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.     

Evolutionary relationships in the genus Bordetella

The nucleotide sequence of the pertussis toxin operon of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica, has shown that the last two species contain many common mutations and are likely to derive from a common ancestor (Arico and Rappuoli, 1987). To elucidate further the evolutionary relationships between the Bordetella species, we have cloned and sequenced the promoter region and the gene coding for the S1 subunit of pertussis toxin from additional B. pertussis strains, such as the type strain BP 18323 and two recent clinical isolates, namely strain BP 13456 from Sweden and strain BP SA1 from Italy. While the strains BP SA1 and BP 13456 are shown to differ from the published B. pertussis sequences by only one base pair, the type strain BP 18323 contains a total of 11 base-pair substitutions. Remarkably, 9 of the 11 substitutions found in BP 18323 are also common to B. parapertussis and B. bronchiseptica, strongly suggesting that this strain derives from the same ancestor as B. parapertussis and B. bronchiseptica. Computer analysis of the sequence data allows the construction of an evolutionary ‘tree’ showing that the B. pertussis strains are very homogeneous and significantly distant from B. parapertussis and B. bronchiseptica. Therefore the proposed conversion from B. parapertussis to B. pertussis appears highly improbable.     

The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis

Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS O-antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide O-antigen units, which are subsequently assembled to form polymerized O-antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single O-antigen-like structure and that B. pertussis LPS is similar to semi-rough LPS seen in some mutants of enteric bacteria.     

Production and characterization of recombinant pertactin, fimbriae 2 and fimbriae 3 from Bordetella pertussis

Background  

Bordetella pertussis is a causative agent of pertussis or whooping cough in humans. Pertactin (Prn), fimbriae 2 (Fim2) and fimbriae 3 (Fim3) of B. pertussis are important virulence factors and immunogens which have been included in some acellular pertussis vaccines. In this present study, we cloned, expressed and purified Prn, Fim2 and Fim3, respectively. The immunogeniCity and protective efficacy of the three recombinant proteins (rPrn, rFim2 and rFim3) were investigated in mouse model.     

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.     

Acellular pertussis vaccination facilitates Bordetella parapertussis infection in a rodent model of bordetellosis

Despite over 50 years of population-wide vaccination, whooping cough incidence is on the rise. Although Bordetella pertussis is considered the main causative agent of whooping cough in humans, Bordetella parapertussis infections are not uncommon. The widely used acellular whooping cough vaccines (aP) are comprised solely of B. pertussis antigens that hold little or no efficacy against B. parapertussis. Here, we ask how aP vaccination affects competitive interactions between Bordetella species within co-infected rodent hosts and thus the aP-driven strength and direction of in-host selection. We show that aP vaccination helped clear B. pertussis but resulted in an approximately 40-fold increase in B. parapertussis lung colony-forming units (CFUs). Such vaccine-mediated facilitation of B. parapertussis did not arise as a result of competitive release; B. parapertussis CFUs were higher in aP-relative to sham-vaccinated hosts regardless of whether infections were single or mixed. Further, we show that aP vaccination impedes host immunity against B. parapertussis—measured as reduced lung inflammatory and neutrophil responses. Thus, we conclude that aP vaccination interferes with the optimal clearance of B. parapertussis and enhances the performance of this pathogen. Our data raise the possibility that widespread aP vaccination can create hosts more susceptible to B. parapertussis infection.     

Representational difference analysis identifies a strain-specific LPS biosynthesis locus in Bordetella spp.

Bordetella pertussis and B. bronchiseptica are genetically very closely related but differ significantly in their virulence properties. Using Representational Difference Analysis (RDA), 11 DNA fragments specific for B. pertussis Tohama I or B. bronchiseptica BB7865 were identified. All B. bronchiseptica BB7865-derived fragments also hybridized with chromosomal DNA from B. parapertussis but not from the B. pertussis strains Tohama I and W28, underlining the close phylogenetic relationship between B. bronchiseptica and B. parapertussis. The B. pertussis type strain BP18323 is a special case, as it contains DNA sequences characteristic for both B. pertussis and B. bronchiseptica. As demonstrated by pulsed-field gel electrophoresis, several of the BB7865-derived fragments are present on a single 30-kb XbaI fragment. Based on the sequences of putative coding regions, four of these fragments may code for proteins involved in carbohydrate metabolism or transport. In agreement with this notion, a mutant for one of these loci synthesizes a significantly altered lipopolysaccharide that lacks the O-specific side chains. The analysis of the corresponding genomic region in various Bordetella species showed that this locus is present in B. bronchiseptica and B. parapertussis but not in B. pertussis. This confirms that the RDA approach has identified a novel strain-specific LPS biosynthesis locus which accounts for the differences between the LPS structures elaborated by different Bordetella species. Received: 24 February 1999 / Accepted: 17 May 1999     

Increased incidence of pertussis and parapertussis in HIV-1-positive adolescents vaccinated previously with whole-cell pertussis vaccine

We investigated 296 adolescents (11–18 years), who had been immunized previously with the three doses of DPT vaccines. 48 were diagnosed positive for HIV-1. Nasopharyngeal swabs were obtained from 296 adolescents who presented with persistent cough and nasopharyngeal secretions. Nasopharyngeal swabs (calcium alginate) specimens were collected by passing the swabs through the nares into the posterior nasopharynx and rotating the swabs for a few seconds. The swabs were plated for culture of Bordetella organisms in charcoal cephalexin blood agar (CCBA). The CCBA plates were incubated for 2–6 days at 35 °C in a humid aerobic atmosphere. The suspected, shiny (mercury-like) colonies were tested by slide agglutination with antisera to B. pertussis and B. parapertussis, and urease, oxidase activities were performed. Results indicate that out of 48 HIV-1-positive adolescents, 18 had positive cultures for Bordetella organisms (14, Bordetella pertussis, and 4, Bordetella parapertussis). Of 248 HIV-1-negative subjects, 3 had Bordetella organisms (2, Bordetella pertussis, 1, Bordetella bronchiseptica). One of the subjects, a boy, aged 14 years, with Bordetella bronchiseptica had a dog as pet, which was found to be infected. The results indicate that adolescents with HIV-1 infection, despite being vaccinated against pertussis have a higher rate of infection when exposed to pertussis bacteria than HIV-1-negative adolescents. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.     

Isolierung von Bordetella-Phagen aus Bakterienstämen der Impfstoffproduktion

It has been demonstrated that strains of Bordetella pertussis used for vaccine production contain temperate phages. It can be conducted from many experiments performed in our laboratory. that 10–100 phages per 10¹⁰ bacteria are released. However, the production of bacterial mass is not markedly influenced by lysogeny. Strains of Bordetella bronchiseptica used for production of vaccine against Rhinitis atrophicans of pigs have temperate phages too. These phages may cause a complete lysis during a submerse cultivation. The phages of Bordetella pertussis and Bordetella bronchiseptica can be propagted on Bordetella parapertussis.     

Identification and Cloning of waaF (rfaF) from Bordetella pertussis and Use To Generate Mutants of Bordetella spp. with Deep Rough Lipopolysaccharide

A DNA locus from Bordetella pertussis capable of reconstituting lipopolysaccharide (LPS) O-antigen biosynthesis in Salmonella typhimurium SL3789 (rfaF511) has been isolated, by using selection with the antibiotic novobiocin. DNA within the locus encodes a protein with amino acid sequence similarity to heptosyltransferase II, encoded by waaF (previously rfaF) in other gram-negative bacteria. Mutation of this gene in B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by allelic exchange generated bacteria with deep rough LPS phenotypes consistent with the proposed function of the gene as an inner core heptosyltransferase. These are the first LPS mutants generated in B. parapertussis and B. bronchiseptica and the first deep rough mutants of any of the bordetellae.     

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 fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae

Summary The fimD gene of Escherichia coli K12 was shown to be necessary for surface localization of type 1 fimbriae, since deletion of the gene resulted in a virtually bald phenotype. The FimD protein was found to be located in the outer membrane. Expressed alone, this protein had a very deleterious effect on cell growth. The DNA sequence of the fimD gene was determined; the corresponding amino acid sequence of the FimD protein was compared with those of the PapC and FaeD proteins. A deletion derivative of FimD gave clues as to which parts of the protein were necessary for outer membrane integration.     

Bordet‐Gengou agar medium supplemented with albumin‐containing biologics for cultivation of bordetellae

Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections in mammals, including humans, and are generally cultivated on Bordet‐Gengou (BG) agar plates in laboratories. The medium requires animal blood as a supplement for better bacterial growth. However, using blood is problematic, as its constant supply is occasionally difficult because of the limited shelf‐life. This study proposes modified BG agar plates supplemented with bovine serum albumin and fetal bovine serum as a simple and convenient medium that confers sufficient growth of bordetellae.     

Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone–usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.     

一株支气管炎鲍特杆菌国家标准菌种的再鉴定

通过生化试验、质谱鉴定、16S rRNA基因序列分析、全基因组序列测定及生物信息学分析等方法对国家标准菌种CMCC(B)40001进行再鉴定。对菌株的毒力、耐药和MLST进行分析，并对鲍特杆菌（Bordetella spp.）的群体进化进行分析。结合不同方法，CMCC(B)40001再鉴定为支气管炎鲍特杆菌（Bordetella bronchiseptica），且其作为标准菌种能够发挥国家标准描述的质控作用。同时获取该菌株的新ST型，在进化关系上，其与百日咳鲍特杆菌(Bordetella pertussis)和副百日咳鲍特杆菌(Bordetella parapertussis)遗传距离近。本研究再鉴定了国家标准菌种CMCC(B)40001为支气管炎鲍特杆菌，并分析了其遗传特征，为国家标准菌种的应用提供资源描述支持。     

O Antigen Allows B. parapertussis to Evade B. pertussis Vaccine–Induced Immunity by Blocking Binding and Functions of Cross-Reactive Antibodies

Although the prevalence of Bordetella parapertussis varies dramatically among studies in different populations with different vaccination regimens, there is broad agreement that whooping cough vaccines, composed only of B. pertussis antigens, provide little if any protection against B. parapertussis. In C57BL/6 mice, a B. pertussis whole-cell vaccine (wP) provided modest protection against B. parapertussis, which was dependent on IFN-γ. The wP was much more protective against an isogenic B. parapertussis strain lacking O-antigen than its wild-type counterpart. O-antigen inhibited binding of wP–induced antibodies to B. parapertussis, as well as antibody-mediated opsonophagocytosis in vitro and clearance in vivo. aP–induced antibodies also bound better in vitro to the O-antigen mutant than to wild-type B. parapertussis, but aP failed to confer protection against wild-type or O antigen–deficient B. parapertussis in mice. Interestingly, B. parapertussis–specific antibodies provided in addition to either wP or aP were sufficient to very rapidly reduce B. parapertussis numbers in mouse lungs. This study identifies a mechanism by which one pathogen escapes immunity induced by vaccination against a closely related pathogen and may explain why B. parapertussis prevalence varies substantially between populations with different vaccination strategies.     

Representational difference analysis identifies a strain-specific LPS biosynthesis locus in Bordetella spp.

Bordetella pertussis and B. bronchiseptica are genetically very closely related but differ significantly in their virulence properties. Using Representational Difference Analysis (RDA), 11 DNA fragments specific for B. pertussis Tohama I or B. bronchiseptica BB7865 were identified. All B. bronchiseptica BB7865-derived fragments also hybridized with chromosomal DNA from B. parapertussis but not from the B. pertussis strains Tohama I and W28, underlining the close phylogenetic relationship between B. bronchiseptica and B. parapertussis. The B. pertussis type strain BP18323 is a special case, as it contains DNA sequences characteristic for both B. pertussis and B. bronchiseptica. As demonstrated by pulsed-field gel electrophoresis, several of the BB7865-derived fragments are present on a single 30-kb XbaI fragment. Based on the sequences of putative coding regions, four of these fragments may code for proteins involved in carbohydrate metabolism or transport. In agreement with this notion, a mutant for one of these loci synthesizes a significantly altered lipopolysaccharide that lacks the O-specific side chains. The analysis of the corresponding genomic region in various Bordetella species showed that this locus is present in B. bronchiseptica and B. parapertussis but not in B. pertussis. This confirms that the RDA approach has identified a novel strain-specific LPS biosynthesis locus which accounts for the differences between the LPS structures elaborated by different Bordetella species.