Comparative role of immunoglobulin A in protective immunity against the Bordetellae

The genus Bordetella includes a group of closely related mammalian pathogens that cause a variety of respiratory diseases in a long list of animals (B. bronchiseptica) and whooping cough in humans (B. pertussis and B. parapertussis). While past research has examined how these pathogens are eliminated from the lower respiratory tract, the host factors that control and/or clear the bordetellae from the upper respiratory tract remain unclear. We hypothesized that immunoglobulin A (IgA), the predominant mucosal antibody isotype, would have a protective role against these mucosal pathogens. IgA(-/-) mice were indistinguishable from wild-type mice in their control and clearance of B. pertussis or B. parapertussis, suggesting that IgA is not crucial to immunity to these organisms. However, na?ve and convalescent IgA(-/-) mice were defective in reducing the numbers of B. bronchiseptica in the upper respiratory tract compared to wild-type controls. Passively transferred serum from convalescent IgA(-/-) mice was not as effective as serum from convalescent wild-type mice in clearing this pathogen from the tracheae of naive recipient mice. IgA induced by B. bronchiseptica infection predominantly recognized lipopolysaccharide-containing O-antigen, and antibodies against O-antigen were important to bacterial clearance from the trachea. Since an IgA response contributes to the control of B. bronchiseptica infection of the upper respiratory tract, immunization strategies aimed at inducing B. bronchiseptica-specific IgA may be beneficial to preventing the spread of this bacterium among domestic animal populations.     

The O antigen enables Bordetella parapertussis to avoid Bordetella pertussis-induced immunity

Bordetella pertussis and Bordetella parapertussis are closely related endemic human pathogens which cause whooping cough, a disease that is reemerging in human populations. Despite how closely related these pathogens are, their coexistence and the limited efficacy of B. pertussis vaccines against B. parapertussis suggest a lack of cross-protective immunity between the two. We sought to address the ability of infection-induced immunity against one of these pathogens to protect against subsequent infection by the other using a mouse model of infection. Immunity induced by B. parapertussis infection protected against subsequent infections by either species. However, immunity induced by B. pertussis infection prevented subsequent B. pertussis infections but did not protect against B. parapertussis infections. The O antigen of B. parapertussis inhibited binding of antibodies to the bacterial surface and was required for B. parapertussis to colonize mice convalescent from B. pertussis infection. Thus, the O antigen of B. parapertussis confers asymmetrical cross-immunity between the causative agents of whooping cough. We propose that these findings warrant investigation of the relative role of B. parapertussis in the resurgence of whooping cough.     

Comparative toll-like receptor 4-mediated innate host defense to Bordetella infection

Bordetella pertussis, B. parapertussis, and B. bronchiseptica are closely related species associated with respiratory disease in humans and other mammals. While B. bronchiseptica has a wide host range, B. pertussis and B. parapertussis evolved separately from a B. bronchiseptica-like progenitor to naturally infect only humans. Despite very different doubling times in vitro, all three establish similar levels of infection in the mouse lung within 72 h. Recent work has revealed separate roles for Toll-like receptor 4 (TLR4) in immunity to B. pertussis and B. bronchiseptica, while no role for TLR4 during B. parapertussis infection has been described. Here we compared the requirement for TLR4 in innate host defense to these organisms using the same mouse infection model. While B. bronchiseptica causes lethal disease in TLR4-deficient mice, B. pertussis and B. parapertussis do not. Correspondingly, TLR4 is critical in limiting B. bronchiseptica but not B. pertussis or B. parapertussis bacterial numbers during the first 72 h. Interestingly, B. bronchiseptica induces a TLR4-dependent cytokine response that is considerably larger than that induced by B. pertussis or B. parapertussis. Analysis of their endotoxins using RAW cells suggests that B. bronchiseptica lipopolysaccharide (LPS) is 10- and 100-fold more stimulatory than B. pertussis or B. parapertussis LPS, respectively. The difference in LPS stimulus is more pronounced when using HEK293 cells expressing human TLR4. Thus, it appears that in adapting to infect humans, B. pertussis and B. parapertussis independently modified their LPS to reduce TLR4-mediated responses, which may compensate for slower growth rates and facilitate host colonization.     

Clearance of Bordetella parapertussis from the lower respiratory tract requires humoral and cellular immunity

Bordetella parapertussis and Bordetella pertussis are closely related species that cause whooping cough, an acute, immunizing disease. Their coexistence in the same host populations at the same time and vaccine studies showing that B. pertussis vaccines have little effect on B. parapertussis infection or disease suggest that the protective immunity induced by each does not efficiently cross protect against the other. Although the mechanisms of protective immunity to B. pertussis have been well studied, those of B. parapertussis have not. The present study explores the mechanism by which B. parapertussis is cleared from the lower respiratory tract by anamnestic immunity. Serum antibodies are necessary and sufficient for elimination of this bacterium, and CD4(+) T cells, complement, and neutrophils are required for serum antibody-mediated clearance. Mice lacking immunoglobulin A had no defect in their ability to control or clear infection. Interestingly, serum antibody-mediated clearance of B. parapertussis did not require Fc receptors that are required for antibody-mediated clearance of B. pertussis. Together these data support a model for the mechanism of protective immunity to B. parapertussis that is similar but distinct from that of B. pertussis.     

Comparative phenotypic analysis of the Bordetella parapertussis isolate chosen for genomic sequencing

The genomes of three closely related bordetellae are currently being sequenced, thus providing an opportunity for comparative genomic approaches driven by an understanding of the comparative biology of these three bacteria. Although the other strains being sequenced are well studied, the strain of Bordetella parapertussis chosen for sequencing is a recent human clinical isolate (strain 12822) that has yet to be characterized in detail. This investigation reports the first phenotypic characterization of this strain, which will likely become the prototype for this species in comparison with the prototype strains of B. pertussis (Tohama I), B. bronchiseptica (RB50), and other isolates of B. parapertussis. Multiple in vitro and in vivo assays distinguished each species. B. parapertussis was more similar to B. bronchiseptica than to B. pertussis in many assays, including in BvgS signaling characteristics, presence of urease activity, regulation of urease expression by BvgAS, virulence in the respiratory tracts of immunocompromised mice, induction of anti-Bordetella antibodies, and serum antimicrobial resistance. In other assays, B. parapertussis was distinct from all other species (in pigment production) or more similar to B. pertussis (by lack of motility and cytotoxicity to a macrophage-like cell line). These results begin to provide phenotypes that can be related to genetic differences identified in the genomic sequences of bordetellae.     

Detection of Bordetella bronchiseptica by the polymerase chain reaction.

Polymerase chain reaction (PCR) assays were developed that enabled not only discriminative detection of three Bordetella species, B. pertussis, B. parapertussis, and B. bronchiseptica (Bspp PCR), but also specific detection of B. bronchiseptica (Bb PCR). An upstream sequence of the flagellin gene was used as a target DNA region. This sequence contained differences in B. pertussis, B. parapertussis, and B. bronchiseptica DNA. These species could then be differentiated using two different sets of primers, Bspp and Bb. When oligonucleotide Bspp primers were used, PCR products were obtained from the three species of Bordetella. A fragment of the expected size (164 bp) was amplified using B. bronchiseptica and B. parapertussis DNA, but a fragment with a distinct molecular weight was amplified with B. pertussis DNA (195 bp). This Bspp PCR was specific and sensitive, but it could not differentiate between B. parapertussis and B. bronchiseptica. When Bb primers were used, a 237-bp PCR product was detected only from B. bronchiseptica DNA. No PCR products were identified after Bb PCR amplification of DNAs either from B. parapertussis isolates or B. pertussis isolates, nor from other respiratory pathogen DNAs tested. This second PCR assay had a sensitivity limit of less than 10 organisms of B. bronchiseptica after detection with a specific probe. The specificity and the sensitivity of the fla PCR assay were evaluated with purified DNA, as was its capacity for detecting the bacteria in human clinical samples and in lungs of infected mice.     

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.     

Role of Bordetella O antigen in respiratory tract infection

Lipopolysaccharide (LPS), as the major surface molecule of gram-negative bacteria, interacts with the host in complex ways, both inducing and protecting against aspects of inflammatory and adaptive immunity. The membrane-distal repeated carbohydrate structure of LPS, the O antigen, can prevent antibody functions and may vary as a mechanism of immune evasion. Genes of the wbm locus are required for the assembly of O antigen on the animal pathogen Bordetella bronchiseptica and the human pathogen B. parapertussis. However, the important human pathogen B. pertussis lacks these genes and a number of in vitro and in vivo characteristics associated with O antigen in other organisms. To determine the specific functions of O antigen in these closely related Bordetella subspecies, we compared wbm deletion (Deltawbm) mutants of B. bronchiseptica and B. parapertussis in a variety of assays relevant to natural respiratory tract infection. Complement was not activated or depleted by wild-type bordetellae expressing O antigen, but both Deltawbm mutants activated complement and were highly sensitive to complement-mediated killing in vitro. Although the O-antigen structures appear to be substantially similar, the two mutants differed strikingly in their defects within the respiratory tract. The B. parapertussis Deltawbm mutant was severely defective in colonization of the tracheas and lungs of mice, while the B. bronchiseptica Deltawbm mutant showed almost no defect. While in vitro characteristics such as serum resistance may be attributable to O antigen directly, the role of O antigen during infection appears to be more complex, possibly involving factors differing among the closely related bordetellae or different interactions between each one and its host.     

Multiple roles for Bordetella lipopolysaccharide molecules during respiratory tract infection

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B. parapertussis). Deletion of the wlb locus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have used wlb deletion (Deltawlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the Deltawlb mutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5(-/-) mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects. B. pertussis and B. parapertussis Deltawlb mutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, the B. bronchiseptica Deltawlb strain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting that wlb-dependent LPS modifications in B. bronchiseptica modulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.     

Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen

Bordetella pertussis and Bordetella parapertussis are both causative agents of whooping cough outbreaks. Although not expressing the pertussis toxin, B. parapertussis induces, in a murine model, an acute hemorrhagic edematous alveolitis, similar to that observed with B. pertussis. These data suggest that the pertussis toxin may only play an accessory role in the acute pulmonary syndrome observed during Bordetella infection. Both with B. pertussis and B. parapertussis, the ability to induce lethal pulmonary lesions is associated with enhanced in vitro adenylate cyclase expression and activity. We also demonstrate that passive immunization with specific anti-B. pertussis adenylate cyclase antibodies or active immunization with purified B. pertussis secreted adenylate cyclase protect mice against a lethal respiratory challenge with B. pertussis or B. parapertussis. Our results suggest that adenylate cyclase might be the primary cytotoxin responsible for mouse pulmonary lesions during respiratory tract infection with B. pertussis or with the related species B. parapertussis and is a protective antigen of B. pertussis.     

Species specificity of Bordetella adherence to human and animal ciliated respiratory epithelial cells.

Bacteria of the genus Bordetella adhere preferentially to ciliated respiratory epithelial cells. We investigated the specificity of this unique tropism by assessing the concentration-dependent adherence of the three Bordetella species to ciliated cells from different hosts. Bordetella pertussis and Bordetella parapertussis adhere better to human ciliated cells than to those from rabbits, mice, or hamsters. In contrast, Bordetella bronchiseptica demonstrates preferential adherence to nonhuman mammalian ciliated cells of rabbits, mice, and hamsters. There was no attachment of any Bordetella organisms to chicken ciliated cells. These observations suggest that specificities of attachment may explain the marked predominance of B. pertussis as the cause of whooping cough in humans and of B. bronchiseptica as a respiratory pathogen of many nonhuman mammals.     

Role of systemic and mucosal immune responses in reciprocal protection against Bordetella pertussis and Bordetella parapertussis in a murine model of respiratory infection

The roles of systemic humoral immunity, cell-mediated immunity, and mucosal immunity in reciprocal protective immunity against Bordetella pertussis and Bordetella parapertussis were examined by using a murine model of respiratory infection. Passive immunization with serum from mice infected with B. pertussis established protective immunity against B. pertussis but not against B. parapertussis. Protection against B. parapertussis was induced in mice that had been injected with serum from mice infected with B. parapertussis but not from mice infected with B. pertussis. Adoptive transfer of spleen cells from mice infected with B. pertussis or B. parapertussis also failed to confer reciprocal protection. To examine the role of mucosal immunity in reciprocal protection, mice were infected with preparations of either B. pertussis or B. parapertussis, each of which had been incubated with the bronchoalveolar wash of mice that were convalescing after infection with B. pertussis or B. parapertussis. Such incubation conferred reciprocal protection against B. pertussis and B. parapertussis on infected mice. The data suggest that mucosal immunity including secreted immunoglobulin A in the lungs might play an important role in reciprocal protective immunity in this murine model of respiratory infection.     

O antigen protects Bordetella parapertussis from complement

Bordetella pertussis, a causative agent of whooping cough, expresses BrkA, which confers serum resistance, but the closely related human pathogen that also causes whooping cough, Bordetella parapertussis, does not. Interestingly, B. parapertussis, but not B. pertussis, produces an O antigen, a factor shown in other models to confer serum resistance. Using a murine model of infection, we determined that O antigen contributes to the ability of B. parapertussis to colonize the respiratory tract during the first week of infection, but not thereafter. Interestingly, an O antigen-deficient strain of B. parapertussis was not defective in colonizing mice lacking the complement cascade. O antigen prevented both complement component C3 deposition on the surface and complement-mediated killing of B. parapertussis. In addition, O antigen was required for B. parapertussis to systemically spread in complement-sufficient mice, but not complement-deficient mice. These data indicate that O antigen enables B. parapertussis to efficiently colonize the lower respiratory tract by protecting against complement-mediated control and clearance.     

Reciprocal protective immunity against Bordetella pertussis and Bordetella parapertussis in a murine model of respiratory infection

The protective immunity induced by infection with Bordetella pertussis and with Bordetella parapertussis was examined in a murine model of respiratory infection. Convalescent mice that had been infected by aerosol with B. pertussis or with B. parapertussis exhibited a protective immune response against B. pertussis and also against B. parapertussis. Anti-filamentous hemagglutinin (anti-FHA) serum immunoglobulin G (IgG) and anti-FHA lung IgA antibodies were detected in both mice infected with B. pertussis and those infected with B. parapertussis. Antibodies against pertussis toxin (anti-PT) and against killed B. pertussis cells were detected in mice infected with B. pertussis. Pertactin-specific antibodies and antibodies against killed B. parapertussis cells were detected in mice infected with B. parapertussis. Spleen cells from mice infected with B. pertussis secreted interferon-gamma (IFN-gamma) in response to stimulation by FHA or PT. Spleen cells from mice infected with B. parapertussis also secreted IFN-gamma in response to FHA. Interleukin-4 was not produced in response to any of the antigens tested. The profiles of cytokine secretion in vitro revealed induction of a Th1-biased immune response during convalescence from infection by B. pertussis and by B. parapertussis. It is possible that Th1 and Th2 responses against FHA might be related to the reciprocal protection achieved in our murine model.     

Polymorphism of repeated regions of pertactin in Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica

Pertactin is an outer membrane protein expressed by Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica that induces protective immunity to Bordetella infections. The immunodominant and immunoprotective epitopes of pertactin include two repeated regions, I and II. Comparison of these two repeated regions showed that B. parapertussis pertactin is invariant, whereas B. pertussis pertactin varies mostly in region I and B. bronchiseptica pertactin varies in both repeated regions I and II, but mostly in region II. These differences may result from specific characteristics of these Bordetella species.     

Nucleotide sequence homology to pertussis toxin gene in Bordetella bronchiseptica and Bordetella parapertussis.

Multiple strains of Bordetella parapertussis and B. bronchiseptica were examined for the presence of nucleotide sequences which hybridized with a cloned 4.5-kilobase (kb) fragment of B. pertussis DNA containing the genes responsible for pertussis toxin expression. All six B. parapertussis strains tested had nucleic acid sequences that hybridized with the cloned 4.5-kb fragment in Southern blot analyses. When the B. parapertussis DNA was digested with restriction endonuclease PstI, the pattern of hybridization was identical to that obtained with B. pertussis. Only five of the seven B. bronchiseptica strains tested had sequences that hybridized with the 4.5-kb fragment. Three of these B. bronchiseptica strains had a hybridization pattern identical to B. pertussis upon PstI digestion and Southern blot analyses. Two B. bronchiseptica strains were shown to lack a PstI cleavage site downstream from the region analogous to that coding for the pertussis toxin structural genes. Monoclonal antibody analyses were unable to detect pertussis toxin subunits S1 and S2 in Western blots with cellular material or culture supernatant from several B. bronchiseptica and B. parapertussis strains that possessed the DNA homologies. In addition, preliminary Northern hybridizations with RNA isolated from B. bronchiseptica and B. parapertussis strains suggested that the homologous regions were not transcribed. The data show that the gene coding for the toxic component of B. pertussis is common in other Bordetella species, though the gene probably is not expressed.     

Analysis of proteins encoded by the ptx and ptl genes of Bordetella bronchiseptica and Bordetella parapertussis.

Bordetella pertussis is the only bacteria] species which is known to produce pertussis toxin (PT); however, both Bordetella bronchiseptica and Bordetella parapertussis contain regions homologous to the ptx genes of B. pertussis that encode the toxin subunits. After finding that several children with B. parapertussis infections exhibited modest antibody titers to PT, we examined the ptx genes of both B. parapertussis and B. bronchiseptica to determine whether they would encode stable, functional proteins even though their promoters are thought to be inactive under the conditions that have been examined. We inserted a functional promoter directly upstream of the ptx-ptl region of both species and examined culture supernatants of the resulting strains for PT activity. Biologically active PT was found in the culture supernatants of both engineered species. The toxin encoded by the B. parapertussis ptx genes appeared more labile in culture supernatants than did toxin produced by either B. pertussis or the engineered strain of B. bronchiseptica. This lability might be due to the lack of a full-length S2 subunit. We also investigated the ptl genes of these species, which are necessary for the secretion of this toxin, and found that both B. bronchiseptica and B. parapertussis contain at least certain of these genes, including ptlE and ptlF. Moreover, B. bronchiseptica appeared to contain all essential ptl genes since the introduction of a functional promoter directly upstream of the ptx-ptl region resulted in both production and efficient secretion of toxin. These results indicate that despite a number of amino acid changes in the sequences of the toxins, the toxins encoded by B. bronchiseptica and B. parapertussis are active.     

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.     

Ultrastructural analysis of the interactions between Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica and human tracheal epithelial cells

Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica are respiratory pathogens that colonize the respiratory tract of their host after adhesion to the respiratory epithelium. Presently, the intracellular fate of these bacteria in human tracheal epithelial cells was compared by use of transmission electron microscopy. The three species, even when cytotoxic, were taken-up by epithelial cells. Although, some intracellular bacteria appeared morphologically intact and survived a few days inside epithelial cells, most of them appeared quickly degraded, phenomenon which was associated with an intense cell metabolic activity. Even cytotoxic Bordetella species is ultimately killed by human epithelial cells.