Assuntos
Lipopolissacarídeos/química , Neisseria meningitidis/imunologia , Neisseria/imunologia , Infecções por Neisseriaceae/imunologia , Transferases/imunologia , Anticorpos/imunologia , Western Blotting , Sequência de Carboidratos , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Humanos , Lipopolissacarídeos/biossíntese , Dados de Sequência Molecular , Neisseria/classificação , Neisseria/enzimologia , Neisseria/genética , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Infecções por Neisseriaceae/microbiologia , Transferases/classificação , Transferases/genéticaRESUMO
There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on lipooligosaccharides (LOSs) of M. catarrhalis serotypes A, B, and C, each of which was shown to cover a portion of the clinical strains. To generate conserved LOS antigens and eliminate a potential autoimmune response to a similar epitope between M. catarrhalis LOS moiety Galα1-4Galß1-4Glc and human P(k) antigen, two LOS mutants from strain O35E were constructed. Mutant O35Elgt5 or O35EgalE revealed a deletion of one or two terminal galactose residues of wild type O35E LOS. Each LOS molecule was purified, characterized, detoxified, and coupled to tetanus toxoid (TT) to form conjugates, namely dLOS-TT. Three subcutaneous immunizations using dLOS-TT from O35Elgt5 or O35EgalE elicited significant increases (a 729- or 1263-fold above the preimmune serum levels) of serum immunoglobulin (Ig)G against O35E LOS in rabbits with an adjuvant or without an adjuvant (an 140- or 140-fold above the preimmune serum levels). Rabbit antisera demonstrated elevated complement-mediated bactericidal activities against the wild type strain O35E. The rabbit sera elicited by O35Elgt5 dLOS-TT were further examined and showed cross bactericidal activity against all additional 19 M. catarrhalis strains and clinical isolates studied. Moreover, the rabbit sera displayed cross-reactivity not only among three serotype strains but also clinical isolates in a whole-cell enzyme-linked immunosorbent assay (ELISA), which was further confirmed under transmission electron microscopy. In conclusion, O35Elgt5 dLOS-TT may act as a vaccine against most M. catarrhalis strains and therefore can be used for further in vivo efficacy studies.
Assuntos
Vacinas Bacterianas/imunologia , Lipopolissacarídeos/imunologia , Moraxella catarrhalis/imunologia , Infecções por Moraxellaceae/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização Secundária/métodos , Imunoglobulina G/sangue , Injeções Subcutâneas , Lipopolissacarídeos/genética , Moraxella catarrhalis/genética , Infecções por Moraxellaceae/imunologia , Coelhos , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/imunologia , Vacinação/métodos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologiaRESUMO
Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose alpha-chain. The gamma-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl alpha-chain even though previous reports suggested that the presence of an extended alpha-chain hinders C3 PEA substitution of Hep II. Approximately 50% of gamma-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and gamma-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.
Assuntos
Acetilglucosamina/química , Lipopolissacarídeos/química , Neisseria meningitidis/química , Oligossacarídeos/química , Acetilglucosamina/imunologia , Configuração de Carboidratos , Sequência de Carboidratos , Parede Celular/química , Parede Celular/imunologia , Lipopolissacarídeos/imunologia , Vacinas Meningocócicas/química , Vacinas Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Neisseria meningitidis/patogenicidade , Oligossacarídeos/imunologiaRESUMO
We described 2 unusual Neisseria meningitidis strains isolated from epidemiologically unrelated invasive meningococcal disease cases in Ontario, Canada. Both isolates have features typical of serogroup Y N. meningitidis: are of serotype 2c, are of the multi-locus sequence types typical of the serogroup Y strains in Canada, and are genotyped as serogroup Y based on a previously described PCR-ELISA method that detects the serogroup-Y-specific siaD gene. However, both strains were poly-agglutinable in both anti-Y and anti-W135 antisera. Further studies on 1 of these 2 isolates showed the presence of glucose and galactose as well as sialic acids in its purified capsular polysaccharide, suggesting the presence of both serogroup Y and serogroup W135 polysaccharides. Rabbit antisera produced to this strain contained antibodies to both purified serogroup Y and serogroup W135 capsular polysaccharides. Absorption experiments with either serogroup Y or serogroup W135 bacteria confirmed the presence of antibodies to these 2 different polysaccharides. DNA sequencing of the cps operon from both isolates revealed a siaD gene with 99.7% homology to the published siaD sequence from a serogroup Y strain but with 3 point mutations that all resulted in amino acid changes. How these strains may affect results of routine surveillance, PCR diagnosis, and immuno-protection by vaccination are discussed.
Assuntos
Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Substituição de Aminoácidos/genética , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/química , Proteínas de Bactérias/genética , Sequência de Bases , Pré-Escolar , DNA Bacteriano/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Galactose/análise , Genótipo , Glucose/análise , Humanos , Recém-Nascido , Masculino , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Neisseria meningitidis/isolamento & purificação , Ontário , Mutação Puntual , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Sorotipagem , Ácidos Siálicos/análiseRESUMO
Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Three galactosyltransferase genes, lgtB, lgtE and lgtH, responsible for the biosynthesis of LOS oligosaccharide chains, were analysed in five Neisseria species. The function of lgtH in Neisseria meningitidis 6,275 was determined by mutagenesis and chemical characterization of the parent and mutant LOS chains. The chemical characterization included SDS-PAGE, immunoblot, hexose and mass spectrometry analyses. Compared with the parent LOS, the mutant LOS lacked galactose, and its oligosaccharide decreased by three or four sugar units in matrix-assisted laser desorption ionization (MALDI)-MS analysis. The results show that lgtH encodes a beta-1,4-galactosyltransferase, and that the glucose moiety linked to heptose (Hep) in the alpha chain is the acceptor site in the biosynthesis of Neisseria LOS. To understand the sequence diversity and relationships of lgtB, lgtE and lgtH, the entire lgt-1 locus was further sequenced in three N. meningitidis strains and three commensal Neisseria strains, and compared with the previously reported lgt genes from Neisseria species. Comparison of the protein sequences of the three enzymes LgtB, LgtE and LgtH showed a conserved N-terminal region, and a highly variable C-terminal region, suggesting functional constraint for substrate and acceptor specificity, respectively. The analyses of allelic variation and evolution of 23 lgtB, 12 lgtE and 14 lgtH sequences revealed a distinct evolutionary history of these genes in Neisseria. For example, the splits graph of lgtE displayed a network evolution, indicating frequent DNA recombination, whereas splits graphs of lgtB and lgtH displayed star-tree-like evolution, indicating the accumulation of point mutations. The data presented here represent examples of the evolution and variation of prokaryotic glycosyltransferase gene families. These imply the existence of multiple enzyme isoforms for biosynthesis of a great diversity of oligosaccharides in nature.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , N-Acetil-Lactosamina Sintase/genética , Neisseria/genética , Alelos , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Evolução Molecular , Variação Genética , Lipopolissacarídeos/biossíntese , Dados de Sequência Molecular , N-Acetil-Lactosamina Sintase/metabolismo , Neisseria/enzimologia , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Alinhamento de SequênciaRESUMO
Serogroup C Neisseria meningitidis belonging to the electrophoretic type (ET) ET-15, a variant of ET-37, is endemic in Canada. Like other serogroup C ET-37 meningococci, the endemic ET-15 strains are usually found to carry the serotype and serosubtype antigens of 2a:P1.5,2. In 2001, a sudden increase in the number of cases of serogroup C meningococcal disease in Quebec, Canada, was caused by an antigenic variant of the ET-15 strain. This antigenic variant carries the unique serosubtype marker of P1.7,1. Strains of C:2a:P1.7,1 meningococci were not isolated in Canada in large numbers prior to 2001, and the characteristics of these meningococcal strains linked to an outbreak in Quebec, Canada, are described in the present study.
Assuntos
Variação Antigênica , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Neisseria meningitidis Sorogrupo C/classificação , Antígenos de Bactérias/classificação , Proteínas da Membrana Bacteriana Externa/química , Genótipo , Humanos , Lipopolissacarídeos/imunologia , Dados de Sequência Molecular , Neisseria meningitidis Sorogrupo C/genética , Neisseria meningitidis Sorogrupo C/isolamento & purificação , Fenótipo , Porinas/genética , Quebeque/epidemiologia , SorotipagemRESUMO
The O-acetyl groups in meningococcal A and typhoid Vi polysaccharides (PSs) are functional immunogenic epitopes in humans. To quantify and determine the extent of O-acetylation in these and other bacterial vaccine PSs, anion-exchange HPLC methods have been developed for quantification of O-acetyl, N-acetyl, and phosphate groups in the PSs after these groups were hydrolyzed into anions. The O-acetylation in meningococcal A, C, Y and W-135, pneumococcal 9 V and 18C and typhoid Vi PSs were analyzed. The O-acetyl group was selectively released from a PS as acetate by mild alkaline hydrolysis in 10 or 20 mM NaOH at 37 degrees C until maximum release. The acetate in the hydrolysate was then quantified by high-performance anion-exchange chromatography with conductivity detection (HPAEC-CD) after removal of the PS by filtration with a 10,000 molecular-weight-cut-off membrane. Since the extent of O-acetylation on the PSs depends on bacterial species, strains and growth conditions, the N-acetyl group of amino-sugars, phosphate or monosaccharide components of the PSs were also quantified using HPAEC with conductivity or amperometry detection to determine the molar ratios of the O-acetyl group to these components. The average numbers of O-acetyl molecules in one PS repeating unit of the PSs were obtained from the molar ratios. Besides the O-acetyl determination, the pyruvate component in non-O-acetylated pneumococcal type 4 PS was analyzed by the HPAEC method. The HPAEC method can quantify the O-acetyl content in 0.2 microg of the meningococcal C PS and has a sensitivity at least 10 times higher than that of the colorimetric Hestrin assay. The method can be used for routine analysis of O-acetylation of PSs for quality control of vaccine PSs.
Assuntos
Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Acetilação , Cromatografia por Troca Iônica , Condutividade Elétrica , Eletroquímica , Hidrólise , Hidroxilaminas/química , Monossacarídeos/química , Neisseria meningitidis/química , Neisseria meningitidis/imunologia , Fosfatos , Piruvatos/análise , Padrões de Referência , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologiaRESUMO
The gene encoding the outer membrane adhesin/invasin protein OpcA was previously described in the genomes of two pathogenic Neisseria species, N. meningitidis (Nm) and N. gonorrhoeae (Ng). In order to understand the presence or absence of opcA in nonpathogenic Neisseria species, 13 strains of N. polysaccharea (Np), four strains of N. lactamica, three strains of N. subflava and nine strains of other species were examined by DNA hybridization, polymerase chain reaction (PCR) and nucleotide sequencing. The opcA gene was found in two Np strains (85322 and 89357). The Np-opcA gene is a novel member of this gene family with 93% homology to Ng-opcA. Comparison of opcA-surrounding regions among eight Neisseria strains revealed five types of genetic organization at the opcA locus in Neisseria, which result from insertion or deletion of genetic elements at the upstream region of opcA. Comparison of the deduced peptide sequences from two Np strains, two representative Ng strains, two representative Nm strains and 13 Nm sequence variants demonstrates interspecies diversity of the OpcA protein family with conserved transmembrane regions and species-specific polymorphism at the surface-exposed loops and periplasmic turns. Reverse transcription-PCR analysis and Northern blotting showed that Np-opcA was transcribable. From an alignment of the Np-OpcA and Ng-OpcA sequences against the three-dimensional crystal structure of Nm-OpcA we conclude that there is no obvious structural reason why these proteins would not be able to form stable, folded, outer membrane proteins. The data presented here provide additional information for understanding the distribution, variation and expression of opcA in Neisseria.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Neisseria/genética , Sequência de Aminoácidos , DNA Bacteriano/química , DNA Bacteriano/genética , Ordem dos Genes , Genes Bacterianos/genética , Variação Genética , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Neisseria meningitidis is the causative agent of meningococcal sepsis and meningitis. Neisseria polysaccharea is a nonpathogenic species. N. polysaccharea is able to use sucrose to produce amylopectin, a starch-like polysaccharide, which distinguishes it biochemically from the pathogenic species N. meningitidis. The data presented here indicate that this may be an insufficient criterion to distinguish between these two species. The nonencapsulated Neisseria strain 93246 expressed a phenotype of amylopectin production similar to that of N. polysaccharea. However, strain 93246 reacted with N. meningitidis serotype 4 and serosubtype P1.14 monoclonal antibodies and showed the N. meningitidis L1(8) lipo-oligosaccharide immunotype. Further analyses were performed on four genetic loci in strain 93246, and the results were compared with 7 N. meningitidis strains, 13 N. polysaccharea strains, and 2 N. gonorrhoeae strains. Three genetic loci, opcA, siaD, and lgt-1 in strain 93246, were the same as in N. meningitidis. Particularly, the siaD gene encoding polysialyltransferase responsible for biosynthesis of N. meningitidis group B capsule was detected in strain 93246. This siaD gene was inactivated by a frameshift mutation at the poly(C) tract, which makes strain 93246 identical to other nonencapsulated N. meningitidis strains. As expected, the ams gene encoding amylosucrase, responsible for production of amylopectin from sucrose, was detected in strain 93246 and all 13 N. polysaccharea strains but not in N. meningitidis and N. gonorrhoeae strains. These data suggest that strain 93246 is nonencapsulated N. meningitidis but has the ability to produce extracellular amylopectin from sucrose. The gene for amylopectin production in strain 93246 was likely imported from N. polysaccharea by horizontal genetic exchange. Therefore, we conclude that genetic analysis is required to complement the traditional phenotypic classification for the nonencapsulated Neisseria strains.
Assuntos
Amilopectina/biossíntese , Neisseria meningitidis/metabolismo , Sacarose/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/análise , Glucosiltransferases/genética , Humanos , Dados de Sequência Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Fenótipo , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
Neisseria meningitidis shows great variation in expression of structurally different lipooligosaccharides (LOS) on its cell surface. To better understand the LOS diversity that may occur within an individual strain, a group C wild-type strain, BB305-Tr4, and two stable isogenic LOS variants, Tr5 and Tr7, were selected for this study. SDS-PAGE analysis showed a size reduction of Tr5 and Tr7 LOS compared to that of Tr4. Immunoblotting showed that parental Tr4 LOS reacted with L1, L2 and L3,7 antibodies, variant Tr5 LOS with L1 and L6 antibodies, while Tr7 LOS was non-typeable. Genetic analysis showed that the gene organization at the lgt-1 locus in the three strains was lgtZ,C,A,B,H4 in Tr4, lgtZ,C,A,H4 in Tr5 and lgtZ,C,A,H9 in Tr7. The genetic differences in the three strains were consistent with their phenotypic changes. Sequence comparison revealed two independent recombination events. The first was the recombination of repeated DNA fragments in the flanking regions to delete lgtB in Tr5. The second was the recombination of a fragment of two genes, lgtB and lgtH4, to create an inactive lgtH9 allele with a mosaic structure in Tr7. These findings suggest that besides phase variation, homologous recombination can contribute to the genetic diversity of the lgt locus and to the generation of LOS variation in N. meningitidis.
Assuntos
Glicosiltransferases/genética , Lipopolissacarídeos/imunologia , Neisseria meningitidis Sorogrupo C/genética , Neisseria meningitidis Sorogrupo C/imunologia , Sequência de Bases , Eletroforese em Gel de Poliacrilamida/métodos , Variação Genética , Modelos Genéticos , Dados de Sequência Molecular , Neisseria meningitidis Sorogrupo C/patogenicidade , Reação em Cadeia da Polimerase , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Transferases/análiseRESUMO
Lipooligosaccharide (LOS) is a major virulence factor of the pathogenic Neisseria. Nine lgt genes at three chromosomal loci (lgt-1, 2, 3) encoding the glycosyltransferases responsible for the biosynthesis of LOS oligosaccharide chains were examined in 26 Neisseria meningitidis, 51 Neisseria gonorrhoeae and 18 commensal Neisseria strains. DNA hybridization, PCR and nucleotide sequence data were compared to previously reported lgt genes. Analysis of the genetic organization of the lgt loci revealed that in N. meningitidis, the lgt-1 and lgt-3 loci were hypervariable genomic regions, whereas the lgt-2 locus was conserved. In N. gonorrhoeae, no variability in the composition or organization of the three lgt loci was observed. lgt genes were detected only in some commensal Neisseria species. The genetic organization of the lgt-1 locus was classified into eight types and the lgt-3 locus was classified into four types. Two types of arrangement at lgt-1 (II and IV) and one type of arrangement at lgt-3 (IV) were novel genetic organizations reported in this study. Based on the three lgt loci, 10 LOS genotypes of N. meningitidis were distinguished. Phylogenetic analysis revealed a gene cluster, lgtH, which separated from the homologous genes lgtB and lgtE. The lgtH and lgtE genes were mutually exclusive and were located at the same position in lgt-1. The data demonstrated that pathogenic and commensal Neisseria share a common lgt gene pool and horizontal gene transfer appears to contribute to the genetic diversity of the lgt loci in Neisseria.
Assuntos
Genes Bacterianos/genética , Variação Genética/genética , Lipopolissacarídeos/biossíntese , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Genótipo , Glicosiltransferases/genética , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/genética , Neisseria meningitidis/classificação , Neisseria meningitidis/crescimento & desenvolvimento , Filogenia , Polimorfismo Genético/genética , Análise de Sequência de DNARESUMO
The sialylation of lipooligosaccharide (LOS) in Neisseria meningitidis plays a role in the resistance of the organism to killing by normal human serum. The length of the alpha chain extending out from the heptose I [Hep (I)] moiety of LOS influenced sialylation of N. meningitidis LOS in vitro and in vivo. The alpha chain required a terminal Gal and a trisaccharide or longer oligosaccharide to serve as an acceptor for sialylation. The disaccharide lactose (Galbeta1-4Glc) in the alpha chain of immunotype L8 LOS could not function as an acceptor for the sialyltransferase, probably due to steric hindrance imposed by the neighboring Hep (II) with phosphorylethanolamine and another group attached.