Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes

Pertussis toxin, the major virulence factor of Bordetella pertussis, is not produced by the closely related species Bordetella parapertussis and Bordetella bronchiseptica. It is shown here that these two species possess but do not express the complete toxin operon. Nucleotide sequencing of an EcoRI fragment of 5 kilobases comprising the regions homologous to the pertussis toxin genes shows that in this region, B. parapertussis and B. bronchiseptica are 98.5% and 96% homologous, respectively, to B. pertussis. The changes (mostly base pair substitutions) in many cases are identical in B. parapertussis and B. bronchiseptica, suggesting that these two species derive from a common ancestor. Many of the mutations common to B. parapertussis and B. bronchiseptica involve the promoter region, which becomes very inefficient. The S1 subunits of both species, when expressed in Escherichia coli, have the same ADP-ribosylating activity as the S1 subunit from B. pertussis, indicating that the mutations in the S1 gene described here do not affect its function.     

Genetic diversity analysis of isolates belonging to Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species

In this study, Amplified Fragment Length Polymorphism (AFLP) method was used to track differences among human and animal isolates of B. pertussis, B. parapertussis and B. bronchiseptica species. One hundred and sixty representative strains of these species orginated from international and Polish bacterial collections were genotyped according to AFLP involving EcoRI/Msel and SpeI/ApaI restriction/ligation/amplification procedures. This study has confirmed high potential AFLP SpeI/ApaI procedure for intra-species differentiation of B. pertussis and B. bronchiseptica strains. Both AFLP EcoRI/MseI and SpeI/ApaI procedures have been found to be useful for species-specific classification in case of B. pertussis strains. In case of B. bronchiseptica or B. parapertussis species-specific classification, SpeI/ApaI procedure has been found more precise than EcoRI/MseI one.     

Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis

Porins of Salmonella minnesota, R595, were purified by anion exchange chromatography and subsequently isolated in their monomeric form by chromatofocusing. Two forms of porin could be isolated, both with an apparent molecular mass of 37,000, but of differing isoelectric points (pI 4.6 versus pI of 4.9). Porins with pI 4.9 did not contain any detectable LPS, but porins with pI 4.6 were found to contain trace amounts of LPS (1.3 x 10(-4) micrograms LPS/1 microgram porin) as measured using a highly sensitive limulus assay. Unlike the LPS-associated porins the monomeric porins were biologically inert with regard to pore formation, but they were still able to bind C1q, a subcomponent of the first component of complement.     

Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica

The Bordetella pertussis wlb locus (wlb_pe, formerly bpl ) is required for the biosynthesis of a trisaccharide that, when attached to the B. pertussis lipopolysaccharide (LPS) core (band B), generates band A LPS. The equivalent loci in Bordetella bronchiseptica (wlb_br) and Bordetella parapertussis (wlb_pa) were identified and cloned. The wlb_br and wlb_pa loci differ from wlb_pe in that they lack the insertion sequence that defines the right-hand terminus of wlb_pe. Deletion of 12 kb of DNA containing the whole wlb locus (Δwlb) by allelic exchange in each of the three bordetellae had no effect on band B biosynthesis, whereas band A biosynthesis was prevented in B. pertussis and B. bronchiseptica. In B. bronchiseptica and B. parapertussis, Δwlb mutants also lacked O-antigen. Reintroduction of the wlb_pe or wlb_br loci on a shuttle vector into the three Δwlb mutants restored the wild-type LPS phenotype in the B. pertussis and B. bronchiseptica mutants. In the case of B. parapertussis, which normally does not synthesize an apparent band A structure, introduction of the wlb_pe or wlb_br loci now enabled the generation of band A. This suggests that the attachment point for band A trisaccharide on the LPS core is present in B. parapertussis, and further suggests that the wild-type wlb_pa locus is not fully functional. Introduction of the wlb_pa locus into the Δwlb_pe, Δwlb_br and Δwlb_pa mutants had interesting consequences. The B. bronchiseptica and B. parapertussis recipients were now able to biosynthesize O-antigen, but no band A was generated. In the B. pertussis recipient, a truncated band A was expressed consistent with a mutation in the wlbH gene, but on Western blotting the expression of a small amount of full-length band A was also seen. Evidence that the wlbH_pa protein is not fully functional was provided by the failure of the wlb_pa locus to fully complement a B. pertussis wlbH (ΔwlbH_pe) mutant. This was supported by DNA sequence data showing that a single amino acid, conserved between homologous proteins from a range of bacteria, is altered in the B. parapertussis WlbH protein.     

The structure of the carbohydrate backbone of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica.

The structure of the core-lipid A region of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica has been analyzed. Lipopolysaccharides were deacylated using strong alkaline hydrolysis, the products were separated by high performance anion-exchange chromatography and analyzed by NMR and mass spectrometry. The following structure of the products can be deduced from the experimental results: where for the product from Bordetella hinzii N = H, R = H, beta-FucN4N- or partially N-acetylated Sug-(1-3)-beta-FucN4N and for the product from Bordetella bronchiseptica N = alpha-Hep, R = H, beta-FucN4N, beta-FucN4NMe or partially N-acetylated Sug-(1-3)-beta-FucN4N or Sug-(1-3)-beta-FucN4NMe; Sug = 2,3-diamino-2,3, 4-trideoxy-hex-4-enuronopyranosyl.     

In-vitro and in-vivo analysis of the production of the Bordetella type three secretion system effector A in Bordetella pertussis,Bordetella parapertussis and Bordetella bronchiseptica

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are three closely related pathogens.They all possess the gene coding for the Bordetella type three secretion system effector A (bteA) toxin that became a focus of interest since it was demonstrated that B. pertussis Japanese non-vaccine-type isolates produce BteA unlike vaccine-type isolates. We thus explored the in-vitro production of BteA in B. pertussis isolates collected in France during periods of different vaccine policy as well as in B. parapertussis and B. bronchiseptica isolates. We also analyzed the in-vivo induction of anti-BteA antibodies after infection with different isolates of the three species.We produced a recombinant His6-tagged BteA (rBteA) protein. Specific rBteA polyclonal serum was prepared which enabled us to screen Bordetella isolates for in-vitro BteA production: 99.0% (293/296) of tested B. pertussis isolates, including French vaccine strains, and 97.5% (79/81) of B. bronchiseptica isolates produced BteA in-vitro but only the latter was capable of inducing an in-vivo immune response. No in-vitro or in-vivo production of BteA was detected by any of the B. parapertussis isolates tested.     

Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization

The 4.7 Kb EcoRI-fragment of phase I B. pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli. Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B. pertussis strain 475 in phase I, B. pertussis in phase IV, B. parapertussis strains 504 and 17903, B. bronchiseptica strain 214, and B. parapertussis strain 17903 (a convertant obtained by means of B. pertussis phage 134), as well as B. pertussis phage 134. Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb). Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bordetella bronchiseptica infection of rats and mice

Bordetella bronchiseptica has long been associated with respiratory tract infections in laboratory research, food-producing, companion, and wildlife animal species. Its range of distribution also may include humans and contaminated inanimate environmental sources. Natural diseases due to B. bronchiseptica infections in laboratory rats and mice were described before many of the major pathogens of these hosts were discovered. To our knowledge, there are no recent reports of natural disease due to B. bronchiseptica in these species; as a result, some have questioned its role as a natural pathogen in murine hosts. We reviewed occurrence of natural B. bronchiseptica infections and present information gained from recent experimental infection studies in murine hosts. We also discuss the potential impact of natural B. bronchiseptica infections on research and methods of control.     

Failure to convert Bordetella parapertussis to Bordetella pertussis with a pertussis phage

To analyze the described lysogenic conversion of Bordetella parapertussis to a Bordetella pertussis-like form we used the phage 134 to lysogenize a B. parapertussis strain. Southern blot analysis of the isolated ‘lysogens’ showed that they were not true lysogens, but rather chronically infected strains. These pseudo-lysogens did not show any changes in virulence properties compared with the parental strain. The only difference we could show was a change in the LPS-structure: the pseudolysogens had a rough LPS, like B. pertussis, whereas the parental B. parapertussis strain was smooth.     

Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica

Bordetella pertussis and Bordetella bronchiseptica contain nearly identical BvgAS signal-transduction systems that mediate a biphasic transition between virulent (Bvg⁺) and avirulent (Bvg^–) phases. In the Bvg⁺ phase, the two species express a similar set of adhesins and toxins, and in both organisms the transition to the Bvg^– phase occurs in response to the same environmental signals (low temperature or the presence of nicotinic acid or sulphate anion). These two species differ, however, with regard to Bvg^–-phase phenotypes, host specificity, the severity and course of the diseases they cause, and also potentially in their routes of transmission. To investigate the contribution of the virulence-control system to these phenotypic differences, we constructed a chimeric B. bronchiseptica strain containing bvgAS from B. pertussis and compared it with wild-type B. bronchiseptica in vitro and in vivo . The chimeric strain was indistinguishable from the wild type in its ability to express Bvg⁺- and Bvg^–-phase-specific factors. However, although the chimeric strain responded to the same signals as the wild type, it differed dramatically in sensitivity to these signals; significantly more nicotinic acid or MgSO₄ was required to modulate the chimeric strain compared with the wild-type strain. Despite this difference in signal sensitivity, the chimeric strain was indistinguishable from the wild type in its ability to cause respiratory-tract infections in rats, indicating that the bvgAS loci of B. pertussis and B. bronchiseptica are functionally interchangeable in vivo . By exchanging discrete fragments of bvgAS , we found that the periplasmic region of BvgS determines signal sensitivity.     

Toxigenicity conversion by pertussis phages in Bordetella parapertussis

For the first time toxigenicity conversion in B. parapertussis induced by B. pertussis phages was discovered. The clones of B. parapertussis recipient strain No. 17903 used in this study were subjected to lysogenization with 4 B. pertussis phages; as a result, 95% of these clones became immune to the repeated phage infection, developed spontaneous phage production and showed toxic properties (lethal toxicity due to the action of thermolabile and thermostable toxins) characteristic of the donor strains from which B. pertussis phages had been obtained. Differences in the degree of toxicity shown by the converted strains were determined by means of the spleen index. The convertants thus obtained did not possess protective potency.     

Production of biomass and filamentous hemagglutinin by Bordetella bronchiseptica

The mammalian pathogen Bordetella bronchiseptica was grown under controlled batch conditions with glutamate as the primary carbon and nitrogen source. First, a Box-Behnken statistical design quantified the effect of Mg, sulfate, and nicotinate on the antigen filamentous hemagglutinin (FHA) formation. Using lactic acid as a secondary carbon source for pH control, Mg, and SO₄ each negatively affected antigen expression, while nicotinate positively affected antigen expression. Sulfate had a stronger negative effect than Mg with 10 mM eliminating FHA altogether; the highest FHA expression (about 1,000 ng/mL) occurred when either Mg concentration or SO₄ concentration, but not both, was about 0.1 mM. Using two Mg and SO₄ compositions modeled to yield the greatest antigen expression, three other organic acids were compared as the secondary carbon source: acetate, citrate, and succinate. Mixtures of acetate and glutamate resulted in the greatest organic acid consumption, OD, and FHA concentration (about 1,500 ng/mL), although significant acetate accumulated during these batch processes. The mechanism leading to elevated FHA expression when acetate is the secondary carbon source is unknown, particularly since these cultures were most prone to phase shift to Bvg^? cultures.     

Phenotypic and Genomic Analysis of Hypervirulent Human-associated Bordetella bronchiseptica

ABSTRACT: BACKGROUND: B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV [1]. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics. RESULTS: Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates. CONCLUSION: Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage.     

Pseudomonas aeruginosa rhamnolipids disperse Bordetella bronchiseptica biofilms

We have previously reported that the respiratory pathogen Bordetella bronchiseptica can form biofilms in vitro. In this report, we demonstrate the disruption of B. bronchiseptica biofilms by rhamnolipids secreted from Pseudomonas aeruginosa. This suggests that biosurfactants such as rhamnolipids may be utilized as antimicrobial agents for removing Bordetella biofilms.