Structure of the O-specific polysaccharide chain of the lipopolysaccharide of Bordetella hinzii

The lipopolysaccharide of Bordetella hinzii was analyzed after various chemical degradations by NMR spectroscopy and MALDI mass spectrometry, and the following structure of the polysaccharide chain was determined: 4-O-Me-alpha-GalpNAc3NAcAN-(1-->[-->4)-beta-GlcpNAc3NAcAN-(1-->4)-beta-GlcpNAc3NAcAN-(1-->4)-alpha-GalpNAc3NAcAN-(1-](n)-where GlcNAc3NAcAN and GalNAc3NAcAN stand for 2,3-diacetamido-2,3-dideoxy-glucuronamide and -galacturonamide, respectively. The polysaccharide chain is terminated with a 4-O-methylated GalNAc3NAcAN residue and is rather short (n < or = 5).     

The structure of the core-O-chain linkage region of the lipopolysaccharide from Bordetella hinzii

Linkage region between core and the O-chain of the lipopolysaccharide from Bordetella hinzii has been analyzed by NMR and MS analysis of the products, obtained by anhydrous HF treatment or consecutive ammonia and AcOH treatment of the LPS. The following structure of this region was deduced from the experimental results: [structure: see text] This structure is identical to the structure of the respective region of Bordetella parapertussis LPS. Polysaccharide part (PS) consists of not more than 15 2,3-diacetamido-2,3-dideoxyhexuronamides, methylated at the only hydroxyl group of the non-reducing terminal monosaccharide.     

Complete structures of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharides

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria.     

Genetic diversity analysis of isolates belonging to Bordetella genus

In this study, RandomAmplified Polymorphic DNA (RAPD) method was used to track differences among human and animals isolates of eight known species of Bordetella genus. One hundred representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247. Problems with reproducibility and discriminatory power, frequently discussed in literature, have been overcome by precise optimization procedure, which allowed to achieve reliable conditions for Bordetella species analysed. This study proved high potential of RAPD method using primers 1247/1254 for intra-species differentiation of B. bronchiseptica and B. hinzii isolates. Additionally RAPD method has been found to be useful for confirming B. avium and B. holmesii identification.     

The structure of the nonreducing terminal groups in the O-specific polysaccharides from two strains of Bordetella bronchiseptica.

The structures of the polysaccharide chains of the LPS from Bordetella bronchiseptica strains 110H and Bp512 were analysed by NMR spectroscopy and mass spectrometry. The polysaccharides consist of alpha-(1-4)-linked 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid repeating units. Polysaccharides from both strains have 2,3, 4-triamino-2,3,4-trideoxy-alpha-galacturonamide derivatives at their nonreducing ends, a monosaccharide identified for the first time in nature. The polymers from the two strains differ in the nature of the acylation of the amino groups of this monosaccharide. In the strain 110H, the residue is formylated at positions 3 and 4, and has N-formyl-L-alanyl or L-alanyl substituents at N-2. In the strain Bp512, the amino group at position 2 is acetylated, at position 3 it is formylated, and the amino group at position 4 bears a 2-methoxypropionyl substituent. The distribution of the acyl groups was determined from long range 1H-13C correlation (HMBC) NMR spectra. Measurement of the spectra under different pH conditions showed that carboxyl groups of the inner uronic acid residues of the polymeric chain are free, and that carboxyl groups of the terminal residues are amidated. These conclusions were confirmed by the results of mass spectrometric analysis.     

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.     

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.     

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 bronchiseptica responses to physiological reactive nitrogen and oxygen stresses

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.     

The bvgAS locus negatively controls motility and synthesis of flagella in Bordetella bronchiseptica.

The products of the bvgAS locus coordinately regulate the expression of Bordetella virulence factors in response to environmental conditions. We have identified a phenotype in Bordetella bronchiseptica that is negatively controlled by bvg. Environmental signals which decrease (modulate) the expression of bvg-activated genes lead to flagellum production and motility in B. bronchiseptica. Wild-type (Bvg+) strains are motile and produce peritrichous flagella only in the presence of modulating signals, whereas Bvg- (delta bvgAS or delta bvgS) strains are motile in the absence of modulators. The bvgS-C3 mutation, which confers signal insensitivity and constitutive activation of positively controlled loci, eliminates the induction of motility and production of flagellar organelles. The response to environmental signals is conserved in a diverse set of clinical isolates of both B. bronchiseptica and B. avium, another motile Bordetella species; however, nicotinic acid induced motility only in B. bronchiseptica. Purification of flagellar filaments from B. bronchiseptica strains by differential centrifugation followed by CsCl equilibrium density gradient centrifugation revealed two classes of flagellins of Mr 35,000 and 40,000. A survey of clinical isolates identified only these two flagellin isotypes, and coexpression of the two forms was not detected in any strain. All B. avium strains tested expressed a 42,000-Mr flagellin. Amino acid sequence analysis of the two B. bronchiseptica flagellins revealed 100% identity in the N-terminal region and 80% identity with Salmonella typhimurium flagellin. Monoclonal antibody 15D8, which recognizes a conserved epitope in flagellins in members of the family Enterobacteriaceae, cross-reacted with flagellins from B. bronchiseptica and B. avium. Our results highlight the biphasic nature of the B. bronchiseptica bvg regulon and provide a preliminary characterization of the Bvg-regulated motility phenotype.     

Biological properties of lipopolysaccharides from Bordetella species

Biological activities of lipopolysaccharides (LPS) extracted from Bordetella pertussis, B. parapertussis and B. bronchiseptica were compared with those of Escherichia coli LPS. The LPS preparations from B. pertussis showed biological activities comparable to those of E. coli LPS in terms of lethal toxicity in galactosamine-sensitized mice, pyrogenicity in rabbits, mitogenicity in C3H/He spleen cell cultures, macrophage activation, and induction of tumour necrosis factor. All the activities of LPS preparations from B. parapertussis, except mitogenicity, were lower than those of E. coli LPS. LPS from B. parapertussis gave the greatest mitogenic action of all those tested. Biological activities stronger than or comparable to those of E. coli LPS were observed for LPS from B. bronchiseptica.     

Efficacy of commercial vaccines for protecting guinea pigs against Bordetella bronchiseptica pneumonia

Autogenous bacterins are recommended to protect guinea pigs (Cavia porcellus) against pneumonia due to Bordetella bronchiseptica. Bordetella vaccines are available commercially for several other animal species. The substantial antigenic cross-reactivity among Bordetella isolates from various animal species suggests that immunity resulting from use of these vaccines might protect guinea pigs. Groups of ten individually housed Hartley guinea pigs from a colony free of Bordetella were vaccinated with one of two commercial porcine B. bronchiseptica vaccines, a human DPT vaccine (which includes a Bordetella pertussis component), or an autogenous B. bronchiseptica bacterin. Twenty-one days following vaccination, the animals were challenged with an intranasal dose of 10(6) virulent B. bronchiseptica cells. The animals were euthanized and necropsied 15 days after challenge. The nares, nasopharynx, distal trachea and lungs were cultured. All nonvaccinated control animals developed acute signs of pneumonia, while none of the vaccinated animals developed clinical signs of disease or gross lesions. The frequency of B. bronchiseptica isolation from the lungs of animals in each vaccine group was reduced. However, approximately 70% of all animals in each vaccine group harbored B. bronchiseptica in the trachea, and almost all harbored B bronchiseptica in the nares and nasopharynx. The porcine vaccines appeared to afford protection against acute pulmonary disease in the guinea pig.     

Iron starvation regulates the type III secretion system in Bordetella bronchiseptica

The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.     

Limitation of phage multiplication by microbes of the genus Bordetella

Possible causes limiting the multiplication of Bordetella phages or inducing their restriction, such as the influence of lysogenic immunity and the restriction-modification (R-M) system or the incompatibility of the receptor apparatus, have been studied. The limitation of the multiplication of phages by some B. bronchiseptica and B. pertussis strains has been shown to be due to the presence of the R-M system and lysogenic immunity. In five B. bronchiseptica strains and two B. pertussis strains site-specific endonucleases (restrictases) with Hind III specificity have been detected. One B. bronchiseptica strain without the R-M system has been detected. B. bronchiseptica strains producing site-specific endonucleases are practically nonpathogenic for humans, grow in common culture media and selectively produce only one restrictase, type Hind III, which guarantees from the admixture of other specific endonucleases. The B. parapertussis strains under study (altogether 100 strains) have not been found to limit the multiplication of Bordetella test phages. The absence of site-specific endonucleases has also been confirmed biochemically. These strains are recommended as indicator strains for the multiplication of Bordetella phages.     

Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium

We discovered and characterized a temperate transducing bacteriophage (Ba1) for the avian respiratory pathogen Bordetella avium. Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B. avium for lysogeny. Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further. With regard to host range, Ba1 grew on six of nine clinical isolates of B. avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis. Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail. Ba1 readily lysogenized our laboratory B. avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection. DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons. Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized. Transduction frequencies ranged from approximately 2 x 10(-7) to 1 x 10(-8) transductants/PFU depending upon the marker being transduced. UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process. Ba1 may prove to be a useful genetic tool for studying B. avium virulence factors.     

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.     

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.