Detection of Bordetella bronchiseptica strains containing the main agglutinogens of all species of the genus Bordetella

The study of 26 B. bronchiseptica strains with typical morphological and biochemical properties resulted in the detection of 8 strains having the main specific agglutinogens of 3 Bordetella species (serovars) in different combinations. The presence of the agglutinogens was confirmed in the agglutination test and the agglutinin adsorption test with the use of monospecific antisera to the main agglutinogens. The comparison of natural B. bronchiseptica serovars and artificial convertants (resulting from the conversion of B. parapertussis by B. pertussis phages) revealed their identical biochemical activity, their capacity for causing necrosis when injected intradermally into rabbits and for the formation of two types of colonies, differing in size and serological activity. In contrast to B. parapertussis convertants, B. bronchiseptica serovars had no lysogenic properties and were sensitive to B. pertussis and B. bronchiseptica phages.     

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.     

Transparent solid nutrient medium for studying the lytic spectrum of bacteriophages of microbes of the genus Bordetella

A solid, transparent culture medium for the study of the lytic spectrum of the phages, active against B. pertussis and B. bronchiseptica, in respect to homologous and heterologous bacteria of the genus Bordetella has been developed. The Cohen-Wheeler liquid medium with nicotinic acid and nicotinamide added, solidified with agar, is nicotinamide added, solidified with agar, is used as the base of the new medium. This base ensures the growth of B. parapertussis and B. bronchiseptica. To stimulate the growth of B. pertussis, the tissue stimulant of B. pertussis growth (a transparent substrate obtained from the tissue of the large intestine of a rabbit) has been used. With 10% of this stimulant added, B. pertussis cells have been found to preserve their typical morphological and immunobiological properties.     

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.     

Role of pertussigen (pertussis toxin) on the mouse protective activity of vaccines made from Bordetella species

Pertussigen [pertussis toxin (Ptx)] from Bordetella pertussis, when detoxified, induces protection in mice to intracerebral challenge (ic) with virulent B. pertussis. In its native form, minute nonprotective doses promote the development of immunity induced by other antigens of B. pertussis. As little as 4 ng of Ptx, given with a nonprotective dose of 8 X 10(7) killed cells of the phase III Sakairi strain, promoted detectable protection to ic challenge. Native Ptx in doses of 0.4 to 400 ng did not protect mice, and vaccines made from strains not producing Ptx induced only weak protection. The marked enhancing action of Ptx was also observed with 5 micrograms of purified filamentous hemagglutinin and with vaccines made from other species of the Bordetella genus, such as B. parapertussis and B. bronchiseptica, but it was not observed with B. pertussis endotoxin. In addition, Ptx was still effective when given as late as 7 days after the vaccine. Antibodies to surface antigens of the challenge strain were demonstrated in sera of mice immunized with vaccines prepared with the different Bordetella species tested, but antibodies to Ptx were detected only in the sera of mice immunized with the wild-type B. pertussis strains. Glutaraldehyde detoxified Ptx does not have this action. Pretreatment of normal mice with Ptx, also enhanced the protective action of a mouse antiserum to a wild-type strain of B. pertussis. These observations show that antigens other than Ptx are responsible for the protection, and that Ptx acts non-specifically to enhance the mouse protective action of those antigens.     

Growth and siderophore production by Bordetella pertussis under iron-restricted conditions

It has been demonstrated that under iron-restricted conditions Bordetella pertussis can obtain iron from iron-saturated human transferrin. Direct contact between B. pertussis and transferrin was not required as B. pertussis was able to acquire iron from transferrin when they were separated by a dialysis membrane. Siderophore activity was detected in supernatants from iron-restricted cultures of B. pertussis, B. bronchiseptica and B. parapertussis. Siderophores were identified as hydroxamates and were produced by both virulent and avirulent strains of B. pertussis.     

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.     

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.     

Bordetella pertussis bacteriophage

For the first time Bordetella pertussis bacteriophage was isolated, and its presence was confirmed by electron microscopy and by agar layer titration. The lysogenic strains were activated by their treatment with mitomycin C in a dose of 4.5 mg/ml. The phage system of the Bordetella genus, heretofore unknown, has been revealed: Bordetella pertussis phage lyzed all the tested strains of Bordetella parapertussis (25 strains) and could be passaged in these strains. The phage formed turbid and transparent negative colonies 0.1 mm and 0.15 mm in size. The phage titer (e. g., in strain No. 3865) was 1 X 10(10). The lysogenic variants of Bordetella pertussis, capable of spontaneous release of the phage, were obtained. These variants were characterized by changes in some of their phenotypical properties, e.g., the increased content of certain toxic substances and increased virulence.     

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.     

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.     

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.     

bvg Repression of alcaligin synthesis in Bordetella bronchiseptica is associated with phylogenetic lineage.

Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mM MgSO4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated.     

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.     

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.     

A genome-wide screen identifies a Bordetella type III secretion effector and candidate effectors in other species

Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone-effector locus, btcA-bteA, and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.     

Examination of the genus Bordetella for specific human IgA protease activity

Abstract Three laboratory strains and 3 clinical isolates of Bordetella pertussis were found to have no IgA protease activity when incubated with radio-labelled IgA. In addition, no IgA protease activity was detected in the Second British Reference Preparation for Pertussis Vaccine, an acellular vaccine produced in Japan, or one strain each of Bordetella parapertussis and Bordetella bronchiseptica .