Characteristic lipids of Bordetella pertussis: simple fatty acid composition, hydroxy fatty acids, and an ornithine-containing lipid.

The lipids and fatty acids of Bordetella pertussis (phases I to IV) were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry and compared with those of B. parapertussis and B. bronchiseptica. The major lipid components of the three species were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, lysophosphatidylethanolamine, and an ornithine-containing lipid. The ornithine-containing lipid was characteristic of the genus Bordetella. The fatty acid composition of the total extractable cellular lipids of B. pertussis was mostly hexadecanoic and hexadecenoic acids (90%) in a ratio of about 1:1. The hexadecenoic acid of B. pertussis was in the cis-9 form. The fatty acid composition of the residual bound lipids was distinctly different from that of the extractable lipids, and residual bound lipids being mainly 3-hydroxytetradecanoic, tetradecanoic, and 3-hydroxydecanoic acids, with 3-hydroxydodecanoic acid occurring in some strains. It was determined that the 3-hydroxy fatty acids were derived from lipid A. The fatty acid composition of the total extractable cellular lipids of B. parapertussis and B. bronchiseptica, mainly composed of hexadecanoic and heptadecacyclopropanoic acid, differed from that of B. pertussis. Although the fatty acid composition of the residual bound lipids of B. parapertussis was similar to that of the residual bound lipids of B. pertussis, 2-hydroxydodecanoic acid was detected only in the bound lipids of B. bronchiseptica.     

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.     

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.     

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.     

Histamine-sensitizing Factor, Mouse-protective Antigens, and Other Antigens of Some Members of the Genus Bordetella

The three species of the genus Bordetella-B. pertussis, B. parapertussis, and B. bronchiseptica-have many antigens in common. Studies on representative strains of these species have shown that there are only a few specific antigens in each species. Whole-cell vaccines and extracts from B. pertussis contained specific mouse-protective antigen and a histamine-sensitizing factor. In addition, whole-cell vaccines and some saline extracts protected mice against intracranial challenge with B. bronchiseptica. Cells and a saline extract of B. parapertussis also protected against B. bronchiseptica but not against B. pertussis. Whole cells of B. bronchiseptica protected against B. bronchiseptica, but only one of three saline extracts protected against this challenge. Neither whole cells nor saline extracts from B. bronchiseptica protected against B. pertussis. The antigen in B. pertussis responsible for cross-protection against B. bronchiseptica was less resistant to heat than the protective antigen in B. bronchiseptica. Since histamine-sensitizing factor was not detected in B. bronchiseptica or B. parapertussis cells or extracts, this factor is not required to protect mice against B. bronchiseptica challenge. Whether B. pertussis vaccines protected against B. bronchiseptica by a nonspecific mechanism was not established, but it is clear that the specific antigen responsible for protection against B. pertussis was found only in B. pertussis and not in B. bronchiseptica or B. parapertussis.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

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.     

Characterisation of major biological properties of Bordetella bacteriophages

The main biological properties (morphology of negative colonies, parameters of adsorption and single development cycle) of B. pertussis and B. bronchiseptica phages, isolated spontaneously and by induction with mitomycin C, were studied. To compare these characteristics, one B. parapertussis indicator strain was used, and the experiments were carried out under identical conditions. Highly active sera were obtained with the use of complete Freund's adjuvant. B. pertussis phages isolated from the strains of different serovars were serologically related, but not identical, and differed in their constant characterizing their rate of neutralization with homologous antisera. The adsorption of the phages on homologous strains was more intensive than on the cells of B. parapertussis indicator strain. However, the authors failed to observe the further development of the phages in the host cells.     

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.     

Chemical characterization of effective and ineffective strains of Rhizobium leguminosarum bv. viciae

Chemical composition of lipopolysaccharide (LPS) isolated from an effective (97) and ineffective (87) strains of R. l. viciae has been determined. LPS preparations from the two strains contained: glucose, galactose, mannose, fucose, arabinose, heptose, glucosamine, galactosamine, quinovosamine, and 3-N-methyl-3,6-dideoxyhexose, as well as glucuronic, galacturonic and 3-deoxyoctulosonic acid. The following fatty acids were identified: 3-OH 14:0, 3-OH 15:0, 3-OH 16:0, 3-OH 18:0 and 27-OH 28:0. The ratio of 3-OH 14:0 to other major fatty acids in LPS 87 was higher that in LPS 97. SDS/PAGE profiles of LPS indicated that, in lipopolysaccharides, relative content of S form LPS I to that of lower molecular mass (LPS II) was much higher in the effective strain 97 than in 87. All types of polysaccharides exo-, capsular-, lipo, (EPS, CPS, LPS, respectively) examined possessed the ability to bind faba bean lectin. The degree of affinity of the host lectin to LPS 87 was half that to LPS 97. Fatty acids (FA) composition from bacteroids and peribacteroid membrane (PBM) was determined. Palmitic, stearic and hexadecenoic acids were common components found in both strains. There was a high content of unsaturated fatty acids in bacteroids as well as in PBM lipids. The unsaturation index in the PBM formed by strain 87 was lower than in the case of strain 97. Higher ratio of 16:0 to 18:1 fatty acids was characteristic for PMB of the ineffective strain.     

Differentiation between B. pertussis and B. parapertussis according to their fatty acid composition

The fatty acid composition of 3 B. pertussis strains and 2 B. parapertussis strains grown on casein-carbon agar (CCA) with 8% of sheep blood added and without blood, as well as B. parapertussis strain grown on beef-extract agar (BEA) has been studied by gas chromatography. The fatty acid profiles characteristic of B. pertussis and B. parapertussis were greatly different, as B. parapertussis has a considerable amount of methylene-hexadenocanoic acid, while containing less hexadecenoic and octadecanoic acids and more tetradecanoic acid. The fatty acid composition of 2- to 5-day Bordetella cultures grown on CCA with and without blood has no essential differences. Differences in the content of various fatty acids in B. parapertussis grown on CCA and BEA had no essential influence on the fatty acid profile. The specificity of the fatty acid composition of B. pertussis and B. parapertussis allows to use this characteristics for their differentiation.     

Lipopolysaccharides from Mesorhizobium huakuii and Mesorhizobium ciceri: chemical and immunological comparative data

Lipopolysaccharides of two Mesorhizobium species of different host specificity were compared: M. huakuii and M. ciceri. M. huakuii sp. was represented by five strains with special consideration of M. huakuii IFO 15243(T). SDS/PAGE profiles revealed that all M. huakuii LPS preparations contained low molecular mass fractions (LPS-II) of the same molecular size. All of lipopolysaccharides contained high molecular mass fractions (LPS-I). However, the high molecular mass fraction from each strain possessed an individual molecular size distribution pattern. The crossreactivity of blotted lipopolysaccharides with rabbit polyclonal antibodies against Mesorhizobium huakuii IFO 15243(T) whole bacteria indicated the presence of common epitope(s) within the investigated Mesorhizobium huakuii strains. Moreover, LPS from M. huakuii S52 also reacted with anti M. ciceri HAMBI 1750 serum showing that there are epitopes common for different mesorhizobial species. LPS isolated from Mesorhizobium huakuii strain IFO 15243(T) contained neutral sugars: L-6-deoxytalose, L-rhamnose, D-galactose and D-glucose, aminosugars:D-quinovosamine, D-glucosamine, D-2,3-diamino-2,3-dideoxyglucose and D-galacturonic and D-glucuronic acids. In the LPS preparation, fatty acids typical for Mesorhizobium strains were detected. 3-Hydroxydodecanoic, 3-hydroxy-iso-tridecanoic, 3-hydroxyeicosanoic, 3-hydroxyheneicosanoic and 3-hydroxydocosenoic acids were the major amide linked fatty acids, while iso -heptadecanoic, eicosanoic, docosenoic, as well as 27-hydroxyoctacosanoic and 27-oxooctacosanoic acids were the dominant ester linked fatty residues.     

Genetic diversity analysis of isolates belonging to Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species

In this study, Amplified Fragment Length Polymorphism (AFLP) method was used to track differences among human and animal isolates of B. pertussis, B. parapertussis and B. bronchiseptica species. One hundred and sixty representative strains of these species orginated from international and Polish bacterial collections were genotyped according to AFLP involving EcoRI/Msel and SpeI/ApaI restriction/ligation/amplification procedures. This study has confirmed high potential AFLP SpeI/ApaI procedure for intra-species differentiation of B. pertussis and B. bronchiseptica strains. Both AFLP EcoRI/MseI and SpeI/ApaI procedures have been found to be useful for species-specific classification in case of B. pertussis strains. In case of B. bronchiseptica or B. parapertussis species-specific classification, SpeI/ApaI procedure has been found more precise than EcoRI/MseI one.     

Analysis of fatty acids from lipopolysaccharides of Bacteroides thetaiotaomicron and Bacteroides fragilis]

The aim of this study was to determine and to compare fatty acids occurring in lipopolysaccharides (LPS) isolated from B. thetaiotaomicron and B. fragilis strains of different origin. Lipopolysaccharides of three B. thetaiotaomicron strains and four B. fragilis strains were isolated by phenol-water extraction according to the procedure of Westphal and Jann (1965). Water-phase LPS fractions were then treated with nucleases and purified by ultracentrifugation as described by Gmeiner (1975). Fatty acid methyl esters, obtained by methanolysis of LPS, were analysed in gas-liquid chromatography combined with mass spectrometry (GLC-MS). Trimethylsilylated hydroxyl groups of fatty acid methyl esters were identified with GLC-MS using a method of selective ion monitoring (SIM). Lipopolysaccharides of B. thetaiotaomicron and B. fragilis strains contained long-chain (15-18 carbon atoms) fatty acids. The broad spectrum of simple long-chain and branched-chain fatty acids as well as 3-hydroxy fatty acids were detected. The main fatty acid of analyzed bacterial species was 3-hydroxy-hexadecanoic acid (3OH C16:0). Several 3-hydroxy fatty acids were detected in LPS of examined strains. Fatty acids occurring in LPS of B. thetaiotaomicron and B. fragilis strains appeared to be qualitatively similar. Quantitative differences in fatty acids composition of lipopolysaccharides isolated from strains of different origin were observed.     

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.     

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.     

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.     

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.     

IgA protease activity of microbes in the genus Bordetella

Monoclonal IgA paraproteins of subclasses 1 and 2, isolated from the sera of myeloma patients, were incubated for 4, 24, 48 and 72 hours with B. pertussis, B. parapertussis, B. bronchiseptica cultures, as well as Haemophilus influenzae strain. The fragmentation of IgA was studied by immunielectrophoresis with antisera to alpha-chain, to Fab alpha + Fc alpha, to Fab alpha and with antisera to light chains corresponding to the type of paraprotein. B. pertussis and B. parapertussis were found to have subclass-unspecific IgA protease which splitted off a cathode fragment, similar to Fab-fragment and, probably, corresponding to the variable domain of alpha-chain (Fv), after 48-hour incubation. Similar IgA protease was detected in H. influenzae, found to have classical IgA1 protease as well. All Bordetella species under study splitted off anode components from IgA paraproteins of both subclasses. These components, containing the determinants of heavy and light IgA chains, were either IgA - alpha I-antitrypsin complexes or some IgA fragments with high electrophoretic motility. None of the strains under study splitted monoclonal IgG.     

Lipopolysaccharides from Bordetella pertussis and Bordetella parapertussis differently modulate human dendritic cell functions resulting in divergent prevalence of Th17-polarized responses

Bordetella pertussis and B. parapertussis are the etiological agents of pertussis, yet the former has a higher incidence and is the cause of a more severe disease, in part due to pertussis toxin. To identify other factors contributing to the different pathogenicity of the two species, we analyzed the capacity of structurally different lipooligosaccharide (LOS) from B. pertussis and LPS from B. parapertussis to influence immune functions regulated by dendritic cells. Either B. pertussis LOS and B. parapertussis LPS triggered TLR4 signaling and induced phenotypic maturation and IL-10, IL-12p40, IL-23, IL-6, and IL-1beta production in human monocyte-derived dendritic cells (MDDC). B. parapertussis LPS was a stronger inducer of all these activities as compared with B. pertussis LOS, with the notable exception of IL-1beta, which was equally produced. Only B. parapertussis LPS was able to induce IL-27 expression. In addition, although MDDC activation induced by B. parapertussis LPS was greatly dependent on soluble CD14, B. pertussis LOS activity was CD14-independent. The analysis of the intracellular pathways showed that B. parapertussis LPS and B. pertussis LOS equally induced IkappaBalpha and p38 MAPK phosphorylation, but B. pertussis LOS triggered ERK1/2 phosphorylation more rapidly and at higher levels than B. parapertussis LPS. Furthermore, B. pertussis LOS was unable to induce MyD88-independent gene induction, which was instead activated by B. parapertussis LPS, witnessed by STAT1 phosphorylation and induction of the IFN-dependent genes, IFN regulatory factor-1 and IFN-inducible protein-10. These differences resulted in a divergent regulation of Th cell responses, B. pertussis LOS MDDC driving a predominant Th17 polarization. Overall, the data observed reflect the different structure of the two LPS and the higher Th17 response induced by B. pertussis LOS may contribute to the severity of pertussis in humans.