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.     

A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens

The Bordetella BvgAS signal-transduction system has traditionally been viewed as mediating a transition between two distinct phenotypic phases: the Bvg⁺ phase, characterized by the expression of adhesins and toxins, and the Bvg⁻ phase, characterized by motility in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis . In B. bronchiseptica , the Bvg⁺ phase is necessary and sufficient for respiratory tract colonization whereas the Bvg⁻ phase is required for growth under nutrient-limiting conditions. This report describes the characterization of a mutant that is locked in a Bvg-intermediate (Bvgⁱ) phase. The mutation conferring this phenotype, designated bvgS -I1, results in a threonine-to-methionine substitution near the primary site of phosphorylation in BvgS. Compared to its Bvg⁺-phase-locked parent, the Bvgⁱ mutant displays increased resistance to nutrient limitation and reduced virulence. Molecular analyses indicate that the mutant has lost the ability to express a subset of Bvg⁺-phase factors and has gained the ability to express factors unique to the Bvgⁱ phase. Although identified by mutation, this work indicates that the Bvgⁱ phase is expressed by wild-type B. bronchiseptica in response to certain (semi-modulating) environmental conditions. The identification of Bvgⁱ-specific antigens suggests the existence of a new class of Bvg-regulated genes. We hypothesize that BvgAS is capable of mediating the expression of a spectrum of phenotypic phases in response to the various environments encountered as Bordetella travels within and between mammalian hosts.     

Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetella pertussis.

The ptl locus of Bordetella pertussis contains eight open reading frames which are predicted to encode proteins (PtlA to PtlH) that are essential for secretion of pertussis toxin from the bacterium and which are members of a family of transport proteins found in other types of bacteria. We have detected PtlE, PtlF, and PtlG in immunoblots of extracts of B. pertussis by using antibodies raised to fusion proteins consisting of maltose-binding protein and the individual Ptl proteins. These proteins have apparent molecular weights similar to those predicted by DNA sequence analysis. Cell fractionation studies indicated that all three Ptl proteins are associated with the membranes of B. pertussis, suggesting that the Ptl proteins form a gate or channel which facilitates transport of pertussis toxin. Cell extracts of other Bordetella spp. were probed with antibodies to Ptl proteins for the presence of these transport proteins. Neither Bordetella parapertussis nor Bordetella bronchiseptica contained detectable levels of PtlE or PtlF. This lack of detectable Ptl protein may provide an explanation for previous observations which indicated that introduction of the genes encoding pertussis toxin subunits from B. pertussis into other Bordetella spp. results in production of the toxin but not secretion of the toxin.     

Genetic diversity and relationships in populations of Bordetella spp

Genetic diversity in 60 strains of three nominal Bordetella species recovered from humans and other mammalian hosts was assessed by analyzing electrophoretically demonstrable allelic variation at structural genes encoding 15 enzymes. Eleven of the loci were polymorphic, and 14 distinctive electrophoretic types, representing multilocus genotypes, were identified. The population structure of Bordetella spp. is clonal, and genetic diversity is relatively limited compared with most other pathogenic bacteria and is insufficient to justify recognition of three species. All isolates of Bordetella parapertussis were of one electrophoretic type, which was closely similar to 9 of the 10 electrophoretic types represented by isolates of Bordetella bronchiseptica. Bordetella pertussis 18-323, which is used in mouse potency tests of vaccines, is more similar genetically to isolates of B. bronchiseptica and B. parapertussis than to other isolates currently assigned to the species B. pertussis. Apart from strain 18-323, the isolates of B. pertussis represented only two closely related clones, and all isolates of B. pertussis from North America (except strain 18-323) were genotypically identical. Strain Dejong, which has been classified as B. bronchiseptica, was strongly differentiated from all of the other Bordetella isolates examined.     

The type III secreted protein BspR regulates the virulence genes in Bordetella bronchiseptica

Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.     

Tracheobronchial washings from seven vertebrate species as growth media for the four species of Bordetella

Abstract The four species of Bordetella differed in their ability to grow at 37°C in membrane-filtered tracheobronchial washings (TBW) from seven vertebrate species, including their natural hosts. From washed inocula of approximately 2×10³ colony-forming units per ml (cfu ml⁻¹), Bordetella bronchiseptica and B. avium grew much better than the other two bordetellae and yielded stationary-phase cultures containing 10⁸−10⁹ cfu ml⁻¹ in most of the TBW samples. These counts were only moderately lower than those attained in CL medium which contains about a 450-times higher concentration of amino acids. B. bronchiseptica and B. avium also grew to a limited extent in phosphate-buffered saline without nutrient supplements. B. parapertussis grew in TBW from man, sheep, rabbit, mouse and chicken, but not in TBW from a dog and a horse or in PBS. B. pertussis grew well in CL medium, but not in PBS or in any of 13 samples of TBW from the seven vertebrate species, which included three samples of lung lavage fluid from human patients. Analysis of the TBW samples for known Bordetella nutrients revealed concentrations of amino acids and nicotinic acid averaging 0.35 mM and 0.56 μg ml⁻ respectively.     

BopC is a novel type III effector secreted by Bordetella bronchiseptica and has a critical role in type III-dependent necrotic cell death

In Bordetella bronchiseptica, the functional type III secretion system (TTSS) is required for the induction of necrotic cell death in infected mammalian cells. To identify the factor(s) involved in necrotic cell death, type III-secreted proteins from B. bronchiseptica were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization tandem mass spectrometry. We identified a 69-kDa secreted protein designated BopC. The gene encoding BopC is located outside of the TTSS locus and is also highly conserved in both Bordetella parapertussis and Bordetella pertussis. The results of a lactate dehydrogenase release assay and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay demonstrated that BopC is required for necrotic cell death. It has been reported that tyrosine-phosphorylated proteins (PY) of host cells are dephosphorylated during B. bronchiseptica infection in a TTSS-dependent manner. We found that BopC is also involved in PY dephosphorylation in infected host cells. It appears that the necrotic cell death triggered by BopC occurs prior to the PY reduction in host cells, because Bordetella-induced cell death was not affected even in the presence of a dephosphorylation inhibitor. Furthermore, a translocation assay showed that the signal sequence for both secretion into culture supernatant and translocation into the host cell is located in 48 amino acid residues of the BopC N terminus. This report reveals for the first time that a novel type III effector, BopC, is required for the induction of necrotic cell death during Bordetella infection.     

Identification of a Bordetella pertussis bvg-regulated porin-like protein.

Bordetella pertussis 18323 produces a bvg-regulated 39.1-kDa porin-like protein, OmpQ. OmpQ had 61% similarity to the major porin of B. pertussis and contains conserved regions common to both the neisserial and enteric porin families. The results of Southern blot analysis indicate that strains of Bordetella parapertussis and Bordetella bronchiseptica but not Bordetella avium contain this gene.     

Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica

Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed.     

Truncated mutants of the colicin A lysis protein are acylated and processed when overproduced

Abstract Bordetella calmodulin-like protein was purified from culture supernatant fluid of B. pertussis, B. parapertussis and B. bronchiseptica by successive chromatography on hydroxyapatite, Toyopearl HW-50F and QAE-Toyopearl 550C columns. The purified calmodulin-like protein appeared to be homogeneous by SDS-polyacrylamide gel electrophoresis. The apparent molecular mass of calmodulin-like protein on SDS-polyacrylamide gel electrophoresis was 10 kDa, which was smaller than bovine brain calmodulin (17 kDa). The purified calmodulin-like protein activated both Bordetella adenylate cyclase and mammalian phosphodiesterase in a Ca²⁺-dependent manner. This activation was inhibited by calmodulin antagonists. The calmodulin-like protein, like calmodulin, was retained by a hydrophobic resin in the presence of Ca²⁺ and eluted by the addition of EDTA. These results indicated that the Bordetella calmodulin-like protein is closely related to calmodulin. As a putative calmodulin the extracellular calmodulin may be involved in Bordetella pathogenesis.     

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.     

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 (Aricò 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.     

Molecular evidence for the lysogenic state of microorganisms belonging to the genus Bordetella and characterization of Bordetella parapertussis temperate bacteriophage 66(2.2)

A new bacteriophage phiK of microorganisms belonging to the genus Bordetella was isolated from cells of the earlier characterized strains 66(2-2) (1 and 2) obtained upon phage conversion of B. parapertussis 17903 cells by B. pertussis bacteriophage phi134. Bacteriophage phiK is identical to previously described Bordetella bacteriophages phiT, phi134, and phi214 in morphology and some biological properties but has a permuted genome different from all other phages. DNA of bacteriophage phiK is not integrated in the chromosome of B. parapertussis 17903, similar to DNA of bacteriophages phiT, phi134, and phi214 that are not integrated into B. pertussis and B. bronchiseptica chromosomes, but may be present in a small part of the bacterial population as linear plasmids. Sequences homologous to DNA of bacteriophage phiK were detected in the chromosome of strain 66(2-2) (1 and 2) and in chromosomes of all tested strains B. pertussis and B. bronchiseptica. Prophage integration in chromosomes of microorganisms of the genus Bordetella may vary in different bacterial strains and species. An assumption about abortive lysogeny of B. parapertussis bacteria for phiK phage and of B. bronchiseptica for closely related phages phiT, phi134, and phi214 has been advanced. The possibility of involvement of B. pertussis insertion sequences in the formation of the chromosomal structure in 66(2-2) convertants and in phage genomes is considered.     

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.     

Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus

Bordetella bronchiseptica is a common respiratory tract pathogen of many mammalian species. Nucleotide sequences from the locus involved in coordinate regulation of B. pertussis virulence factors, vir, were shown to have a high degree of homology to chromosomal DNA from virulent (Vir+) and avirulent (Vir-) strains of B. bronchiseptica. Small deletions, 50 bp to 500 bp, within the vir locus were found in some of the Vir- phase variants. The vir locus and the adjacent 5' portion of the fhaB structural gene were cloned from the parental Vir+ B. bronchiseptica strain on a 23.5 kb BamHI fragment. Restriction enzyme mapping of the cloned B. bronchiseptica vir locus revealed similarities with and differences from the previously cloned B. pertussis vir locus. The cloned B. bronchiseptica vir locus complemented spontaneous Vir- variants of Bordetella pertussis and B. bronchiseptica as well as vir::Tn5 mutants of B. pertussis. Comparison of various functions of the vir loci of B. bronchiseptica and B. pertussis revealed some interesting differences in the coordinate regulation of virulence factors.     

Agglutination of Bordetella species by lectins

Phase I cells of Bordetella pertussis but not those of B. parapertussis, B. bronchiseptica or B. avium were agglutinated by Limulus polyphemus lectin. Most strains of B. pertussis but not those of the other species were also agglutinated by Helix pomatia lectin. In precipitation reactions between lectins and purified Bordetella lipopolysaccharide (LPS) preparations a similar pattern occurred. Lectin agglutination provides a rapid presumptive method for the differentiation of B. pertussis from B. parapertussis and other Bordetella species.     

Adaptive evolution of the Bordetella autotransporter pertactin

The virulence factor pertactin is expressed by the closely related pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Pertactin is an autotransporter involved in adherence of Bordetella species to the lung epithelium of mammalian hosts, and it is an important component of most current acellular pertussis vaccines. These three species produce immunologically distinct pertactin molecules, resulting in a lack of cross-protection against B. parapertussis and probably also against B. bronchiseptica. Variation in pertactin is not only inter-specific, but also occurs between isolates from the same species. Knowledge about codons that are under positive selection could facilitate the development of more broadly protective vaccines. Using different nucleotide substitution models, pertactin genes from B. bronchiseptica, B. parapertussis and B. pertussis were compared, and positively selected codons were identified using an empirical Bayesian approach. This approach yielded 15 codons predicted to be under diversifying selection pressure. These results were interpreted in an immunological context and may help in improving future pertussis vaccines.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

Role of a putative polysaccharide locus in Bordetella biofilm development

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by Bordetella spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc), we provide evidence for the production of poly-beta-1,6-GlcNAc by various Bordetella species (Bordetella bronchiseptica, B. pertussis, and B. parapertussis) and its role in their biofilm development. We have investigated the role of a Bordetella locus, here designated bpsABCD, in biofilm formation. The bps (Bordetella polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-beta-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the bps locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of Bordetella biofilms.     

Bordetella bronchiseptica expresses the fimbrial structural subunit gene fimA.

The differential host species specificities of Bordetella pertussis, B. parapertussis, and B. bronchiseptica might be explained by polymorphisms in adherence factor genes. We have found that B. parapertussis and B. bronchiseptica, unlike B. pertussis, contain a full-length gene for the fimbrial subunit FimA. B. bronchiseptica expresses fimA in a BvgAS-dependent fashion.