Pertactin contributes to shedding and transmission of Bordetella bronchiseptica.

Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis.

Bordetella bronchiseptica exploits the complex life cycle of Dictyostelium discoideum as an amplifying transmission vector.

Multiple lines of evidence suggest that Bordetella species have a significant life stage outside of the mammalian respiratory tract that has yet to be defined. The Bordetella virulence gene (BvgAS) two-component system, a paradigm for a global virulence regulon, controls the expression of many "virulence factors" expressed in the Bvg positive (Bvg+) phase that are necessary for successful respiratory tract infection. A similarly large set of highly conserved genes are expressed under Bvg negative (Bvg-) phase growth conditions; however, these appear to be primarily expressed outside of the host and are thus hypothesized to be important in an undefined extrahost reservoir. Here, we show that Bvg- phase genes are involved in the ability of Bordetella bronchiseptica to grow and disseminate via the complex life cycle of the amoeba Dictyostelium discoideum. Unlike bacteria that serve as an amoeba food source, B. bronchiseptica evades amoeba predation, survives within the amoeba for extended periods of time, incorporates itself into the amoeba sori, and disseminates along with the amoeba. Remarkably, B. bronchiseptica continues to be transferred with the amoeba for months, through multiple life cycles of amoebae grown on the lawns of other bacteria, thus demonstrating a stable relationship that allows B. bronchiseptica to expand and disperse geographically via the D. discoideum life cycle. Furthermore, B. bronchiseptica within the sori can efficiently infect mice, indicating that amoebae may represent an environmental vector within which pathogenic bordetellae expand and disseminate to encounter new mammalian hosts. These data identify amoebae as potential environmental reservoirs as well as amplifying and disseminating vectors for B. bronchiseptica and reveal an important role for the Bvg- phase in these interactions.

Characterization of a Unique Bordetella bronchiseptica vB_BbrP_BB8 Bacteriophage and Its Application as an Antibacterial Agent.

Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been postulated that some hypervirulent B. bronchiseptica isolates may be responsible for undiagnosed respiratory infections in humans. B. bronchiseptica was shown to acquire antibiotic resistance genes from other bacterial genera, especially Escherichia coli. Here, we present a new B. bronchiseptica lytic bacteriophage-vB_BbrP_BB8-of the Podoviridae family, which offers a safe alternative to antibiotic treatment of B. bronchiseptica infections. We explored the phage at the level of genome, physiology, morphology, and infection kinetics. Its therapeutic potential was investigated in biofilms and in an in vivo Galleria mellonella model, both of which mimic the natural environment of infection. The BB8 is a unique phage with a genome structure resembling that of T7-like phages. Its latent period is 75 ± 5 min and its burst size is 88 ± 10 phages. The BB8 infection causes complete lysis of B. bronchiseptica cultures irrespective of the MOI used. The phage efficiently removes bacterial biofilm and prevents the lethality induced by B. bronchiseptica in G. mellonella honeycomb moth larvae.

Bordetella Colonization Factor A (BcfA) Elicits Protective Immunity against Bordetella bronchiseptica in the Absence of an Additional Adjuvant.

Bordetella bronchiseptica is an etiologic agent of respiratory diseases in animals and humans. Despite the widespread use of veterinary B. bronchiseptica vaccines, there is limited information on their composition and relative efficacy and on the immune responses that they elicit. Furthermore, human B. bronchiseptica vaccines are not available. We leveraged the dual antigenic and adjuvant functions of Bordetella colonization factor A (BcfA) to develop acellular B. bronchiseptica vaccines in the absence of an additional adjuvant. BALB/c mice immunized with BcfA alone or a trivalent vaccine containing BcfA and the Bordetella antigens FHA and Prn were equally protected against challenge with a prototype B. bronchiseptica strain. The trivalent vaccine protected mice significantly better than the canine vaccine Bronchicine and provided protection against a B. bronchiseptica strain isolated from a dog with kennel cough. Th1/17-polarized immune responses correlate with long-lasting protection against bordetellae and other respiratory pathogens. Notably, BcfA strongly attenuated the Th2 responses elicited by FHA and Prn, resulting in Th1/17-skewed responses in inherently Th2-skewed BALB/c mice. Thus, BcfA functions as both an antigen and an adjuvant, providing protection as a single-component vaccine. BcfA-adjuvanted vaccines may improve the efficacy and durability of vaccines against bordetellae and other pathogens.

Immunogenicity of recombinant outer membrane porin protein and protective efficacy against lethal challenge with Bordetella bronchiseptica in rabbits.

AIMS: The outer membrane porin protein (OMPP) of Bordetella bronchiseptica is an important adhesion factor and protective immunogen. The aim of this study was to verify the immunogenicity of recombinant OMPP and its protective efficacy against a lethal challenge with B. bronchiseptica in rabbits. METHODS AND RESULTS: Soluble rOMPP was successfully expressed in Escherichia coli, and the purified recombinant protein was mixed with the ISA 201 VG adjuvant to prepare a subunit vaccine for B. bronchiseptica. Rabbits were immunized with the rOMPP subunit vaccine and then infected with the virulent B. bronchiseptica strain QDBb01. Rabbits immunized with the subunit vaccine were completely protected compared to the control group, and the protective effect was obviously better than that of the inactivated whole-cell vaccine. Moreover, analysis of the immunization duration showed that the rOMPP subunit vaccine provided immune protection for at least 4 months after the second immunization. CONCLUSIONS: The rOMPP subunit vaccine completely protected rabbits from a subsequent B. bronchiseptica challenge. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will provide key information for the development of a safe and effective recombinant subunit vaccine against B. bronchiseptica in rabbits.

Development of macrolide resistance in Bordetella bronchiseptica is associated with the loss of virulence.

Background: Why resistance to specific antibiotics emerges and spreads rapidly in some bacteria confronting these drugs but not others remains a mystery. Resistance to erythromycin in the respiratory pathogens Staphylococcus aureus and Streptococcus pneumoniae emerged rapidly and increased problematically. However, resistance is uncommon amongst the classic Bordetella species despite infections being treated with this macrolide for decades. Objectives: We examined whether the apparent progenitor of the classic Bordetella spp., Bordetella bronchiseptica, is able to rapidly generate de novo resistance to antibiotics and, if so, why such resistance might not persist and propagate. Methods: Independent strains of B. bronchiseptica resistant to erythromycin were generated in vitro by successively passaging them in increasing subinhibitory concentrations of this macrolide. Resistant mutants obtained were evaluated for their capacity to infect mice, and for other virulence properties including adherence, cytotoxicity and induction of cytokines. Results: B. bronchiseptica rapidly developed stable and persistent antibiotic resistance de novo. Unlike the previously reported trade-off in fitness, multiple independent resistant mutants were not defective in their rates of growth in vitro but were consistently defective in colonizing mice and lost a variety of virulence phenotypes. These changes rendered them avirulent but phenotypically similar to the previously described growth phase associated with the ability to survive in soil, water and/or other extra-mammalian environments. Conclusions: These observations raise the possibility that antibiotic resistance in some organisms results in trade-offs that are not quantifiable in routine measures of general fitness such as growth in vitro, but are pronounced in various aspects of infection in the natural host.

Membrane Vesicles Derived from Bordetella bronchiseptica: Active Constituent of a New Vaccine against Infections Caused by This Pathogen.

Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.

The Bordetella Bps Polysaccharide Is Required for Biofilm Formation and Enhances Survival in the Lower Respiratory Tract of Swine.

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Additionally, B. bronchiseptica is capable of establishing long-term or chronic infections in swine. Bacterial biofilms are increasingly recognized as important contributors to chronic bacterial infections. Recently the polysaccharide locus bpsABCD has been demonstrated to serve a critical role in the development of mature biofilms formed by the sequenced laboratory strain of B. bronchiseptica We hypothesized that swine isolates would also have the ability to form mature biofilms and the bpsABCD locus would serve a key role in this process. A mutant containing an in-frame deletion of the bpsABCD structural genes was constructed in a wild-type swine isolate and found to be negative for poly-N-acetylglucosamine (PNAG)-like material by immunoblot assay. Further, the bpsABCD locus was found to be required for the development and maintenance of the three-dimensional structures under continuous-flow conditions. To investigate the contribution of the bpsABCD locus to the pathogenesis of B. bronchiseptica in swine, the KM22Δbps mutant was compared to the wild-type swine isolate for the ability to colonize and cause disease in pigs. The bpsABCD locus was found to not be required for persistence in the upper respiratory tract of swine. Additionally, the bpsABCD locus did not affect the development of anti-Bordetella humoral immunity, did not contribute to disease severity, and did not mediate protection from complement-mediated killing. However, the bpsABCD locus was found to enhance survival in the lower respiratory tract of swine.

Bordetella bronchiseptica pneumonia in a patient with lung cancer; a case report of a rare infection.

BACKGROUND: Bordetella bronchiseptica (B.bronchiseptica) is a frequent cause of respiratory infections in animals but rarely causes serious infection in humans. We present a rare case of B. bronchiseptica pneumonia in a patient with lung cancer. CASE PRESENTATION: A 52-year-old white male with non small cell lung cancer developed fever during treatment with nivolumab. A persistent productive cough and a deterioration in his clinical condition led to his hospitalization for evaluation. Bronchoscopy was performed and a diagnosis of B. bronchiseptica pneumonia was made. The infection was successfully managed by antiobiotic therapy. CONCLUSIONS: B. bronchiseptica is a pathogen that can cause serious infection in humans, especially in immunocompromised or immunoincompetent individuals. In our patient it showed unusual resistance to cephalosporins and poor sensitivity to amikacin. To our knowledge this is the first case of such an infection in a lung cancer patient undergoing treatment with nivolumab. When B. bronchiseptica is identified, the possibility of a nosocomial transmission must be considered.

[Construction and characterization of an hcp mutant of swine Bordetella bronchiseptica].

Objective: We constructed Bordetella bronchiseptica QH0814 hcp mutant to characterize its pathogenicity.[Methods] Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD50) and the infection capacity. Methods: Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD50) and the infection capacity. Results: There was no significant variation of the growth rate between the mutant and the parental strain. Compared with the parental strain, the adherence ability of the mutant did not change remarkably. However, the invasion ability decreased significantly. Mice lethal test showed that the LD50 of the mutant was higher than that of the parental strain. Correspondingly, the bacterial colonization of the mutant in mice blood, lung and liver was much less than that of the parental strain. Conclusion: The knocking-out of the hcp gene had no influence on bacterial growth, but it could attenuate significantly the invasion and colonization of the bacterium. Therefore, the gene may play a role in the pathogenesis of Bordetella bronchiseptica.

Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.

Interspecies variations in Bordetella catecholamine receptor gene regulation and function.

Bordetella bronchiseptica can use catecholamines to obtain iron from transferrin and lactoferrin via uptake pathways involving the BfrA, BfrD, and BfrE outer membrane receptor proteins, and although Bordetella pertussis has the bfrD and bfrE genes, the role of these genes in iron uptake has not been demonstrated. In this study, the bfrD and bfrE genes of B. pertussis were shown to be functional in B. bronchiseptica, but neither B. bronchiseptica bfrD nor bfrE imparted catecholamine utilization to B. pertussis. Gene fusion analyses found that expression of B. bronchiseptica bfrA was increased during iron starvation, as is common for iron receptor genes, but that expression of the bfrD and bfrE genes of both species was decreased during iron limitation. As shown previously for B. pertussis, bfrD expression in B. bronchiseptica was also dependent on the BvgAS virulence regulatory system; however, in contrast to the case in B. pertussis, the known modulators nicotinic acid and sulfate, which silence Bvg-activated genes, did not silence expression of bfrD in B. bronchiseptica. Further studies using a B. bronchiseptica bvgAS mutant expressing the B. pertussis bvgAS genes revealed that the interspecies differences in bfrD modulation are partly due to BvgAS differences. Mouse respiratory infection experiments determined that catecholamine utilization contributes to the in vivo fitness of B. bronchiseptica and B. pertussis. Additional evidence of the in vivo importance of the B. pertussis receptors was obtained from serologic studies demonstrating pertussis patient serum reactivity with the B. pertussis BfrD and BfrE proteins.

Diversity of secretion systems associated with virulence characteristics of the classical bordetellae.

Secretion systems are key virulence factors, modulating interactions between pathogens and the host's immune response. Six potential secretion systems (types 1-6; T1SS-T6SS) have been discussed in classical bordetellae, respiratory commensals/pathogens of mammals. The prototypical Bordetella bronchiseptica strain RB50 genome seems to contain all six systems, whilst two human-restricted subspecies, Bordetella parapertussis and Bordetella pertussis, have lost different subsets of these. This implicates secretion systems in the divergent evolutionary histories that have led to their success in different niches. Based on our previous work demonstrating that changes in secretion systems are associated with virulence characteristics, we hypothesized there would be substantial divergence of the loci encoding each amongst sequenced strains. Here, we describe extensive differences in secretion system loci; 10 of the 11 sequenced strains had lost subsets of genes or one entire secretion system locus. These loci contained genes homologous to those present in the respective loci in distantly related organisms, as well as genes unique to bordetellae, suggesting novel and/or auxiliary functions. The high degree of conservation of the T3SS locus, a complex machine with interdependent parts that must be conserved, stands in dramatic contrast to repeated loss of T5aSS 'autotransporters', which function as an autonomous unit. This comparative analysis provided insights into critical aspects of each pathogen's adaptation to its different niche, and the relative contributions of recombination, mutation and horizontal gene transfer. In addition, the relative conservation of various secretion systems is an important consideration in the ongoing search for more highly conserved protective antigens for the next generation of pertussis vaccines.

SET-domain bacterial effectors target heterochromatin protein 1 to activate host rDNA transcription.

Transcription of rRNA genes (rDNAs) in the nucleolus is regulated by epigenetic chromatin modifications including histone H3 lysine (de)methylation. Here we show that LegAS4, a Legionella pneumophila type IV secretion system (TFSS) effector, is targeted to specific rDNA chromatin regions in the host nucleolus. LegAS4 promotes rDNA transcription, through its SET-domain (named after Drosophila Su(var)3-9, enhancer of zeste [E(z)], and trithorax [trx]) histone lysine methyltransferase (HKMTase) activity. LegAS4's association with rDNA chromatin is mediated by interaction with host HP1α/γ. L. pneumophila infection potently activates rDNA transcription in a TFSS-dependent manner. Other bacteria, including Bordetella bronchiseptica and Burkholderia thailandensis, also harbour nucleolus-localized LegAS4-like HKMTase effectors. The B. thailandensis type III effector BtSET promotes H3K4 methylation of rDNA chromatin, contributing to infection-induced rDNA transcription and bacterial intracellular replication. Thus, activation of host rDNA transcription might be a general bacterial virulence strategy.

Resident microbiota affect Bordetella pertussis infectious dose and host specificity.

Before contacting host tissues, invading pathogens directly or indirectly interact with host microbiota, but the effects of such interactions on the initial stages of infection are poorly understood. Bordetella pertussis is highly infectious among humans but requires large doses to colonize rodents, unlike a closely related zoonotic pathogen, Bordetella bronchiseptica, raising important questions about the contributions of bacterial competition to initial colonization and host selection. We observed that <100 colony-forming units (CFU) of B. bronchiseptica efficiently infected mice and displaced culturable host microbiota, whereas 10 000 CFU of B. pertussis were required to colonize murine nasal cavities and did not displace host microorganisms. Bacteria isolated from murine nasal cavities but not those from the human lower respiratory tract limited B. pertussis growth in vitro, indicating that interspecies competition may limit B. pertussis colonization of mice. Further, a broad-spectrum antibiotic treatment delivered before B. pertussis inoculation reduced the infectious dose to <100 CFU, and reintroduction of single Staphylococcus or Klebsiella species was sufficient to inhibit B. pertussis colonization of antibiotic-treated mice. Together, these results reveal that resident microorganisms can prevent B. pertussis colonization and influence host specificity, and they provide rationale for manipulating microbiomes to create more-accurate animal models of infectious diseases.

Comparative analyses of a cystic fibrosis isolate of Bordetella bronchiseptica reveal differences in important pathogenic phenotypes.

Bordetella bronchiseptica is a Gram-negative bacterium that infects and causes disease in a wide variety of animals. B. bronchiseptica also infects humans, thereby demonstrating zoonotic transmission. An extensive characterization of human B. bronchiseptica isolates is needed to better understand the distinct genetic and phenotypic traits associated with these zoonotic transmission events. Using whole-genome transcriptome and CGH analysis, we report that a B. bronchiseptica cystic fibrosis isolate, T44625, contains a distinct genomic content of virulence-associated genes and differentially expresses these genes compared to the sequenced model laboratory strain RB50, a rabbit isolate. The differential gene expression pattern correlated with unique phenotypes exhibited by T44625, which included lower motility, increased aggregation, hyperbiofilm formation, and an increased in vitro capacity to adhere to respiratory epithelial cells. Using a mouse intranasal infection model, we found that although defective in establishing high bacterial burdens early during the infection process, T44625 persisted efficiently in the mouse nose. By documenting the unique genomic and phenotypic attributes of T44625, this report provides a blueprint for understanding the successful zoonotic potential of B. bronchiseptica and other zoonotic bacteria.

Evidence for phenotypic bistability resulting from transcriptional interference of bvgAS in Bordetella bronchiseptica.

Bordetella species cause respiratory infections in mammals. Their master regulatory system BvgAS controls expression of at least three distinct phenotypic phases in response to environmental cues. The Bvg⁺ phase is necessary and sufficient for respiratory infection while the Bvg⁻ phase is required for survival ex vivo. We obtained large colony variants (LCVs) from the lungs of mice infected with B. bronchiseptica strain RBX9, which contains an in-frame deletion mutation in fhaB, encoding filamentous haemagglutinin. RBX9 also yielded LCVs when switched from Bvg⁻ phase conditions to Bvg⁺ phase conditions in vitro. We determined that LCVs are composed of both Bvg⁺ and Bvg⁻ phase bacteria and that they result from defective bvgAS positive autoregulation. The LCV phenotype was linked to the presence of a divergent promoter 5' to bvgAS, suggesting a previously undescribed mechanism of transcriptional interference that, in this case, leads to feedback-based bistability (FBM). Our results also indicate that a small proportion of RBX9 bacteria modulates to the Bvg⁻ phase in vivo. In addition to providing insight into transcriptional interference and FBM, our data provide an example of an in-frame deletion mutation exerting a 'polar' effect on nearby genes.

An improved recombination-based in vivo expression technology-like reporter system reveals differential cyaA gene activation in Bordetella species.

Bordetella pertussis and Bordetella bronchiseptica rely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg(-) phase, expression of vrg genes, including those required for motility in B. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg(+) phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg(+) phase during respiratory infection, Bvg-regulated gene activation in B. bronchiseptica has not been investigated in vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation both in vitro and in vivo. Using pGFLIP, we demonstrated that cyaA, considered to be a "late" Bvg(+) phase gene, is activated substantially earlier in B. bronchiseptica than B. pertussis following a switch from Bvg(-) to Bvg(+) phase conditions. We show that the altered activation of cyaA is not due to differences in the cyaA promoter or in the bvgAS alleles of B. bronchiseptica compared to B. pertussis, but appears to be species specific. Finally, we used pGFLIP to show that flaA remains repressed during infection, confirming that B. bronchiseptica does not modulate to the Bvg(-) phase in vivo.

Swine atrophic rhinitis caused by pasteurella multocida toxin and bordetella dermonecrotic toxin.

Atrophic rhinitis is a widespread and economically important swine disease caused by Pasteurella multocida and Bordetella bronchiseptica. The disease is characterized by atrophy of the nasal turbinate bones, which results in a shortened and deformed snout in severe cases. P. multocida toxin and B. bronchiseptica dermonecrotic toxin have been considered to independently or cooperatively disturb the osteogenesis of the turbinate bone by inhibiting osteoblastic differentiation and/or stimulating bone resorption by osteoclasts. Recently, the intracellular targets and molecular actions of both toxins have been clarified, enabling speculation on the intracellular signals leading to the inhibition of osteogenesis.

Network model of immune responses reveals key effectors to single and co-infection dynamics by a respiratory bacterium and a gastrointestinal helminth.

Co-infections alter the host immune response but how the systemic and local processes at the site of infection interact is still unclear. The majority of studies on co-infections concentrate on one of the infecting species, an immune function or group of cells and often focus on the initial phase of the infection. Here, we used a combination of experiments and mathematical modelling to investigate the network of immune responses against single and co-infections with the respiratory bacterium Bordetella bronchiseptica and the gastrointestinal helminth Trichostrongylus retortaeformis. Our goal was to identify representative mediators and functions that could capture the essence of the host immune response as a whole, and to assess how their relative contribution dynamically changed over time and between single and co-infected individuals. Network-based discrete dynamic models of single infections were built using current knowledge of bacterial and helminth immunology; the two single infection models were combined into a co-infection model that was then verified by our empirical findings. Simulations showed that a T helper cell mediated antibody and neutrophil response led to phagocytosis and clearance of B. bronchiseptica from the lungs. This was consistent in single and co-infection with no significant delay induced by the helminth. In contrast, T. retortaeformis intensity decreased faster when co-infected with the bacterium. Simulations suggested that the robust recruitment of neutrophils in the co-infection, added to the activation of IgG and eosinophil driven reduction of larvae, which also played an important role in single infection, contributed to this fast clearance. Perturbation analysis of the models, through the knockout of individual nodes (immune cells), identified the cells critical to parasite persistence and clearance both in single and co-infections. Our integrated approach captured the within-host immuno-dynamics of bacteria-helminth infection and identified key components that can be crucial for explaining individual variability between single and co-infections in natural populations.