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.     

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.     

Identification of alcaligin as the siderophore produced by Bordetella pertussis and B. bronchiseptica.

The siderophores produced by iron-starved Bordetella pertussis and B. bronchiseptica were purified and were found to be identical. Using mass spectrometry and proton nuclear magnetic resonance, we determined that the siderophore produced by these organisms was identical to alcaligin, a siderophore produced by Alcaligenes denitrificans.     

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.     

Use of Bordetella bronchiseptica and Bordetella pertussis as live vaccines and vectors for heterologous antigens

Bordetella pertussis and Bordetella bronchiseptica are respiratory pathogens of humans and animals respectively. Unlike many bacteria, they are able to efficiently colonise healthy ciliated respiratory mucosa. This characteristic of Bordetella spp. can potentially be exploited to develop efficient live vaccines and vectors for delivery of heterologous antigens to the respiratory tract. Here we review the progress in this area.     

Characterization of fimN, a new Bordetella bronchiseptica major fimbrial subunit gene

Fimbrial proteins play an important role in the binding of Bordetella bronchiseptica to mammalian cells, an event that is key to the pathogenesis of this organism. The fimbrial phenotype of B. bronchiseptica isolates is usually defined serologically by Fim2 and Fim3 antigens. In this study, a previously unidentified fimbrial gene, fimN, was cloned and sequenced. The identity of fimN is based on several observations. The predicted FimN protein has 59.4 and 52. 2% homology with B. bronchiseptica Fim2 and Fim3, respectively, and is similar in size to these fimbriae. fimN, expressed as a recombinant protein, is recognized by mAb prepared against Fim2 from Bordetella pertussis. The fimN promoter region contains a stretch of cytosine residues similar in length to those of other fimbrial genes expressed by Bordetella species. It also has an activator binding region, upstream from the C-stretch, that closely resembles a corresponding bvg regulated region in fim2, fim3, and fimX. The fimN gene was isolated from a cosmid prepared with B. bronchiseptica genomic DNA that restored normal properties of cellular adhesion to an adhesion deficient strain of B. bronchiseptica. As such, FimN may be a previously overlooked fimbrial antigen and may play an important role in the pathogenicity of B. bronchiseptica.     

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.     

Purification, spectroscopic analysis and biological activity of the macrocyclic dihydroxamate siderophore alcaligin produced by Bordetella pertussis and Bordetella bronchiseptica

Hydroxamate siderophores of virulent Bordetella pertussis and Bordetella bronchiseptica strains were purified using a simple large-scale isolation procedure, and identified by various spectroscopic techniques as the macrocyclic dihydroxamate siderophore trivially known as alcaligin, 1,8(S),11,18(S)-tetrahydroxy-1,6,11,16-tetraazacycloeicosane-2,5,12,15-tetrone, which was previously isolated from the taxonomically-related bacterial species Alcaligenes denitrificans subsp. xylosoxydans. Alcaligin purified from iron-depleted cultures of B. pertussis and B. bronchiseptica exhibited specific growth-promoting activity under iron-restricted conditions for Bordetella indicator strains, and were active in [⁵⁵Fe]ferric alcaligin transport assays. Evidence suggests that several C ₂-symmetric conformations of alcaligin exist simultaneously in both methanolic and aqueous solution.     

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.     

Characterization of a highly conserved island in the otherwise divergent Bordetella holmesii and Bordetella pertussis genomes

The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.     

Characterization of Proteus mirabilis Precocious Swarming Mutants: Identification of rsbA, Encoding a Regulator of Swarming Behavior

Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull’s-eye colony often associated with cultures of P. mirabilis. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.     

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter.

Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough. However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields. To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer. The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized. The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B. pertussis grown for 24 h. The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test. These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B. bronchiseptica background.     

Identification of a Structural Gene Encoding a Metallothionein-like Domain that Includes a Putative Regulator Protein for Streptomyces Protease Gene Expression

An open reading frame (termed ORF-PR) encoding a metallothionein-like domain-including protein was found upstream of a previously identified Streptomyces chymotrypsin-type protease gene (sam-P20). Promoter and terminator activities of ORF-PR were detected using the promoterless Streptomyces tyrosinase gene as a reporter gene and expression of ORF-PR was supposed to occur before that of sam-P20 gene. Frameshift mutation anaysis showed that the ORF-PR product might act as a repressive regulator of the sam-P20 gene.