Bordetella holmesii Lipopolysaccharide Hide and Seek Game with Pertussis: Structural Analysis of the O-Specific Polysaccharide and the Core Oligosaccharide of the Type Strain ATCC 51541.

Whooping cough is a highly contagious disease caused predominantly by Bordetella pertussis, but it also comprises of a pertussis-like illness caused by B. holmesii. The virulence factors of B. holmesii and their role in the pathogenesis remain unknown. Lipopolysaccharide is the main surface antigen of all Bordetellae. Data on the structural features of the lipopolysaccharide (LPS) of B. holmesii are scarce. The poly- and oligosaccharide components released by mild acidic hydrolysis of the LPS were separated and investigated by 1H and 13C NMR spectroscopy, mass spectrometry, and chemical methods. The structures of the O-specific polysaccharide and the core oligosaccharide of B. holmesii ATCC 51541 have been identified for the first time. The novel pentasaccharide repeating unit of the B. holmesii O-specific polysaccharide has the following structure: {→2)-α-l-Rhap-(1→6)-α-d-Glcp-(1→4)-[ß-d-GlcpNAc-(1→3]-α-d-Galp-(1→3)-α-d-GlcpNAc-(1→}n. The SDS-PAGE and serological cross-reactivities of the B. holmesii LPS suggested the similarity between the core oligosaccharides of B. holmesii ATCC 51541 and B. pertussis strain 606. The main oligosaccharide fraction contained a nonasaccharide. The comparative analysis of the NMR spectra of B. holmesii core oligosaccharide fraction with this of the B. pertussis strain 606 indicated that the investigated core oligosaccharides were identical.

A comparative study of the complete lipopolysaccharide structures and biosynthesis loci of Bordetella avium, B. hinzii, and B. trematum.

A dozen species of human and animal pathogens have been described to date in the Bordetella genus, with the majority being respiratory tract pathogens. Bordetella avium lipopolysaccharides have been shown to be important virulence factors for this bird pathogen. B. hinzii is closely related to the B. avium species, but has also been isolated from humans. B. trematum is associated to ear and blood infections in humans. Its lipid A structure, the biological active moiety of LPS, was found to be closely related to those of B. avium and B. hinzii. It is important to unveil the subtle structural modifications orchestrated during the LPS biosynthetic pathway to better understand host adaptation. The present data are also important in the context of deciphering the virulence pathways of this important genus containing the major pathogens B. pertussis and B. parapertussis, responsible for whooping cough. We recently reported the isolated lipid A structures of the three presented species, following the previously identified O-chain structures. In the present study, we provide details on the free and O-chain-linked core oligosaccharides which were required to characterize the complete LPS structures. Data are presented here in relation to relevant biosynthesis genes. The present characterization of the three species is well illustrated by Matrix Assisted Laser Desorption Mass Spectrometry experiments, and data were obtained mainly on native LPS molecules for the first time.

Mechanism of single- and double-sided inhibition of dual topology fluoride channels by synthetic monobodies.

The Fluc family of proteins comprises small, electrodiffusive fluoride channels, which prevent accumulation of toxic F- ions in microorganisms. Recent crystal structures have confirmed their unusual architecture, in which a pair of antiparallel subunits convenes to form a dimer with a twofold symmetry axis parallel to the plane of the membrane. These structures have also revealed the interactions between Fluc channels and several different fibronectin domain monobodies that inhibit Fluc-mediated F- currents; in all structures, each channel binds to two monobodies symmetrically, one on either side of the membrane. However, these structures do not reveal the mechanism of monobody inhibition. Moreover, the results appear to diverge from a recent electrophysiological study indicating that monobody binding is negatively cooperative; that is, a bound monobody on one side of a Fluc channel decreases the affinity of an oppositely bound monobody by ∼10-fold. In this study, we reconcile these observations by probing the mechanism of monobody binding and its negative cooperativity using electrophysiological experiments in planar lipid bilayers. Our results indicate that monobody inhibition occurs via a pore-blocking mechanism and that negative cooperativity arises from electrostatic repulsion between the oppositely bound monobodies. A single glutamate residue, on a loop of the monobody that extends into the channel interior, is responsible for negatively cooperative binding. This glutamate side chain also confers voltage dependence and sensitivity to the concentration of trans-F- ion to monobody binding. Neutralization by mutation to glutamine abolishes these electrostatic effects. Monobodies that are amenable to cocrystallization with Fluc channels lack an analogous negatively charged side chain and bind independently to opposite sides of the channel. Thus, this work reveals the source of voltage dependence and negative cooperativity of monobody binding to Fluc channels along with the pore-blocking mechanism.

Structure activity characterization of Bordetella petrii lipid A, from environment to human isolates.

Bordetella petrii, a facultative anaerobic species, is the only known member of the Bordetella genus with environmental origin. However it was also recently isolated from humans. The structures of the B. petrii lipid A moieties of the endotoxins were characterized here for the first time for an environmental strain and compared to that of human isolates. Characterization was achieved using chemical analyses, gas chromatography-mass spectrometry, and Matrix Assisted Laser Desorption Ionisation mass spectrometry. The analyses revealed that the different lipid A structures contain a common bisphosphorylated ß-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide as well at the C-3' in ester linkages. Similar to Bordetella pertussis and Bordetella bronchiseptica lipids A, the hydroxytetradecanoic acid at the C-2' position was substituted by tetradecanoic acid. Unlike B. pertussis, the hydroxytetradecanoic acid at the C-2 position was substituted with either 12:0 or 14:0 and/or their 2-OH forms. Depending on the environmental or human origin the structures differed in the length and degree of fatty acid acylation and impacted the IL-6 and TNF-α inflammatory responses tested. In one isolate we showed the presence at the C-3 position of the short-chain 10:0(3-OH), which according to our previous analyses is more characteristic of the human pathogens in the genus like B. pertussis and Bordetella parapertussis.

Extracellular polysaccharide from Bordetella species reduces high glucose-induced macrophage apoptosis via regulating interaction between caveolin-1 and TLR4.

Microphage apoptosis is a critical event in atherosclerotic lesions in patients with diabetes. In the present investigation, high glucose treatment inhibited Akt phosphorylation and activated caspase 3 in primary peritoneal macrophage, leading to cell apoptosis. Hypoxia prolonged macrophage survival in high glucose condition. Extracellular polysaccharide from Bordetella species (EPS) further decreased cell apoptosis in response to high glucose during hypoxia. Under high glucose and hypoxic condition, EPS treatment promoted caveolin-1 phosphorylation by recognizing TLR4. Caveolin-1 phosphorylation elevated membrane Glut1 level to accelerate glucose consumption, which should be the reason for protective effect of EPS on macrophage exposed to high glucose. Further investigation demonstrated that TLR4-dependent caveolin-1 phosphorylation induced by EPS promoted association of caveolin-1 with TLR4, which should be critical for activation of TLR4 signaling pathway.

Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

Characterization of the N-terminal domain of BteA: a Bordetella type III secreted cytotoxic effector.

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s) and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA) in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287) and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR) experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.

High yield recombinant expression, characterization and homology modeling of two types of cis-epoxysuccinic acid hydrolases.

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form D: (-)-tartaric acid or L: (+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[D: ] and CESH[L: ], producing D: (-)- and L: (+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver-Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[L: ] showed much higher catalytic efficiency than CESH[D: ], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[L: ] and CESH[D: ] have different structural folds and potential active site residues. CESH[L: ] adopted a typical α/ß-hydrolase fold with a cap domain and a core domain, whereas CESH[D: ] possessed a unique TIM barrel fold composed of 8 α-helices and 8 ß-strands, and 2 extra short α-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[D: ], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.

Structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry.

Mass spectrometric studies are now playing a leading role in the elucidation of lipopolysaccharide (LPS) structures through the characterization of antigenic polysaccharides, core oligosaccharides and lipid A components including LPS genetic modifications. The conventional MS and MS/MS analyses together with CID fragmentation provide additional structural information complementary to the previous analytical experiments, and thus contribute to an integrated strategy for the simultaneous characterization and correct sequencing of the carbohydrate moiety.

Genomic analysis of the adenylate cyclase-hemolysin C-terminal region of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica.

Adenylate cyclase-hemolysin plays an important role in the virulence of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species. Its C-terminal region carries protective epitopes and receptor binding site for human cells. Genomic analyses of this region indicate no polymorphism in B. pertussis and B. parapertussis regions, but substantial variability in B. bronchiseptica that might be linked to the various niches of this species.

Bordetella hinzii septicemia in association with Epstein-Barr virus viremia and an Epstein-Barr virus-associated diffuse large B-cell lymphoma.

We report the 1st case of Bordetella hinzii septicemia associated with Epstein-Barr virus viremia and lymphoma. B. hinzii identification necessitated cellular fatty acid analysis by gas-liquid chromatography and 16S rRNA gene sequencing. Isolates were resistant to many antimicrobials. Resistance and diagnostic challenges complicated management and contributed to mortality.

RTX cytotoxins recognize beta2 integrin receptors through N-linked oligosaccharides.

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated beta(2) integrin receptor CD11b/CD18 (alpha(M)beta(2), Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosidase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharide complex, whereas saccharides absent from integrin oligosaccharide chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the beta(2) integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli alpha-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharide chains of beta(2) integrin receptors. This sets a new paradigm for action of RTX cytotoxins.

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.

Metabolism of 4-amino-3-hydroxybenzoic acid by Bordetella sp. strain 10d: A different modified meta-cleavage pathway for 2-aminophenols.

Bordetella sp. strain 10d metabolizes 4-amino-3-hydroxybenzoic acid via 2-hydroxymuconic 6-semialdehyde. Cell extracts from 4-amino-3-hydroxybenzoate-grown cells showed high NAD(+)-dependent 2-hydroxymuconic 6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, 4-oxalocrotonate decarboxylase, and 2-oxopent-4-enoate hydratase activities, but no 2-hydroxymuconic 6-semialdehyde hydrolase activity. These enzymes involved in 4-amino-3-hydroxybenzoate metabolism were purified and characterized. When 2-hydroxymuconic 6-semialdehyde was used as substrate in a reaction mixture containing NAD(+) and cell extracts from 4-amino-3-hydroxybenzoate-grown cells, 4-oxalocrotonic acid, 2-oxopent-4-enoic acid, and 4-hydroxy-2-oxovaleric acid were identified as intermediates, and pyruvic acid was identified as the final product. A complete pathway for the metabolism of 4-amino-3-hydroxybenzoic acid in strain 10d is proposed. Strain 10d metabolized 2-hydroxymuconic 6-semialdehyde derived from 4-amino-3-hydroxybenzoic acid via a dehydrogenative route, not via a hydrolytic route. This proposed metabolic pathway differs considerably from the modified meta-cleavage pathway of 2-aminophenol and those previously reported for methyl- and chloro-derivatives.

Structure of the Bordetella trematum LPS O-chain subunit.

Analysis of the O-chain subunit of the lipopolysaccharide (LPS, endotoxin) isolated from Bordetella trematum, a recently identified human pathogen, was undertaken. The polysaccharide (PS) moiety was shown to contain only two O-chain subunits, which differed in the anomeric bond of their first sugar. A trisaccharide fragment resulting from the cleavage of a FucNAc glycosidic bond was isolated after treatment of the PS with anhydrous HF. Nitrous deamination of the LPS led to the release of the following heptasaccharide corresponding to two trisaccharide subunits linked to an anhydromannitol residue. beta-ManNAc3NAmA-(1-4)-beta-ManNAc3NAmA-(1-3)-alpha-FucNAc-(1-4)-beta-ManNAc3NAmA-(1-4)-beta-ManNAc3NAmA-(1-3)-beta-FucNAc-(1-6)-2,5-anhManol.

Comparison of bipA alleles within and across Bordetella species.

The Bordetella BvgAS signal transduction system controls the expression of at least three phenotypic phases, the Bvg(+) or virulent phase, the Bvg(-) or avirulent phase, and the Bvg(i) or Bvg intermediate phase, which has been hypothesized to be important for transmission. bipA, the first identified Bvg(i)-phase gene, encodes a protein with similarity to the well-characterized bacterial adhesins intimin and invasin. Proteins encoded by the bipA genes present in Bordetella pertussis Tohama I and Bordetella bronchiseptica RB50 differ in the number of 90-amino-acid repeats which they possess and in the sequence of the C-terminal domain. To investigate the possibility that bipA alleles segregate according to host specificity and to gain insight into the role of BipA and the Bvg(i) phase in the Bordetella infectious cycle, we compared bipA alleles across members of the B. bronchiseptica cluster, which includes both human-infective (B. pertussis and B. parapertussis(hu)) and non-human-infective (B. bronchiseptica and B. parapertussis(ov)) strains. bipA genes were present in most, but not all, strains. All bipA genes present in B. bronchiseptica strains were identical to bipA of RB50 (at least with regard to the DNA sequence of the 3' C-terminal-domain-encoding region, the number of 90-amino-acid repeats encoded, and expression patterns). Although all bipA genes present in the other Bordetella strains were identical in the 3' C-terminal-domain-encoding region to bipA of B. pertussis Tohama I, they varied in the number of 90-amino-acid repeats that they encoded and in expression level. Notably, the genes present in B. parapertussis(hu) strains were pseudogenes, and the genes present in B. parapertussis(ov) strains were expressed at significantly reduced levels compared with the levels in B. pertussis and B. bronchiseptica strains. Our results indicate that there is a correlation between specific bipA alleles and specific hosts. They also support the hypothesis that both horizontal gene transfer and fine-tuning of gene expression patterns contribute to the evolution of host adaptation in lineages of the B. bronchiseptica cluster.

Structural characterization of the O-chain polysaccharide isolated from Bordetella avium ATCC 5086: variation on a theme(1).

The O-chain polysaccharide (O-PS) of Bordetella avium was isolated from the lipopolysaccharide by mild acid hydrolysis to remove the lipid A, followed by hydrofluorolysis to remove the lipopolysaccharide core oligosaccharide leaving a residual O-PS for structural analysis. High resolution (1)H and (13)C NMR and MALDI studies showed the O-chain to be a polymer composed of 1,4-linked 2-acetamidino-3-[3-hydroxybutanamido]-2,3-dideoxy-beta-D-glucopyranosyluronic acid residues.

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).

Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides.

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The polysaccharides isolated from its endotoxin (lipopolysaccharide, LPS) were investigated using chemical analyses, NMR, gas-liquid chromatography/mass spectrometry and mass spectrometry by plasma desorption, matrix-assisted laser desorption/ionization and electrospray. The following structure for the O-chain-free LPS was deduced from the experimental results: carbohydrate structure [see text] Mass spectrometry and serology revealed that the O-chains were different from the homopolymer common to Bordetella bronchiseptica and Bordetella parapertussis strains and were composed of a trisaccharide repeating unit. Masses up to 8 kDa were obtained for native LPS molecular species.