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 extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches.

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

Cyclic-di-GMP signalling regulates motility and biofilm formation in Bordetella bronchiseptica.

The signalling molecule bis-(3'-5')-cyclic-dimeric guanosine monophosphate (c-di-GMP) is a central regulator of diverse cellular functions, including motility, biofilm formation, cell cycle progression and virulence, in bacteria. Multiple diguanylate cyclase and phosphodiesterase-domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) modulate the levels of the second messenger c-di-GMP to transmit signals and obtain such specific cellular responses. In the genus Bordetella this c-di-GMP network is poorly studied. In this work, we evaluated the expression of two phenotypes in Bordetella bronchiseptica regulated by c-di-GMP, biofilm formation and motility, under the influence of ectopic expression of Pseudomonas aeruginosa proteins with EAL or GGDEF domains that regulates the c-di-GMP level. In agreement with previous reports for other bacteria, we observed that B. bronchiseptica is able to form biofilm and reduce its motility only when GGDEF domain protein is expressed. Moreover we identify a GGDEF domain protein (BB3576) with diguanylate cyclase activity that participates in motility and biofilm regulation in B. bronchiseptica. These results demonstrate for the first time, to our knowledge, the presence of c-di-GMP regulatory signalling in B. bronchiseptica.

Transcriptome profiling reveals stage-specific production and requirement of flagella during biofilm development in Bordetella bronchiseptica.

We have used microarray analysis to study the transcriptome of the bacterial pathogen Bordetella bronchiseptica over the course of five time points representing distinct stages of biofilm development. The results suggest that B. bronchiseptica undergoes a coordinately regulated gene expression program similar to a bacterial developmental process. Expression and subsequent production of the genes encoding flagella, a classical Bvg(-) phase phenotype, occurs and is under tight regulatory control during B. bronchiseptica biofilm development. Using mutational analysis, we demonstrate that flagella production at the appropriate stage of biofilm development, i.e. production early subsequently followed by repression, is required for robust biofilm formation and maturation. We also demonstrate that flagella are necessary and enhance the initial cell-surface interactions, thereby providing mechanistic information on the initial stages of biofilm development for B. bronchiseptica. Biofilm formation by B. bronchiseptica involves the production of both Bvg-activated and Bvg-repressed factors followed by the repression of factors that inhibit formation of mature biofilms.

Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice.

Bacteria form complex and highly elaborate surface adherent communities known as biofilms which are held together by a self-produced extracellular matrix. We have previously shown that by adopting a biofilm mode of existence in vivo, the gram negative bacterial pathogens Bordetella bronchiseptica and Bordetella pertussis are able to efficiently colonize and persist in the mammalian respiratory tract. In general, the bacterial biofilm matrix includes polysaccharides, proteins and extracellular DNA (eDNA). In this report, we investigated the function of DNA in Bordetella biofilm development. We show that DNA is a significant component of Bordetella biofilm matrix. Addition of DNase I at the initiation of biofilm growth inhibited biofilm formation. Treatment of pre-established mature biofilms formed under both static and flow conditions with DNase I led to a disruption of the biofilm biomass. We next investigated whether eDNA played a role in biofilms formed in the mouse respiratory tract. DNase I treatment of nasal biofilms caused considerable dissolution of the biofilm biomass. In conclusion, these results suggest that eDNA is a crucial structural matrix component of both in vitro and in vivo formed Bordetella biofilms. This is the first evidence for the ability of DNase I to disrupt bacterial biofilms formed on host organs.

A simple system for converting lacZ to gfp reporter fusions in diverse bacteria.

We describe new plasmids that facilitate the rapid conversion of lacZ fusions to gfp transcriptional fusions in bacteria. The exchange is based on a double recombination between lacZ sequences in a suicide vector and the recipient chromosome. The suicide vector is a mobilizable, conditionally replicative plasmid that contains the gene for gfp with flanking lacZ homology and is derived from a broad host range plasmid that has been successfully used in a wide range of bacterial species. The technique was used to convert lacZ reporter fusions to gfp fusions in Escherichia coli, Bordetella bronchiseptica and Agrobacterium tumefaciens. Green fluorescent protein expression in the new recombinants reflected the beta galactosidase expression in the parent strains. GFP is particularly useful for rapid quantification of gene expression in real time and in single cells. As a demonstration of an application of this system, we studied the induction of virE transcription by the VirA/VirG two-component system in A. tumefaciens in response to various levels of phenolic inducer. Analysis of GFP fluorescence in single cells revealed that at intermediate levels of inducer the population of cells was remarkably heterogeneous. The tools described here will be useful for general studies of transcriptional regulation as well as for applications that require spatial and temporal identification of gene expression, such as in the study of biofilms, and interactions between bacteria and their environment.

[Alginate gel microchip for real-time monitoring of intracellular processes in bacterial and yeast cells].

A method of alginate-based hydrogel cell microchip manufacturing is proposed. The development of mild conditions for cell immobilization in microvolumes of non-toxic alginate gel allows extending the range of microorganisms used. Different approaches to cell analysis using microchip have been approved in pilot studies. By the example of Escherichia coli, Bordetella bronchiseptica and Saccharomyces cerevisiae it is shown that cell microchip can be successfully applied for monitoring of nucleic acid and protein synthesis in growing cells simultaneously using two fluorescent dyes. The influence of chloramphenicol on the nucleic acids and protein synthesis in five bacterial strains has been studied on the microchip. The microchip was also applied for the analysis of inducible fluorescent protein EGFP synthesis in E. coli cells, the correlation between the level of EGFP synthesis and concentration of the inductor in the medium has been established.

The Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica.

Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg+ phase, a nonvirulent Bvg- phase, and an intermediate Bvgi phase. Genes expressed in the Bvg+ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvgi phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvgi phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvgi phase in B. bronchiseptica.

Bordetella bronchiseptica has a BvgAS-controlled cytotoxic effect upon interaction with epithelial cells.

The interaction between the respiratory pathogen Bordetella bronchiseptica and epithelial cells was studied. After 2-3 h, B. bronchiseptica strains exerted a strong cytotoxic effect on HeLa cells which was evident by rounding of the cells and detachment from the substrate and which ultimately resulted in total disintegration of the host cell. Production of this cytotoxic activity appeared to be regulated by the BvgAS sensory transduction system, which coordinately regulates many B. bronchiseptica virulence factors, since bacteria cultured in the presence of sulfate anions, inhibitors of the BvgAS response, did not exhibit this effect. Furthermore, spontaneous phase variants of B. bronchiseptica strains adhered to HeLa cells but were not cytotoxic. The cytotoxic component is presumably not secreted because the bacterial culture supernatant was not cytotoxic for HeLa cells. Besides HeLa cells other human epithelial cell lines such as Chang cells, larynx HEp-2 cells and lung NCI-H292 cells were sensitive to the cytotoxic activity of B. bronchiseptica. These results suggest the presence of a novel BvgAS-regulated virulence factor in B. bronchiseptica.

Invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells.

We have studied the interaction between the respiratory pathogen Bordetella bronchiseptica and murine spleen dendritic cells, important antigen-presenting cells that are found in the airway epithelium. Wild-type B. bronchiseptica 5376 attached very efficiently to dendritic cells, whereas the bvg mutant ATCC 10580, wild-type strain BB7865, and its spontaneous delta bvgS mutant BB7866 bound less efficiently. However, all tested B. bronchiseptica strains were able to invade dendritic cells and survive intracellularly for at least 72 h. These results suggest that bvg-independent or bvg-downregulated products are involved in the uptake and intracellular survival. Transmission electron microscopic analysis revealed that bacteria grew and replicated intracellularly and were present in typical phagosomes, which fused with lysosomes during the initial infection period. However, in later infection stages some bacteria seemed to escape into an unfused endocytic compartment, where individual bacteria were tightly surrounded by a membrane. The in vitro interaction of B. bronchiseptica with dendritic cells reported here may be relevant to natural infections caused by this organism that lead to chronicity or an altered immune response.