Bordetella bronchiseptica is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of B. bronchiseptica strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that cyaA, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the cya locus, which encodes, activates, and secretes CyaA, was replaced by an operon (ptp) predicted to encode peptide transport proteins. Other B. bronchiseptica strains from the same phylogenetic lineage as that of strain 253 also lacked the cya locus, contained the ptp genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the ptp genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of B. bronchiseptica.

Type VI Secretion Systems (T6SSs) have been identified in numerous Gram-negative pathogens, but the lack of a natural host infection model has limited analysis of T6SS contributions to infection and pathogenesis. Here, we describe disruption of a gene within locus encoding a putative T6SS in Bordetella bronchiseptica strain RB50, a respiratory pathogen that circulates in a broad range of mammals, including humans, domestic animals, and mice. The 26 gene locus encoding the B. bronchiseptica T6SS contains apparent orthologs to all known core genes and possesses thirteen novel genes. By generating an in frame deletion of clpV, which encodes a putative ATPase required for some T6SS-dependent protein secretion, we observe that ClpV contributes to in vitro macrophage cytotoxicity while inducing several eukaryotic proteins associated with apoptosis. Additionally, ClpV is required for induction of IL-1β, IL-6, IL-17, and IL-10 production in J774 macrophages infected with RB50. During infections in wild type mice, we determined that ClpV contributes to altered cytokine production, increased pathology, delayed lower respiratory tract clearance, and long term nasal cavity persistence. Together, these results reveal a natural host infection system in which to interrogate T6SS contributions to immunomodulation and pathogenesis.

Interleukin-1 receptor-deficient (IL-1R؊/؊ ) mice are healthy despite being colonized by commensal microbes but are defective in defenses against specific pathogens, suggesting that IL-1R-mediated effects contribute to immune responses against specific pathogenic mechanisms. To better define the role of IL-1R in immunity to respiratory infections, we challenged IL-1R؊/؊ mice with Bordetella pertussis and Bordetella parapertussis, the causative agents of whooping cough. Following inoculation with B. pertussis, but not B. parapertussis, IL-1R ؊/؊ mice showed elevated bacterial numbers and more extensive inflammatory pathology than wild-type mice. Acellular B. pertussis vaccines were not efficiently protective against B. pertussis in IL-1R ؊/؊ mice. B. pertussisstimulated dendritic cells from IL-1R؊/؊ mice produced higher levels of tumor necrosis factor alpha (TNF-␣) and IL-6 than wild-type cells. Moreover, elevated levels of gamma interferon (IFN-␥) and TNF-␣ but lower levels of IL-10 were detected during B. pertussis infection in IL-1R

Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO2. Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO2 conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO2 was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO2. The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO2, indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO2 concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response.

BackgroundThe cell envelope of a bacterial pathogen can be damaged by harsh conditions in the environment outside a host and by immune factors during infection. Cell envelope stress responses preserve the integrity of this essential compartment and are often required for virulence. Bordetella species are important respiratory pathogens that possess a large number of putative transcription factors. However, no cell envelope stress responses have been described in these species. Among the putative Bordetella transcription factors are a number of genes belonging to the extracytoplasmic function (ECF) group of alternative sigma factors, some of which are known to mediate cell envelope stress responses in other bacteria. Here we investigate the role of one such gene, sigE, in stress survival and pathogenesis of Bordetella bronchiseptica.ResultsWe demonstrate that sigE encodes a functional sigma factor that mediates a cell envelope stress response. Mutants of B. bronchiseptica strain RB50 lacking sigE are more sensitive to high temperature, ethanol, and perturbation of the envelope by SDS-EDTA and certain β-lactam antibiotics. Using a series of immunocompromised mice deficient in different components of the innate and adaptive immune responses, we show that SigE plays an important role in evading the innate immune response during lethal infections of mice lacking B cells and T cells. SigE is not required, however, for colonization of the respiratory tract of immunocompetent mice. The sigE mutant is more efficiently phagocytosed and killed by peripheral blood polymorphonuclear leukocytes (PMNs) than RB50, and exhibits decreased cytotoxicity toward macrophages. These altered interactions with phagocytes could contribute to the defects observed during lethal infection.ConclusionsMuch of the work on transcriptional regulation during infection in B. bronchiseptica has focused on the BvgAS two-component system. This study reveals that the SigE regulon also mediates a discrete subset of functions associated with virulence. SigE is the first cell envelope stress-sensing system to be described in the bordetellae. In addition to its role during lethal infection of mice deficient in adaptive immunity, our results indicate that SigE is likely to be important for survival in the face of stresses encountered in the environment between hosts.