Bordetella pertussis and Bordetella bronchiseptica establish respiratory infections with notorious efficiency. Our previous studies showed that the fhaB genes of B. pertussis and B. bronchiseptica, which encode filamentous hemagglutinin (FHA), are functionally interchangeable and provided evidence that FHA-deficient B. bronchiseptica induces more inflammation in the lungs of mice than wild-type B. bronchiseptica. We show here that the robust inflammatory response to FHA-deficient B. bronchiseptica is characterized by the early and sustained influx of interleukin-17 (IL-17)-positive neutrophils and macrophages and, at 72 h postinoculation, IL-17-positive CD4(+) T cells, suggesting that FHA allows the bacteria to suppress the development of an IL-17-mediated inflammatory response. We also show that the cyaA genes of B. pertussis and B. bronchiseptica, which encode adenylate cyclase toxin (ACT), are functionally interchangeable and that ACT, specifically its catalytic activity, is required for B. bronchiseptica to resist phagocytic clearance but is neither required for nor inhibitory of the induction of inflammation if bacteria are present in numbers sufficient to persist during the first 3 days postinoculation. Incubation of bone marrow-derived macrophages with a ΔcyaA strain caused decreased production of IL-1β and increased production of tumor necrosis factor alpha (TNF-α) and IL-12, while incubation with a ΔcyaA ΔfhaB strain caused increased production of IL-23. These data suggest that FHA and ACT both contribute to suppress the recruitment of neutrophils and the development of an IL-17-mediated immune response. To our knowledge, this is the first demonstration of a microbial pathogen suppressing IL-17-mediated inflammation in vivo as a strategy to evade innate immunity.

ABSTRACT We describe the serendipitous discovery of BatB, a classical-type Bordetella autotransporter (AT) protein with an ∼180-kDa passenger domain that remains noncovalently associated with the outer membrane. Like genes encoding all characterized protein virulence factors in Bordetella species, batB transcription is positively regulated by the master virulence regulatory system BvgAS. BatB is predicted to share similarity with immunoglobulin A (IgA) proteases, and we showed that BatB binds Ig in vitro. In vivo, a Bordetella bronchiseptica ΔbatB mutant was unable to overcome innate immune defenses and was cleared from the lower respiratory tracts of mice more rapidly than wild-type B. bronchiseptica. This defect was abrogated in SCID mice, suggesting that BatB functions to resist clearance during the first week postinoculation in a manner dependent on B- and T-cell-mediated activities. Taken together with the previous demonstration that polymorphonuclear neutrophils (PMN) are critical for the control of B. bronchiseptica in mice, our data support the hypothesis that BatB prevents nonspecific antibodies from facilitating PMN-mediated clearance during the first few days postinoculation. Neither of the strictly human-adapted Bordetella subspecies produces a fully functional BatB protein; nucleotide differences within the putative promoter region prevent batB transcription in Bordetella pertussis, and although expressed, the batB gene of human-derived Bordetella parapertussis (B. parapertussishu) contains a large in-frame deletion relative to batB of B. bronchiseptica. Taken together, our data suggest that BatB played an important role in the evolution of virulence and host specificity among the mammalian-adapted bordetellae.

ABSTRACT The Bordetella BvgAS virulence control system is prototypical of phosphorelays that use a polydomain sensor and a response regulator to control gene expression in response to environmental cues. BvgAS controls the expression of at least three distinct phenotypic phases (Bvg−, Bvgi, and Bvg+) by differentially regulating the expression of at least four classes of genes. Among the loci regulated by BvgAS is bvgAS itself. We investigated the role of autoregulation in the ability of BvgAS to control multiple gene expression patterns in a temporal and steady-state manner by constructing Bordetella bronchiseptica strains in which the bvgAS promoter was replaced with constitutively active promoters. Our results show that positive autoregulation of bvgAS transcription is required for the temporal expression of multiple phenotypic phases that occurs in response to a shift from Bvg−-phase conditions to Bvg+-phase conditions. Autoregulation was also shown to contribute to steady-state regulation; it influences the sensitivity of the system in response to subtle differences in signal intensity. In addition, considered in relation to BvgA and BvgS activities demonstrated in vitro, our results provide insight into how BvgA and BvgS function mechanistically.

The BvgAS two-component system controls virulence in the human respiratory pathogen Bordetella pertussis, the etiological agent of whooping cough. BvgAS is unlike orthodox two-component signal transduction systems in that it employs a four step phosphorelay from the sensor protein BvgS to the response regulator BvgA, instead of the more common two step phosphotransfer. Further, B. pertussis displays at least three distinct phenotypic phases, each characterized by maximal expression of some genes and minimal expression of others. In vitro experiments are modeled by ordinary differential equations of chemical kinetics in order to obtain kinetic parameter estimates. Completed versions of the model are then simulated using deterministic (ODE), stochastic (Gillespie's algorithm) or multiscale (tau leaping, slow-scale SSA or hybrid) chemical kinetics algorithms, depending on what is appropriate. Preliminary results indicate that the full complexity of the three phenotypic phases of B. pertussis cannot be achieved without incorporating the phosphorelay (i.e. by simple two-step phosphotransfer) or BvgAS autoregulation in the model

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

SummaryThe Bordetella bipA gene is expressed maximally when the BvgAS phosphorelay is semi‐active, i.e. in the Bvg‐intermediate (Bvgi) phase. We used a BvgA‐FeBABE cleavage approach together with site‐directed mutagenesis and bipA–lacZ fusion analyses to determine precisely where BvgA‐phosphate (BvgA∼P) binds at the bipA promoter and how that binding contributes to the complex transcription pattern displayed by bipA. BvgA∼P bound with high affinity and cooperatively with RNAP to sequences at the bipA promoter immediately 5′ to and overlapping those bound by RNAP to activate transcription under Bvgi phase conditions. bipA therefore, like fhaB, appears to be similar to classical class‐II promoters with regard to the mechanism by which its transcription is activated. BvgA∼P bound with relatively low affinity to sequences immediately 3′ of those bound by RNAP at the bipA promoter and this binding mediated repression of bipA transcription under Bvg+ phase conditions. BvgA∼P binding to these sequences occurred simultaneously, if not cooperatively, with RNAP, indicating that BvgA∼P represses bipA expression by inhibiting transcription initiation and/or elongation, rather than by competing with RNAP for binding. As bipA is the first Bvgi phase gene to be characterized, and the first gene shown to be repressed by BvgA∼P directly, our results will provide a basis for comparison as additional Bvg‐regulated genes are identified and characterized.