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Found 1 structure.
Displayed structure 1
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Structure type: homopolymer
Trivial name: perosamine homopolymer, perosamine
Compound class: O-polysaccharide, O-antigen
Contained glycoepitopes: IEDB_131172,IEDB_134281,IEDB_1397515,IEDB_2116320,IEDB_434547,IEDB_628715
Brucella spp. are Gram-negative, facultative intracellular bacteria that cause a zoonotic world-wide disease. As in other Gram-negative bacteria, its S-LPS (smooth lipopolysaccharide) is a major determinant of virulence. The Brucella melitensis 16M LPS O-antigen is a homopolymer of 4-formamido-4,6, dideoxymannose. In this study, the previously cloned 14-kb wbk gene cluster was sequenced, and seven open reading frames (ORFs) as well as four insertion sequences were identified. Six of the seven ORFs are homologous to LPS biosynthesis genes from other organisms. The gmd, per and wbkC gene products are predicted to be involved in 4-formamido-4,6,dideoxymannose synthesis. By deletion experiments, we demonstrated that the putative formyltransferase WbkC is absolutely required for the O-side-chain production. The wbkA gene product is similar to several mannosyltransferases and is probably involved in the polymerisation of the B. melitensis O-side-chain. We also identified two genes (wzm and wzt) encoding proteins with high similarity to several two-component ABC (ATP-binding cassette) transporters. Their implication in O-antigen translocation across the inner membrane was confirmed by gene replacement. Finally, no function has been assigned to the wbkB gene either by homology search or functionally, because deletion of wbkB did not interfere with the O-antigen structure. The seven ORFs have a low G + C content, indicating that they might have been acquired by lateral transfer from a progenitor with more A + T rich DNA
Lipopolysaccharide, biosynthesis, genetic, O-antigen, O antigen, O-side-chain, region, Brucella, ABC transporter, Brucella melitensis, lipopolysaccharide O-antigen, LPS genetics
NCBI PubMed ID: 11081580Serological cross-reactivity among intact lipopolysaccharides (LPS) from O1 Vibrio cholerae Inaba O-form (Inaba), Yersinia enterocolitica O9 (O9), non-O1 V. cholerae serogroup Hakata (Hakata) and Vibrio bio-serogroup 1875 Variant (1875 Variant) (all of which share Inaba antigen factor C), as well as a total of six kinds of chemically modified LPS (three from O9 and three from Inaba) was demonstrated by passive hemolysis and passive hemolysis inhibition by using these LPS as antigen for sensitizing sheep red blood cells and as inhibitor. These intact as well as chemically modified LPS contained, in their O polysaccharide chain, α(1→2)-linked linear perosamine (4-amino-4,6-dideoxy-D-manno-pyranose) homopolymers with different N-acyl groups: their acyl groups comprise 3-deoxy-L-glycero-tetronyl (Inaba LPS), formyl (O9 LPS), 3-hydroxypropionyl (1875 Variant LPS), acetyl (Hakata LPS and artificially introduced into Inaba and O9 LPS), propionyl and butyryl (both artificially introduced into Inaba and O9 LPS) groups. N-Deacylation of the α(l→2)-linked N-(3-deoxy-L-glycero-tetronyl)perosamine homopolymer of Inaba and the N-formyl one of O9 LPS resulted in virtual elimination of their serological reactivity with both homologous and heterologous antisera. Furthermore, when the resultant NH2 groups of the N-deacylated perosamine homopolymers of both LPS were N-acylated with acetyl, propionyl or butyryl groups, they markedly recovered both of their serological reactivities. These results are compatible with the interpretation that the Inaba antigen factor C possessed by the four bacteria is substantially related to the common presence of N-acyl groups, regardless of their identity, residing in the perosamine residues constituting the O polysaccharide chain of their LPS. It was also indicated that the group antigen factor A of O1 V. cholerae is substantially related to the 3-deoxy-L-glycerotetronyl groups residing in the perosamine homopolymer of Inaba LPS.
antigen, LPS, O-antigen, Vibrio, Vibrio cholerae O1
NCBI PubMed ID: 7538078An antigenic O-chain polysaccharide fragment derived from Brucella abortus lipopolysaccharide was labeled with 14.8 +/- 1.8 (n = 5) and 52.3 +/- 2.4 (n = 3) micromol of fluorescein/g of polysaccharide (designated FL1 and FL2, respectively) for use in investigating the binding of O-chain to a specific murine antibody YsT9 under equilibrium conditions. Upon binding to YsT9, the fluorescence of FL1 and FL2 was quenched 45-57% with no shift in the excitation and emission spectra, and polarization of fluorescence increased by 300-335%. With fluorescence quenching and polarization as sensitive signals for antibody-bound labeled O-chains, the equilibrium constants for binding of FL1, FL2, and unlabeled O-chain to YsT9 were determined to be within a similar order (1.5 x 10(7) to 2.0 x 10(7) M-1) using a nonlinear curve fitting approach rather than Scatchard analysis. These results indicated that covalent attachment of fluorescein groups to the O-chain did not influence the recognition of the YsT9-defined epitope by the antibody. The reversibility of the O-chain-antibody reaction was also demonstrated by showing a rapid depolarization of the labeled O-chain-antibody complex in the presence of unlabeled O-chain, suggesting that this displacement experiment could be exploited to quantify the Brucella polysaccharide antigen. The study described here provides a useful model for characterization of the complex formation between a carbohydrate-binding protein and a carbohydrate ligand and also for the design of a homogeneous assay system to quantitate antigens or antibodies of clinical interest.
Lipopolysaccharide, monoclonal antibodies, Brucella, Brucella abortus, binding, fluorescence, polarization, quantitative
NCBI PubMed ID: 9006923Studies on the molecular genetics of bacterial LPS serve at least two main purposes: (i) to help develop an understanding of the biology, biochemistry and genetics of this bacterial surface macromolecule, and (ii) to provide a basis for both vaccine development and virulence experiments. Both of these goals have been the driving force in studies of Yersinia LPS carried out during the last decade. Here we will review the progress made in the molecular genetics and biochemistry of Yersinia LPS. A deep understanding has been achieved with respect to Y. enterocolitica serotype O:3, reaching as far as a detailed analysis of the gene clusters directing the biosynthesis of the outer core oligosaccharide and of the O-ag. The O-ag gene clusters of Y. enterocolitica serotype O:8 and Y. pseudotuberculosis serotypes O:2a and O:5a have also been cloned and partially characterized LPS biosynthesis of these Yersinia species includes examples of the two major variations recognized in the biosynthesis of this macromolecule: (i) homopolymeric or O-antigen polymerase-independent biosynthesis, and (ii) heteropolymeric or O-antigen polymerase-dependent biosynthesis.
Lipopolysaccharide, genetic, gene, genetics, O-antigen, biochemistry, Yersinia, molecular genetics
NCBI PubMed ID: 9048864Brucella native haptens (NHs) extracted with hot water from smooth (S)-type B. abortus and B. melitensis were purified to high levels of serological activity and compared with the polysaccharide obtained by acid hydrolysis (PS) of the S lipopolysaccharide (S-LPS). By 13C nuclear magnetic resonance analysis, NHs showed the spectrum of a homopolymer of a-1,2- or a-1,2- plus a-1,3-linked 4-formamido-4,6-dideoxy-D-mannose (N-formylperosamine) previously reported for the LPS O chain. However, while PS contained up to 0.6% 3-deoxy-D-manno-2-octulosonate, this LPS-core marker was absent from NH. High performance liquid chromatography and thin-layer chromatography showed heterogeneity in NH purified from whole cells but not in PS. By immunoprecipitation, polysaccharides indistinguishable from NH were demonstrated in extracts obtained with phenol-water, saline at 60 C, and ether-water treatments, and none of these treatments caused S-LPS hydrolysis detectable with antibodies to the O chain and lipid A. Two lines of evidence showed that NH was in the cell surface. First, NH became biotinylated when B. abortus live cells were labelled with biotin-hydrazide, and the examination of cell fractions and electron microscopy sections with streptavidin-peroxidase and streptavidin-coloidal gold, respectively, showed that labelling was extrinsic. Moreover, whereas only traces of NH were found in cytosols, the amount of NH was enriched in cell envelopes and in the outer membrane blebs spontaneously released by brucellae during growth. Interactions between NH and S-LPS were observed in crude cell extracts, and such interactions could be reconstituted by using purified NH and LPS. The results demonstrate that NH is not a hydrolytic product of S-LPS and suggest a model in which LPS-independent O-type polysaccharides (NH) are intertwined with the O chain in the outer membrane of S-type brucellae.
Lipopolysaccharide, LPS, characterization, polysaccharide, polysaccharides, Brucella, Brucella abortus, hapten, Brucella melitensis, membrane, outer membrane, native, Haptens, O-type, smooth
NCBI PubMed ID: 8576040Smooth Brucella strains are classified into three serotypes, i.e., A+M-, A-M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface of Brucella spp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominant Brucella strains and to cross-reacting Yersinia enterocolitica O:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of all Brucella species and biovars including the live vaccine strains by analyzing the levels of MAb binding to whole Brucella cells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for each Brucella species, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding to B. suis biovar 2 strains and bound at lower titers to B. suis biovar 3 and B. neotomae than to the other Brucella strains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees by Brucella spp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities and Brucella serotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding to Y. enterocolitica O:9, this MAb bound strongly to Y. enterocolitica O:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level to B. suis biovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all to B. suis biovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smooth Brucella infection in mice. As shown in the present study the occurrence of Brucella strains apparently completely devoid of one specific C O-PS epitope (e.g., B. suis biovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.
strain, characterization, epitope, O-polysaccharide, O polysaccharide, specific, surface, Brucella, Yersinia enterocolitica, assay, enzyme-linked immunosorbent assay, flow cytometry, heterogeneity, immunosorbent
NCBI PubMed ID: 9801349no abstract
Lipopolysaccharide, biosynthesis, antigen, lipopolysaccharides, structure, core, gene, strain, characterization, serotype, O-antigen, O antigen, cell, cloning, cluster, gene cluster, cell wall, PAGE, Yersinia pseudotuberculosis, function, genus, bacteriophage, Yersinia, Yersinia enterocolitica, Yersinia pestis, influence, Bacteriophages, growth, temperature
NCBI PubMed ID: 12756758Brucella is a facultative intracellular bacterium responsible for brucellosis. Virulence factors involved in Brucella replication and Brucella's strategies to circumvent the immune response are under investigation. VirB proteins that form the type IV secretion system and that are involved in intracellular replication are considered as one of Brucella's virulence factors. In addition to this secretion system, bacterial outer membrane components have also been described as being implicated in Brucella survival in the host. For example, this bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. These properties make lipopolysaccharide an important virulence factor for Brucella survival and replication in the host
Lipopolysaccharide, Bacterial, host, virulence, form, property, type, factor, protein, response, bacteria, Brucella, intracellular, component, resistance, case, membrane, proteins, immune response, outer membrane, virulence factor, immune, France, secretion, antimicrobial, brucellosis, survival
NCBI PubMed ID: 15694858This review summarizes data on the composition and structure of the O-antigens, the polysaccharide chains of the outer-membrane lipopolysaccharides (LPS) of Gram-negative bacteria defining the immunospecificity of these microbial cells. Special reference is given to some structural features of the O-antigens, such as the presence of unique monosaccharides and noncarbohydrate components, masked regularity, and the occurrence in one microorganism of LPS with structurally different polysaccharide chains. Antigenic relationships between microorganisms belonging to different taxonomic groups are discussed.
structure, O-antigen, chemical composition, bacterial lipopolysaccharides, Salmonella livingstone C1
NCBI PubMed ID: 7533007The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-α-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.
NCBI PubMed ID: 2413867The phenol-phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotype O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S-type lipopolysaccharide with a linear O-antigenic polysaccharide of 1,2-linked 4,6-dideoxy-4-formamido-α-D-mannopyranosyl units. The serological cross-reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl residues in their respective O-antigenic chains.
NCBI PubMed ID: 6199199Antigenic phenol-phase soluble lipopolysaccharide isolated from Brucella abortus 1119-3 by hot phenol-water extraction was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, controlled hydrolysis, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as an unbranched linear homopolymer of 1,2-linked 4,6-dideoxy-4-formamido-α-D-mannopyranosyl residues. The serological reactivity of bovine antiserum to B. abortus 1119-3 with the lipopolysaccharides of Yersinia enterocolitica serotype O:9 and Vibrio cholerae species has now been related to the occurrence of 1,2-linked N-acylated 4-amino-4,6-dideoxy-α-D-mannopyranosyl units in the O-chain polysaccharides of their lipopolysaccharides.
NCBI PubMed ID: 6437981Synthetic oligosaccharides and polysaccharides of known structure have been covalently attached to carrier protein and enzymes for use in a strategy to generate and select monoclonal antibodies with well defined carbohydrate binding profiles. Direct competitive binding assays utilizing antibody bound to a solid phase and saccharide - enzyme conjugates provided convenient and reliable measurement of specificity . Protocols were developed to prepare these carbohydrate - enzyme conjugates. Antibodies specific for the Brucella A and M polysaccharide/antigen were selected and characterized by these techniques. The crystal structure of an Fab fragment derived from one antibody has been solved at 2.7A resolution. Preliminary crystal structure data in conjunction with the amino acid sequence of light and Fd polypeptide chains implicate tyrosine, arginine, asparagine, glutamine and aspartic acid as the residues that contact antigen. A second a antibody - antigen system involving the Shigella flexneri Y antigen has been studied by a similar approach, although in this case two distinct antibodies have been subjected to detailed binding studies with oligosaccharide fragments of the polysaccharide repeating unit and specifically modified derivatives. These data support the contention that the principal polar interactions between an oligosaccharide and its antibody involve a small group of hydroxyl residues and that specifically deoxygenated oligosaccharide inhibitors can have association constants significantly higher than the natural oligosaccharide.
Journal NLM ID: 0376514The O-polysaccharide (O-PS; O-antigen) of bacterial lipopolysaccharides is made up of repeating units of one or more sugar residues and displays remarkable structural diversity. Despite the structural variations, there are only three strategies for O-PS assembly. The ATP-binding cassette (ABC)-transporter-dependent mechanism of O-PS biosynthesis is widespread. The Escherichia coli O9a and Klebsiella pneumoniae O2a antigens provide prototypes, which are distinguished by the fine details that link glycan polymerization and chain termination at the cytoplasmic face of the inner membrane to its export via the ABC transporter. Here, we describe the current understanding of these processes. Since glycoconjugate assembly complexes that utilize an ABC transporter-dependent pathway are widespread among the bacterial kingdom, the models described here are expected to extend beyond O-PS biosynthesis systems
Lipopolysaccharide, O-polysaccharide, ATP-binding cassette transporter, Escherichia coli O9a, Klebsiella pneumoniae O2a
NCBI PubMed ID: 22475157The lipopolysaccharide (LPS) is the major constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. Its lipid A moiety is embedded in the membrane and serves as an anchor for the rest of the LPS molecule. The outermost repetitive glycan region of the LPS is linked to the lipid A through a core oligosaccharide (OS), and is designated as the O-specific polysaccharide (O-polysaccharide, OPS) or O-antigen. The O-antigen is the most variable portion of the LPS and provides serological specificity, which is used for bacterial serotyping. The OPS also provides protection to the microorganisms from host defenses such as complement mediated killing and phagocytosis, and is involved in interactions of bacteria with plants and bacteriophages. Studies of the OPSs ranging from the elucidation of their chemical structures and conformations to their biological and physico-chemical properties help improving classification schemes of Gram-negative bacteria. Furthermore, these studies contributed to a better understanding of the mechanisms of pathogenesis of infectious diseases, as well as provided information to develop novel vaccines and diagnostic reagents.
Lipopolysaccharide, synthesis, lipopolysaccharides, structure, Bacterial, host, O-antigen, O antigen, cell, O antigens, O-antigens, chemical, interaction, cells, PDF, chemical synthesis, biogenesis
Publication DOI: 10.1007/978-3-7091-0733-1_3O-Specific polysaccharides of Brucella contain two antigenic determinants, called A and M. Most of the strains express epitope A with a small amount of epitope M, whereas Brucella melitensis strain 16M expresses longer polymer consisting mostly of M-type epitopes. Proposed explanation was that epitope A is defined by 1-2-linked homopolymer of N-formylperosamine (Rha4NFo), while epitope M is a pentasaccharide with four 2- and one 3-substituted Rha4NFo. We reinvestigated both types of structures by 2D NMR and showed that M-epitope is a tetrasaccharide, missing one of the 2-linked Rha4NFo as compared to the previously proposed structure. Polysaccharide from B. melitensis 16M contains a fragment of 1-2-linked polymer, capped with M-type polymer. Other strains contain one or two M-type units at the non-reducing end of the 1-2-linked O-chain.
NMR, LPS, structure, O-antigens, O-specific, O-specific polysaccharide, Brucella, MS, Brucella melitensis
NCBI PubMed ID: 23664729The brucellae are Gram-negative bacteria that cause an important zoonosis. Studies with the main Brucella species have shown that the O-antigens of the Brucella smooth lipopolysaccharide are α-(1→2) and α-(1→3)-linked N-formyl-perosamine polysaccharides that carry M, A and C (A = M, A>M and A
Lipopolysaccharide, O-antigen, epitope, monoclonal antibodies, typing, Brucella suis
NCBI PubMed ID: 23335981Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract
Lipopolysaccharide, lipid A, Brucella, Brucella abortus, MALDI-TOF MS, brucellosis, Brucella suis
NCBI PubMed ID: 28924631Brucellosis is a serious zoonotic bacterial disease that is ranked by the World Health Organization among the top seven "neglected zoonoses" that threaten human health and cause poverty. It is a costly, highly contagious disease that affects ruminants, cattle, sheep, goats, and other productive animals such as pigs. Symptoms include abortions, infertility, decreased milk production, weight loss, and lameness. Brucellosis is also the most common bacterial disease that is transmitted from animals to humans, with approximately 500 000 new human cases each year. Detection and slaughter of infected animals is required to eradicate the disease, as vaccination alone is currently insufficient. However, as the most protective vaccines compromise serodiagnosis, this creates policy dilemmas, and these often result in the failure of eradication and control programs. Detection of antibodies to the Brucella bacterial cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is used in diagnosis of this disease, and the same molecule contributes important protective efficacy to currently deployed veterinary whole-cell vaccines. This has set up a long-standing paradox that while Brucella OPS confers protective efficacy to vaccines, its presence results in similar antibody profiles in infected and vaccinated animals. Consequently, differentiation of infected from vaccinated animals (DIVA) is not possible, and this limits efforts to combat the disease. Recent clarification of the chemical structure of Brucella OPS as a block copolymer of two oligosaccharide sequences has provided an opportunity to utilize unique oligosaccharides only available via chemical synthesis in serodiagnostic tests for the disease. These oligosaccharides show excellent sensitivity and specificity compared with the native polymer used in current commercial tests and have the added advantage of assisting discrimination between brucellosis and infections caused by several bacteria with OPS that share some structural features with those of Brucella. During synthesis and immunochemical evaluation of these synthetic antigens, it became apparent that an opportunity existed to create a polysaccharide-protein conjugate vaccine that would not create antibodies that give false positive results in diagnostic tests for infection. This objective was reduced to practice, and immunization of mice showed that antibodies to the Brucella A antigen could be developed without reacting in a diagnostic test based on the M antigen. A conjugate vaccine of this type could readily be developed for use in humans and animals. However, as chemical methods advance and modern methods of bacterial engineering mature, it is expected that the principles elucidated by these studies could be applied to the development of an inexpensive and cost-effective vaccine to combat endemic brucellosis in animals.
O-polysaccharide, Brucella, vaccine
NCBI PubMed ID: 29219305Brucellosis is a bacterial zoonosis of worldwide distribution caused by bacteria of the genus Brucella. In Brucella abortus and Brucella melitensis, the major species infecting domestic ruminants, the smooth lipopolysaccharide (S-LPS) is a virulence factor. This S-LPS carries a N-formyl-perosamine homopolymer O-polysaccharide that is the major antigen in serodiagnostic tests and is required for virulence. We report that the Brucella O-PS can be structurally and antigenically modified using wbdR, the acetyl-transferase gene involved in N-acetyl-perosamine synthesis in Escherichia coli O157:H7. Brucella constructs carrying plasmidic wbdR expressed a modified O-polysaccharide but were unstable, a problem circumvented by inserting wbdR into a neutral site of chromosome II. As compared to wild-type bacteria, both kinds of wbdR constructs expressed shorter O-polysaccharides and NMR analyses showed that they contained both N-formyl and N-acetyl-perosamine. Moreover, deletion of the Brucella formyltransferase gene wbkC in wbdR constructs generated bacteria producing only N-acetyl-perosamine homopolymers, proving that wbdR can replace for wbkC. Absorption experiments with immune sera revealed that the wbdR constructs triggered antibodies to new immunogenic epitope(s) and the use of monoclonal antibodies proved that B. abortus and B. melitensis wbdR constructs respectively lacked the A or M epitopes, and the absence of the C epitope in both backgrounds. The wbdR constructs showed resistance to polycations similar to that of the wild-type strains but displayed increased sensitivity to normal serum similar to that of a per R mutant. In mice, the wbdR constructs produced chronic infections and triggered antibody responses that can be differentiated from those evoked by the wild-type strain in S-LPS ELISAs. These results open the possibilities of developing brucellosis vaccines that are both antigenically tagged and lack the diagnostic epitopes of virulent field strains, thereby solving the diagnostic interference created by current vaccines against Brucella.
antigen, Brucella, acetyltransferase, lipopolysaccharide (LPS), virulence factor, brucellosis, bacterial pathogenesis, vaccine development
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