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.     

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization.

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP. In the present study, we show that active immunization with purified B. pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection. Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract. Furthermore, B. parapertussis AC-Hly or AC are also protective antigens against B. parapertussis colonization; their protective activities are equivalent to that of the whole-cell vaccine. These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model. If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage.     

Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different.     

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.     

The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin.

The adenylate cyclase-hemolysin of Bordetella pertussis consists of a cell-invasive N-terminal adenylate cyclase domain linked to a C-terminal RTX hemolysin containing extensive glycine-rich repeats. The toxin is an essential virulence factor required in the initial stages of infection. Adenylate cyclase-hemolysin was also shown to be a potent vaccinating antigen inducing protection against B. pertussis colonization of the mouse respiratory tract. This protective activity depends on a posttranslational fatty-acylation modification. We used a set of deletion derivatives of the recombinant adenylate cyclase-hemolysin to localize the protective epitopes on the 1,706-residue toxin. We show that specific anti-adenylate cyclase-hemolysin antibodies present in the sera of B. pertussis-infected mice and humans are directed predominantly against the modification-and-repeat portion of the toxin, contained in the last 800 residues of the adenylate cyclase-hemolysin. These antibodies appear to recognize conformational epitopes present only in a structure formed by the intact C-terminal half of the toxin. There was no correlation between the capacity of the truncated adenylate cyclase-hemolysin derivatives to induce both toxin-neutralizing antibodies upon immunization of mice and protective immunity. However, only the truncated proteins which were recognized by the sera of infected mice and humans and which had their last 800 residues intact had the capacity to induce protection of mice against colonization by B. pertussis. This indicates that the structure of the modification-and-repeat region of adenylate cyclase-hemolysin is critical for its protective activity.     

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.     

Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection.

Among virulence factors synthesized and secreted by Bordetella pertussis, pertussis toxin (PTX) and the bifunctional adenylate cyclase-hemolysin (AC-Hly) are able to invade mammalian cells and to impair intracellular functions. Moreover, both proteins are protective antigens in murine intracerebral and respiratory models. In order to study their in vivo properties, different B. pertussis mutants, deficient in AC-Hly expression or secretion, or producing modified AC-Hly devoid of either adenylate cyclase or hemolytic activities, were constructed and examined. The in vivo properties of the mutants were compared to PTX deficient strains, using the murine respiratory model. We show that lack of PTX as well as adenylate cyclase or hemolytic activities results in avirulence. Furthermore, we show that mutants lacking adenylate cyclase or hemolytic activities were unable to multiply as fast as the parental strains and PTX mutants during the first 5 days following infection. Thus, both adenylate cyclase and hemolytic activities are required by B. pertussis to initiate infection.     

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.     

Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins.

Bordetella pertussis expresses factors such as filamentous hemagglutinin, agglutinogens, pertactin, and pertussis toxin, which participate in bacterial adhesion; pertussis toxin, dermonecrotic toxin, lipopolysaccharide, and tracheal cytotoxin, which are responsible for toxic effects; and adenylate cyclase-hemolysin, which is required to initiate infection. By using a murine respiratory model, we showed that the RGD sequences of filamentous hemagglutinin and pertactin are important for bacterial persistence. However, mutants deficient in filamentous hemagglutinin and agglutinogens or in pertactin and the RGD sequence of filamentous hemagglutinin behaved as did wild-type B. pertussis, i.e., induced bronchopneumonia, alveolitis, and an influx of macrophages, lymphocytes, and polymorphonuclear leukocytes into bronchoalveolar lavage fluids. These results suggest that these adhesins are not involved in the induction of pulmonary lesions following infection. The intensity of inflammation was markedly reduced after infection with mutants deficient in either hemolytic activity or pertussis toxin expression, whereas a mutant devoid of adenylate cyclase activity behaved as did the avirulent mutant. Pertussis toxin and adenylate cyclase-hemolysin may act indirectly by altering immune cell functions and thus allowing other factors, such as filamentous hemagglutinin, agglutinogens, and pertactin, to trigger adhesion and lipopolysaccharide, dermonecrotic toxin, and tracheal cytotoxin to induce their toxic effects. However, it is possible that pertussis toxin is also responsible for the induction of some pulmonary alterations.     

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.     

Suppression of serum antibody responses by pertussis toxin after respiratory tract colonization by Bordetella pertussis and identification of an immunodominant lipoprotein

Pertussis toxin (PT), a virulence factor secreted by Bordetella pertussis, contributes to respiratory tract infection and disease caused by this pathogen. By comparing a wild-type (WT) B. pertussis strain to a mutant strain with an in-frame deletion of the ptx genes encoding PT (DeltaPT), we recently found that the lack of PT confers a significant defect in respiratory tract colonization in mice after intranasal inoculation. In this study, we analyzed serum antibody responses in mice infected with the WT or DeltaPT strain and found that infection with the DeltaPT strain elicited greater responses to several B. pertussis antigens than did infection with the WT, despite the lower colonization level achieved by the DeltaPT strain. The same enhanced antibody response was observed after infection with a strain expressing an enzymatically inactive PT; but this response was not observed after infection with B. pertussis mutant strains lacking filamentous hemagglutinin or adenylate cyclase toxin, nor when purified PT was administered with the DeltaPT inoculum, indicating a specific role for PT activity in this immunosuppressive effect. In particular, there were consistent strong serum antibody responses to one or more low-molecular-weight antigens after infection with the DeltaPT strain. These antigens were Bvg independent, membrane localized, and also expressed by the closely related pathogens Bordetella parapertussis and Bordetella bronchiseptica. Two-dimensional gel electrophoresis and mass spectrometry were used to identify one of the immunodominant low-molecular-weight antigens as a protein with significant sequence homology to peptidoglycan-associated lipoprotein in several other gram-negative bacterial species. However, a serum antibody response to this protein alone did not protect mice against respiratory tract infection by B. pertussis.     

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.     

Human Bordetella bronchiseptica infection related to contact with infected animals: persistence of bacteria in host.

Within a period of 2 1/2 years, Bordetella bronchiseptica was isolated four times from a 79-year-old woman with bronchopneumonia. We have demonstrated by pulsed-field gel electrophoresis that this infection was related to contact with infected rabbits. The initial human B. bronchiseptica isolate had a phenotype characteristic of usual B. bronchiseptica clinical isolates; it produced toxin and adhesins, such as adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin, and was able to induce lethality in a murine respiratory model. By contrast, although the three successive human isolates produced adhesins, they did not express adenylate cyclase-hemolysin and were unable to induce lethality. This implies that adenylate cyclase-hemolysin is required to induce lethality. We suggest that B. bronchiseptica may persist in the host, with expression of adenylate cyclase-hemolysin being essential for the initiation of infection and expression of adhesins being essential for persistence.     

CyaC-mediated activation is important not only for toxic but also for protective activities of Bordetella pertussis adenylate cyclase-hemolysin.

Bordetella pertussis adenylate cyclase-hemolysin (AC-Hly), encoded by the cyaA gene, belongs to the RTX family of toxins with extensive glycine-rich repeats in the carboxy-terminal portion. AC-Hly possesses both adenylate cyclase toxic and hemolytic activities that depend on a posttranslational modification mediated by the product of the cyaC gene. An improved system for AC-Hly synthesis and activation in Escherichia coli was developed. The results show that with purified AC-Hly (i) increased expression of the cyaC gene leads to a higher proportion of activated AC-Hly, (ii) the increase in protective activity of the activated recombinant AC-Hly correlates with the increase in its invasive and hemolytic activities, and (iii) the activated recombinant AC-Hly, but not the nonactivated recombinant AC-Hly, is a protective antigen against B. pertussis infection in a murine respiratory model. This suggests that possibly an immunodominant epitope required for protective activity is linked to the CyaC-mediated modification. Surprisingly, the protective and hemolytic activities of activated recombinant AC-Hly were lower than those of AC-Hly produced by B. pertussis, while its invasive activity was higher. This indicates that the modification of AC-Hly in B. pertussis and that in E. coli may differ.     

Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin.

Bordetella pertussis, the causative agent of whooping cough, has been shown recently to enter and survive in epithelial cells and macrophages in vitro. In the present study, we show that B. pertussis is cytotoxic for J774A.1 cells, a monocyte-macrophage cell line, and for murine alveolar macrophages. We demonstrate that cell cytotoxicity mediated by B. pertussis occurred through apoptosis, as shown by changes in nuclear morphology and by host cell DNA fragmentation. Parental strains and a mutant deficient in pertussis toxin expression are able to induce apoptosis, whereas avirulent mutant or adenylate cyclase-hemolysin-deficient mutants are not cytotoxic. Both adenylate cyclase and hemolytic activities are required for programmed cell death. These results show that induction of apoptosis is dependent on the expression of adenylate cyclase-hemolysin. The infection of murine alveolar macrophages in primary culture with B. pertussis leads to apoptosis, suggesting that this process might be relevant in vivo. The ability of B. pertussis to promote cell death may be important for the initiation of infection, bacterial survival, and escape of the host immune response.     

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.     

Role of adhesins and toxins in invasion of human tracheal epithelial cells by Bordetella pertussis

Bordetella pertussis, the agent of whooping cough, can invade and survive in several types of eukaryotic cell, including CHO, HeLa 229, and HEp-2 cells and macrophages. In this study, we analyzed bacterial invasiveness in nonrespiratory human HeLa epithelial cells and human HTE and HAE0 tracheal epithelial cells. Invasion assays and transmission electron microscopy analysis showed that B. pertussis strains invaded and survived, without multiplying, in HTE or HAE0 cells. This phenomenon was bvg regulated, but invasive properties differed between B. pertussis strains and isolates and the B. pertussis reference strain. Studies with B. pertussis mutant strains demonstrated that filamentous hemagglutinin, the major adhesin, was involved in the invasion of human tracheal epithelial cells by bacteria but not in that of HeLa cells. Fimbriae and pertussis toxin were not found to be involved. However, we found that the production of adenylate cyclase-hemolysin prevents the invasion of HeLa and HTE cells by B. pertussis because an adenylate cyclase-hemolysin-deficient mutant was found to be more invasive than the parental strain. The effect of adenylate cyclase-hemolysin was mediated by an increase in the cyclic AMP concentration in the cells. Pertactin (PRN), an adhesin, significantly inhibited the invasion of HTE cells by bacteria, probably via its interaction with adenylate cyclase-hemolysin. Isolates producing different PRNs were taken up similarly, indicating that the differences in the sequences of the PRNs produced by these isolates do not affect invasion. We concluded that filamentous hemagglutinin production favored invasion of human tracheal cells but that adenylate cyclase-hemolysin and PRN production significantly inhibited this process.     

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.     

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.     

Synergistic effect of Bordetella pertussis lymphocytosis-promoting factor on protective activities of isolated Bordetella antigens in mice.

The effect of low levels of added lymphocytosis-promoting factor (LPF) on the ability of several antigenic preparations isolated from Bordetella pertussis and other bacteria to protect mice against intracerebral infection with B. pertussis was examined. LPF was found to enhance the protective activities of filamentous hemagglutinin, 22S antigen, and fimbriae isolated from B. pertussis. Outer membrane protein preparations from phase I B. pertussis which had LPF removed by haptoglobin affinity columns or inactivated by glutaraldehyde, sodium dodecyl sulfate, or Formalin had reduced protective activities but were made fully protective by the readdition of LPF. Similarly, outer membrane protein preparations from Bordetella bronchiseptica, Bordetella parapertussis, or phase IV B. pertussis lacking LPF were protective only when low levels of LPF were added to the preparations. Outer membrane protein preparations from Neisseria gonorrhoeae or Escherichia coli were nonprotective even in the presence of added LPF. The purified LPF by itself was nonprotective unless treated with glutaraldehyde. LPF that had been detoxified with glutaraldehyde was, however, ineffective at enhancing the protective activity of antigenic preparations. The synergistic effect of LPF is discussed in relation to its known biological properties.