Structure of the Bordetella pertussis gene coding for the serotype 3 fimbrial subunit

Bordetella pertussis strains contain at least three distinct genes coding for fimbrial subunits, designated fim2, fim3, and fimX. The sequences of the fim2 and fimX genes have been published. Here we present the sequence of the fim3 gene. Proximal and distal to the fim3 gene, regions were observed that could function as rho-independent terminators, suggesting that the gene is not part of a larger operon. Comparison of the putative promoter regions of the fim2 and fim3 genes revealed a conserved region containing a stretch of approximately 13 C's. This region may be involved in fimbrial phase variation. A comparison of the deduced amino acid sequences of the three fimbrial subunits revealed conserved, variable, and hypervariable regions. The hypervariable regions coincided with predicted antigenic determinants. Peptides derived from the conserved regions may be incorporated into a future pertussis vaccine to induce antibodies which confer protection against strains producing different fimbrial serotypes.     

Sequential activation and environmental regulation of virulence genes in Bordetella pertussis.

Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level.     

Characterization of a Bordetella pertussis fimbrial gene cluster which is located directly downstream of the filamentous haemagglutinin gene

The biosynthesis of fimbriae is a complex process requiring multiple genes which are generally found clustered on the chromosome. In Bordetella pertussis, only major fimbrial subunit genes have been identified, and no evidence has yet been found that they are located in a fimbrial gene cluster. To locate additional genes involved in the biosynthesis of B. pertussis fimbriae, we used TnphoA mutagenesis. A PhoA+ mutant (designated B176) was isolated which was affected in the production of both serotype 2 and 3 fimbriae. Cloning and sequencing of the DNA region harbouring the transposon insertion revealed the presence of at least three additional fimbrial genes, designated fimB, fimC and fimD. The transposon was found to be located in fimD. Analysis of PhoA activity indicated that the fimbrial gene cluster was positively regulated by the bvg locus. A potential binding site for BvgA was observed upstream of fimB. FimB showed homology with the so-called chaperone-like fimbrial proteins, while FimC was homologous with a class of fimbrial proteins located in the outer membrane and presumed to be involved in transport and anchorage of fimbrial subunits. An insertion mutation in fimB abolished the expression of fimbrial subunits, implicating this gene in the biosynthesis of both serotype 2 and 3 fimbriae. Upstream of fimB a pseudogene (fimA) was observed which showed homology with the three major fimbrial subunit genes, fim2, fim3 and fimX. The construction of a phylogenetic tree suggested that fimA may be the primordial major fimbrial subunit gene from which the other three were derived by gene duplication. Interestingly, the fimbrial gene cluster was found to be located directly downstream from the gene coding for the filamentous haemagglutinin, an important B. pertussis adhesin, possibly suggesting co-operation between the two loci in the pathogenesis of pertussis.     

Structural and genetic analysis of the bvg locus in Bordetella species

The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.     

Differential response of the bvg virulence regulon of Bordetella pertussis to MgSO4 modulation.

Magnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon.     

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.     

Serotype variation in Bordetella pertussis is governed by cis-acting elements

Bordetella pertussis contains two genes encoding the serospecific fimbrial subunit proteins 2 and 3 which are assembled into completed fimbriae, which elicit the formation of agglutinating antibodies. Expression of these agglutinogens can vary independently of each other. A gene library from a B. pertussis strain (fimbrial serotype 0.3) was probed with an oligonucleotide probe specific for fimbrial subunit genes. Three homologous genetic loci were identified; an active fim 3 gene, an inactive fim 2 gene and an unknown fim-homologous region. The fim 3 gene carried on a cosmid produced agglutinating fimbrial structures in B. parapertussis and in variants of B. pertussis which had lost the capacity to produce the agglutinogen. This indicated that cis-acting factors are associated with serotype variation in B. pertussis rather than the production of trans-acting repressor molecules.     

Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein

We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis . The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal σ factors of a number of divergent prokaryotes. It is larger than most σ factors identified to date, having a molecular mass of 81.3 kDa. We have designated this factor sigma 80. In a heterologous complementation assay, B. pertussis rpoD was able to complement the Escherichia coli rpoD temperature-sensitive mutant UQ285. Furthermore, B. pertussis rpoD conferred better specificity to the E. coli RNA polymerase, allowing increased expression of the B. pertussis virulence-associated fha promoter, but could not activate the ptx and cya promoters in the E. coli UQ285 strains carrying the B. pertussis bvg locus. We discuss the implications of these results on the mechanisms involved in the activation of virulence-associated promoters.