In a mouse model of respiratory tract infection by Bordetella pertussis, bacteria multiply in the airways over the first week and are then cleared over the next 3–4 weeks by the host immune response. Pertussis toxin (PT), a virulence factor secreted exclusively by B. pertussis, promotes bacterial growth in the airways by suppression and modulation of host immune responses. By comparison of wild type and PT-deficient strains, we examined the role of PT in modulating airway cytokine and chemokine responses affecting neutrophil recruitment during B. pertussis infection in mice. We found that, despite early inhibition of neutrophil recruitment by PT, high numbers of neutrophils were recruited to the airways by 4 days post-infection with the wild type strain, but not with the PT-deficient strain, and that this correlated with upregulation of neutrophil-attracting chemokine gene expression. In addition, there was similar upregulation of genes expressing the cytokines IL-17A (IL-17), TNF-α and IFN-γ, indicating a mixed Th1/Th17 response. Expression of IL-6, a cytokine involved in Th17 induction, was upregulated earlier than the IL-17 response. We showed that PT, rather than bacterial numbers, was important for induction of these responses. Flow cytometric analysis revealed that the IL-17-producing cells were macrophages and neutrophils as well as T cells, and were present predominantly in the airways rather than the lung tissue. Antibody neutralization of IL-17 significantly reduced chemokine gene expression and neutrophil recruitment to the airways, but only modestly increased peak bacterial loads. These data indicate that PT stimulates inflammatory responses by induction of Th1- and Th17-associated cytokines, including IL-17, during B. pertussis infection in mice, but a role for IL-17 in protection against the infection remains to be established.

Pertussis is an acute respiratory disease of humans caused by the bacterium Bordetella pertussis. Pertussis toxin (PT) plays a major role in the virulence of this pathogen, including important effects that it has soon after inoculation. Studies in our laboratory and other laboratories have indicated that PT inhibits early neutrophil influx to the lungs and airways in response to B. pertussis respiratory tract infection in mice. Previous in vitro and in vivo studies have shown that PT can affect neutrophils directly by ADP ribosylating G i proteins associated with surface chemokine receptors, thereby inhibiting neutrophil migration in response to chemokines. However, in this study, by comparing responses to wild-type (WT) and PT-deficient strains, we found that PT has an indirect inhibitory effect on neutrophil recruitment to the airways in response to infection. Analysis of lung chemokine expression indicated that PT suppresses early neutrophil recruitment by inhibiting chemokine upregulation in alveolar macrophages and other lung cells in response to B. pertussis infection. Enhancement of early neutrophil recruitment to the airways in response to WT infection by addition of exogenous keratinocyte-derived chemokine, one of the dominant neutrophil-attracting chemokines in mice, further revealed an indirect effect of PT on neutrophil chemotaxis. Additionally, we showed that intranasal administration of PT inhibits lipopolysaccharide-induced chemokine gene expression and neutrophil recruitment to the airways, presumably by modulation of signaling through Toll-like receptor 4. Collectively, these results demonstrate how PT inhibits early inflammatory responses in the respiratory tract, which reduces neutrophil influx in response to B. pertussis infection, potentially providing an advantage to the pathogen in this interaction.

Previously we found that pertussis toxin (PT), an exotoxin virulence factor produced by Bordetella pertussis, plays an important early role in colonization of the respiratory tract by this pathogen, using a mouse intranasal infection model. In this study, we examined the early role played by another exotoxin produced by this pathogen, adenylate cyclase toxin (ACT). By comparing a wild-type strain to a mutant strain (⌬CYA) with an in-frame deletion of the cyaA gene encoding ACT, we found that the lack of ACT confers a significant peak (day 7) colonization defect (1 to 2 log 10 ). In mixed-infection experiments, the ⌬CYA strain was significantly outcompeted by the wild-type strain, and intranasal administration of purified ACT did not increase colonization by ⌬CYA. These data suggest that ACT benefits the bacterial cells that produce it and, unlike PT, does not act as a soluble factor benefiting the entire infecting bacterial population. Comparison of lower respiratory tract infections over the first 4 days after inoculation revealed that the colonization defect of the PT deletion strain was apparent earlier than that of ⌬CYA, suggesting that PT plays an earlier role than ACT in the establishment of B. pertussis infection. Examination of cells in the bronchoalveolar lavage fluid of infected mice revealed that, unlike PT, ACT does not appear to inhibit neutrophil influx to the respiratory tract early after infection but may combat neutrophil activity once influx has occurred.Bordetella pertussis, a gram-negative bacterial pathogen of the human respiratory tract, secretes at least two protein toxins, pertussis toxin (PT) and adenylate cyclase toxin (ACT), that are important virulence factors in mouse models of infection. PT is an AB 5 toxin that is uniquely produced by B. pertussis. It comprises an enzymatically active A subunit (S1) that ADP ribosylates the alpha subunit of heterotrimeric Gi proteins in mammalian cells (17,22) and a B heteropentamer that binds unidentified glycoconjugate receptors on cells (1, 32). ADP ribosylation of G proteins by PT causes a wide range of effects on signaling pathways in mammalian cells (25) and is responsible for the systemic symptoms of pertussis disease, such as lymphocytosis, insulinemia, and histamine sensitivity (21,23,24). Recently, by comparing a wild-type strain to a mutant strain with an in-frame deletion of the genes encoding PT (⌬PT), we found that PT is an important colonization factor for B. pertussis lower respiratory tract infection and that PT plays an early role in this host-pathogen interaction, including delaying the recruitment of neutrophils to the site of infection (2). We also found that PT acts as a soluble factor that can enhance B. pertussis respiratory tract colonization, even when administered 14 days prior to bacterial inoculation (2). PT also suppresses serum antibody responses to B. pertussis after respiratory tract infection (3) and may play multiple immunosuppressive roles in the host-pathogen interaction.ACT is a single 177-kDa polypeptide with ...

Pertussis is an acute respiratory disease caused by the bacterium Bordetella pertussis, for which humans are the only known reservoir. During infection, B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play roles in promoting bacterial growth during early infection in a mouse model. Furthermore, in vitro and in vivo studies suggest that PT and ACT affect neutrophil chemotaxis and/or function, thereby altering the innate immune response. In this study we depleted animals of neutrophils to investigate whether neutrophils play a protective role during B. pertussis infection in mice. In addition, by infection with toxin-deficient strains, we investigated whether neutrophils are the main targets for PT and/or ACT activity in promoting bacterial growth. Surprisingly, we found no role for neutrophils during B. pertussis infection in naïve mice. However, in previously infected (immune) mice or in mice receiving immune serum, we observed a significant role for neutrophils during infection. Furthermore, in this immune mouse model our evidence indicates that neutrophils appear to be the main target cells for ACT, but not for PT.

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 (⌬PT), 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 ⌬PT strain and found that infection with the ⌬PT strain elicited greater responses to several B. pertussis antigens than did infection with the WT, despite the lower colonization level achieved by the ⌬PT 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 ⌬PT 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 ⌬PT 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.Pertussis toxin (PT) is an important virulence factor produced exclusively by Bordetella pertussis, a gram-negative bacterial pathogen that colonizes the human respiratory tract and causes a disease known as whooping cough or pertussis. PT is a member of the AB 5 structural class of bacterial toxins (41, 43), consisting of an enzymatically active A subunit (S1) that ADP-ribosylates the alpha subunit of the Gi family of heterotrimeric G proteins in mammalian cells (17, 30) and a pentameric B oligomer that binds unidentified glycoconjugate receptors on cells (5, 48). PT activity has a wide range of effects on signaling pathways and normal function in mammalian cells (35,49). The administration of purified PT to experimental animals can reproduce almost all of the systemic symptoms associated with human pertussis disease, such as histamine sensitivity, lymphocytosis, and insulinemia (29, 31, 32), but its role in respiratory tract colonization and disease is uncertain. Several studies of mice intranasally infected with bacteria have shown that PT plays a role in the overall respiratory tract infection by B. pertussis (2,10,45,46). Recently, it was found that PT is a significant colonization factor for resp...

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