mediated signal transduction in B. pertussis pathogenesis, we constructed Bvg⁺ and Bvg⁻ phase-locked mutants and compared them with the wild type for their ability to colonize the respiratory tracts of mice. Our results show that the Bvg⁺ phase of B. pertussis is necessary and sufficient for respiratory infection. By constructing a strain with a deletion in the bvgR regulatory locus, we also show that ectopic expression of Bvg⁻ phase phenotypes decreases the efficiency of colonization, underscoring the importance of Bvg-mediated repression of gene expression in vivo. Finally, we show that the virulence defect present in strain SK6 cannot be attributed to the vrg6 mutation. These data contradict an in vivo role for the Bvg⁻ phase of B. pertussis.     

Phenotypic modulation of the virulent Bvg phase is not required for pathogenesis and transmission of Bordetella bronchiseptica in swine

The majority of virulence gene expression in Bordetella is regulated by a two-component sensory transduction system encoded by the bvg locus. In response to environmental cues, the BvgAS regulatory system controls expression of a spectrum of phenotypic phases, transitioning between a virulent (Bvg⁺) phase and a nonvirulent (Bvg⁻) phase, a process referred to as phenotypic modulation. We hypothesized that the ability of Bordetella bronchiseptica to undergo phenotypic modulation is required at one or more points during the infectious cycle in swine. To investigate the Bvg phase-dependent contribution to pathogenesis of B. bronchiseptica in swine, we constructed a series of isogenic mutants in a virulent B. bronchiseptica swine isolate and compared each mutant to the wild-type isolate for its ability to colonize and cause disease. We additionally tested whether a BvgAS system capable of modulation is required for direct or indirect transmission. The Bvg⁻ phase-locked mutant was never recovered from any respiratory tract site at any time point examined. An intermediate phase-locked mutant (Bvgⁱ) was found in numbers lower than the wild type at all respiratory tract sites and time points examined and caused limited to no disease. In contrast, colonization of the respiratory tract and disease caused by the Bvg⁺ phase-locked mutant and the wild-type strain were indistinguishable. The Bvg⁺ phase-locked mutant transmitted to naïve pigs by both direct and indirect contact with efficiency equal to that of the wild-type isolate. These results indicate that while full activation of the BvgAS regulatory system is required for colonization and severe disease, it is not deleterious to direct and indirect transmission. Overall, our results demonstrate that the Bvg⁺ phase is sufficient for respiratory infection and host-to-host transmission of B. bronchiseptica in swine.     

The Bpel locus encodes type III secretion machinery in Bordetella pertussis

Type III secretory genes(Bscl, J, K, L, N and O) have recently been identified in Bordetella bronchiseptica and shown to be under the control of the BvgAS locus. We examined a 35 616 byte DNA sequence amplified from Bordetella pertussis Tohama I for homology with known type III secretory genes in Yersinia spp. and Pseudomonas sppand a total of 20 homologous open reading frames were detected. Putative type III secretion proteins in B. pertussis were designated according to their homology with type III secretion proteins in B. bronchiseptica, Yersinia and Pseudomonas. These ORFs were arranged in two putative operons, which together we have designated as the BpeI locus. The first spans nucleotides 23385–7888 and encodes the putative proteins LcrH1, BopD, BopB, LcfH2, BscI, BscJ, BscK, BscL, BscN, BscO, BscQ, BscR, BscS, BscT, BscU, and BscC, in this order. The second spans nucleotides 23580–29863 and encodes the putative proteins LcrE, LcrD, BscD and BscF, in this order. The homology of these proteins to type III secretory proteins was B. bronchiseptica (73–99%),Yersinia spp. (17–65%), Pseudomonas spp. (18–64%). The B. pertussis proteins were similar to their homologues in B. bronchiseptica, Yersinia and Pseudomonas in terms of length, molecular weight and isoelectric point. Coiled-coil domains were detected in putative translocation proteins, BopB and BopD. BopB and BopD were similar to each other, to the RTX toxin family and to cyaA, cyaB, cyaD and cyaE. The percentage G+C content of the sequence analysed was 66.16%, which is similar to the published percentage G+C (67–70%) for the B. pertussis chromosome.     

The Lipopolysaccharide of Bordetella bronchiseptica Acts as a Protective Shield against Antimicrobial Peptides

Resistance profiles of the two Bordetella species B. bronchiseptica and B. pertussis against various antimicrobial peptides were determined in liquid survival and agar diffusion assays. B. bronchiseptica exhibited significantly higher resistance against all tested peptides than B. pertussis. The most powerful agents acting on B. bronchiseptica were, in the order of their killing efficiencies, cecropin P > cecropin B > magainin-II-amide > protamine > melittin. Interestingly, for B. bronchiseptica, the resistance level was significantly affected by phase variation, as a bvgS deletion derivative showed an increased sensitivity to these peptides. Tn5-induced protamine-sensitive B. bronchiseptica mutants, which were found to be very susceptible to most of the cationic peptides, were isolated. In two of these mutants, the genetic loci inactivated by transposon insertion were identified as containing genes highly homologous to the wlbA and wlbL genes of B. pertussis that are involved in the biosynthesis of lipopolysaccharide (LPS). In agreement with this finding, the two peptide-sensitive mutants revealed structural changes in the LPS, resulting in the loss of the O-specific side chains and the prevalence of the LPS core structure. This demonstrates that LPS plays a major role in the resistance of B. bronchiseptica against the action of antimicrobial peptides and suggests that B. pertussis is much more susceptible to these peptides due to the lack of the highly charged O-specific sugar side chains.     

Identification and evaluation of new target sequences for specific detection of Bordetella pertussis by real-time PCR

A comparative analysis of the Bordetella pertussis, B. bronchiseptica, and B. parapertussis genome assemblies permitted the identification of regions with significant sequence divergence and the design of two new real-time PCR assays, BP283 and BP485, for the specific detection of B. pertussis. The performance characteristics of these two assays were evaluated and compared to those of culture and an existing real-time PCR assay targeting the repetitive element IS481. The testing of 324 nasopharyngeal specimens indicated that, compared to culture, the BP283 assay had a sensitivity and specificity of 100 and 96.8% and the BP485 assay had a sensitivity and specificity of 92.3 and 97.1%. Notably, B. holmesii was isolated from two specimens that were positive by the IS481 assay but negative by the BP283 and BP485 assays. These two assays represent an improvement in specificity over those of PCR assays targeting only IS481 and may be duplexed or used in conjunction with existing PCR assays to improve the molecular detection of B. pertussis.     

Bordetella bronchiseptica flagellin is a proinflammatory determinant for airway epithelial cells

Motility is an important virulence phenotype for many bacteria, and flagellin, the monomeric component of flagella, is a potent proinflammatory factor. Of the three Bordetella species, Bordetella pertussis and Bordetella parapertussis are nonmotile human pathogens, while Bordetella bronchiseptica expresses flagellin and causes disease in animals and immunocompromised human hosts. The BvgAS two-component signal transduction system regulates phenotypic-phase transition (Bvg+, Bvg-, and Bvg(i)) in bordetellae. The Bvg- phase of B. bronchiseptica is characterized by the expression of flagellin and the repression of adhesins and toxins necessary for the colonization of the respiratory tract. B. bronchiseptica naturally infects a variety of animal hosts and constitutes an excellent model to study Bordetella pathogenesis. Using in vitro coculture models of bacteria and human lung epithelial cells, we studied the effects of B. bronchiseptica flagellin on host defense responses. Our results show that B. bronchiseptica flagellin is a potent proinflammatory factor that induces chemokine, cytokine, and host defense gene expression. Furthermore, we investigated receptor specificity in the response to B. bronchiseptica flagellin. Our results show that B. bronchiseptica flagellin is able to signal effectively through both human and mouse Toll-like receptor 5.     

Filamentous Hemagglutinin of Bordetella bronchiseptica Is Required for Efficient Establishment of Tracheal Colonization

Adherence to ciliated respiratory epithelial cells is considered a critical early step in Bordetella pathogenesis. For Bordetella pertussis, the etiologic agent of whooping cough, several factors have been shown to mediate adherence to cells and cell lines in vitro. These putative adhesins include filamentous hemagglutinin (FHA), fimbriae, pertactin, and pertussis toxin. Determining the precise roles of each of these factors in vivo, however, has been difficult, due in part to the lack of natural-host animal models for use with B. pertussis. Using the closely related species Bordetella bronchiseptica, and by constructing both deletion mutation and ectopic expression mutants, we have shown that FHA is both necessary and sufficient for mediating adherence to a rat lung epithelial (L2) cell line. Using a rat model of respiratory infection, we have shown that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting that its role in establishment may be dedicated to overcoming the clearance action of the mucociliary escalator.     

Comparison of bipA alleles within and across Bordetella species

The Bordetella BvgAS signal transduction system controls the expression of at least three phenotypic phases, the Bvg(+) or virulent phase, the Bvg(-) or avirulent phase, and the Bvg(i) or Bvg intermediate phase, which has been hypothesized to be important for transmission. bipA, the first identified Bvg(i)-phase gene, encodes a protein with similarity to the well-characterized bacterial adhesins intimin and invasin. Proteins encoded by the bipA genes present in Bordetella pertussis Tohama I and Bordetella bronchiseptica RB50 differ in the number of 90-amino-acid repeats which they possess and in the sequence of the C-terminal domain. To investigate the possibility that bipA alleles segregate according to host specificity and to gain insight into the role of BipA and the Bvg(i) phase in the Bordetella infectious cycle, we compared bipA alleles across members of the B. bronchiseptica cluster, which includes both human-infective (B. pertussis and B. parapertussis(hu)) and non-human-infective (B. bronchiseptica and B. parapertussis(ov)) strains. bipA genes were present in most, but not all, strains. All bipA genes present in B. bronchiseptica strains were identical to bipA of RB50 (at least with regard to the DNA sequence of the 3' C-terminal-domain-encoding region, the number of 90-amino-acid repeats encoded, and expression patterns). Although all bipA genes present in the other Bordetella strains were identical in the 3' C-terminal-domain-encoding region to bipA of B. pertussis Tohama I, they varied in the number of 90-amino-acid repeats that they encoded and in expression level. Notably, the genes present in B. parapertussis(hu) strains were pseudogenes, and the genes present in B. parapertussis(ov) strains were expressed at significantly reduced levels compared with the levels in B. pertussis and B. bronchiseptica strains. Our results indicate that there is a correlation between specific bipA alleles and specific hosts. They also support the hypothesis that both horizontal gene transfer and fine-tuning of gene expression patterns contribute to the evolution of host adaptation in lineages of the B. bronchiseptica cluster.     

Vir90, a virulence-activated gene coding for a Bordetella pertussis iron-regulated outer membrane protein

Bordetella pertussis undergoes phenotypic changes modulated by the bvgAS locus, which regulates the expression of many genes related to virulence and immunogenicity. We previously reported the N-terminal sequence of a 90 kDa bvg-regulated outer membrane protein (OMP) of B. pertussis (SWISS-PROT accession No. p81549), a novel potential virulence factor that we named Vir90. The open reading frames (ORFs) which potentially code for Vir90 in B. pertussis, B. parapertussis and B. bronchiseptica were identified by computer analysis of the genomic sequences available for the three Bordetella species. Nucleotide sequence analysis of the vir90 upstream region revealed the presence of a putative promoter, a BvgA binding site and a putative Fur binding site. The B. pertussis Vir90 protein showed significant homology with ferrisiderophore receptors from Gram-negative bacteria. An antiserum raised against Vir90His recombinant protein recognized the 90-kDa protein in immunoblots of OMPs from these three virulent Bordetella species. The accumulation of the Vir90 protein increased 4-fold under low iron growth conditions. Therefore, the vir90 gene is expressed in the tested species and its expression is regulated positively by the BvgAS system and negatively under high iron concentration, likely by Fur.     

Immunodominance in Mouse and Human CD4+ T-Cell Responses Specific for the Bordetella pertussis Virulence Factor P.69 Pertactin

P.69 pertactin (P.69 Prn), an adhesion molecule from the causative agent of pertussis, Bordetella pertussis, is present in cellular and most acellular vaccines that are currently used worldwide. Although both humoral immunity and cellular immunity directed against P.69 Prn have been implicated in protective immune mechanisms, the identities of CD4⁺ T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-A^d-restricted B. pertussis conserved CD4⁺ T-cell epitope at the N terminus of P.69 Prn was identified by using a BALB/c T-cell hybridoma. The epitope appeared immunodominant among four other minor strain-conserved P.69 Prn epitopes recognized after vaccination and B. pertussis infection, and it was capable of evoking a Th1/Th17-type cytokine response. B. pertussis P.69 Prn immune splenocytes did not cross-react with natural variants of the epitope as present in Bordetella parapertussis and Bordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4⁺ T cells in an HLA-DQ-restricted manner. During B. pertussis infection, the epitope was associated with a Th1-type CD4⁺ T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4⁺ T-cell immunity after B. pertussis vaccination and infection in mice and, more importantly, in humans. Thus, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance of B. pertussis-specific CD4⁺ T-cell mechanisms in preclinical and clinical vaccine studies.     

The BvgAS two-component system of Bordetella spp.: a versatile modulator of virulence gene expression.

Bordetella pertussis and the closely related species B. parapertussis and B. bronchiseptica colonize the respiratory tract and cause related diseases in man or mammalian species, respectively. Expression of virulence factors by these pathogens is coordinately regulated by the BvgAS two-component system according to changes in the growth conditions. Signal transduction by the BvgAS system is characterized by a complex His-Asp-His-Asp phosphorelay. This system controls the expression of two distinct subsets of genes either in a positive (vag genes) or in a negative (vrg genes) manner. Most of the known virulence factors such as several toxins and adhesins are encoded by vag genes, whereas the functions of most vrg genes and the biological significance of the vrg regulon are not yet clear. This review discusses the current knowledge about the molecular mechanisms of virulence regulation and their relevance for infection by these respiratory pathogens.