RESUMEN
There is a need for next-generation cholera vaccines that provide high-level and durable protection in young children in cholera-endemic areas. A cholera conjugate vaccine (CCV) is in development to address this need. This vaccine contains the O-specific polysaccharide (OSP) of Vibrio cholerae O1 conjugated via squaric acid chemistry to a recombinant fragment of the tetanus toxin heavy chain (OSP:rTTHc). This vaccine has been shown previously to be immunogenic and protective in mice and found to be safe in a recent preclinical toxicological analysis in rabbits. We took advantage of excess serum samples collected as part of the toxicological study and assessed the immunogenicity of CCV OSP:rTTHc in rabbits. We found that vaccination with CCV induced OSP-, lipopolysaccharide (LPS)-, and rTTHc-specific immune responses in rabbits, that immune responses were functional as assessed by vibriocidal activity, and that immune responses were protective against death in an established virulent challenge assay. CCV OSP:rTTHc immunogenicity in two animal model systems (mice and rabbits) is encouraging and supports further development of this vaccine for evaluation in humans.
Asunto(s)
Vacunas contra el Cólera , Cólera , Vibrio cholerae O1 , Niño , Conejos , Humanos , Animales , Ratones , Preescolar , Cólera/prevención & control , Antígenos O , Toxina Tetánica , Vacunas Conjugadas , Inmunoglobulina M , Vacunación , Formación de Anticuerpos , Modelos Animales de Enfermedad , Anticuerpos Antibacterianos , Toxina del CóleraRESUMEN
There is a need for vaccines effective against shigella infection in young children in resource-limited areas. Protective immunity against shigella infection targets the O-specific polysaccharide (OSP) component of lipopolysaccharide. Inducing immune responses to polysaccharides in young children can be problematic, but high level and durable responses can be induced by presenting polysaccharides conjugated to carrier proteins. An effective shigella vaccine will need to be multivalent, targeting the most common global species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. Here we report the development of shigella conjugate vaccines (SCV) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using squaric acid chemistry to result in single point sun-burst type display of OSP from carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. We confirmed structure and demonstrated that these conjugates were recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG responses, as well as rTTHc-specific IgG responses. Vaccination induced serotype-specific bactericidal antibody responses against S. flexneri, and vaccinated animals were protected against keratoconjunctivitis (Sereny test) and intraperitoneal challenge with virulent S. flexneri 2a and 3a, respectively. Our results support further development of this platform conjugation technology in the development of shigella conjugate vaccines for use in resource-limited settings.
Asunto(s)
Disentería Bacilar , Vacunas contra la Shigella , Shigella , Humanos , Niño , Animales , Ratones , Preescolar , Shigella flexneri , Vacunas Conjugadas , Disentería Bacilar/prevención & control , Lipopolisacáridos , Antígenos O , Anticuerpos Antibacterianos , Inmunoglobulina GRESUMEN
There is a need to develop cholera vaccines that are protective in young children under 5 years of age, which induce long-term immunity, and which can be incorporated into the Expanded Programme of Immunization (EPI) in cholera-endemic countries. The degree of protection afforded by currently available oral cholera vaccines (OCV) to young children is significantly lower than that induced by vaccination of older vaccine recipients. Immune responses that protect against cholera target the O-specific polysaccharide (OSP) of Vibrio cholerae, and young children have poor immunological responses to bacterial polysaccharides, which are T cell independent antigens. To overcome this, we have developed a cholera conjugate vaccine (CCV) containing the OSP of V. cholerae O1, the main cause of endemic and epidemic cholera. Here, we describe production of CCV through a scalable manufacturing process and preclinical evaluation of immunogenicity in the presence and absence of aluminum phosphate (alum) as an adjuvant. The vaccine displays V. cholerae O1 Inaba OSP in sun-burst display via single point attachment of core oligosaccharide to a recombinant tetanus toxoid heavy chain fragment (rTTHc). Two different pilot-scale production batches of non-GMP CCV were manufactured and characterized in terms of physico-chemical properties and immunogenicity. In preclinical testing, the vaccine induced OSP- and lipopolysaccharide (LPS)-specific IgG and IgM responses, vibriocidal responses, memory B cell responses, and protection in a V. cholerae O1 challenge model. The addition of alum to the administered vaccine increased OSP-specific immune responses. These results support evaluation of CCV in humans.
Asunto(s)
Vacunas contra el Cólera , Cólera , Vibrio cholerae O1 , Administración Oral , Anticuerpos Antibacterianos , Preescolar , Cólera/prevención & control , Humanos , Inmunoglobulina A , Inmunoglobulina G , Inmunoglobulina M , Células B de Memoria , Vacunas ConjugadasRESUMEN
Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the ß-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a ß-KDO linkage.
RESUMEN
Capsular polysaccharides are important virulence factors in pathogenic bacteria. Characterizing the structural components and biosynthetic pathways for these polysaccharides is key to our ability to design vaccines and other preventative therapies that target encapsulated pathogens. Many gram-negative pathogens such as Neisseria meningitidis and Escherichia coli express acidic capsules. The E. coli K15 serotype has been identified as both an enterotoxigenic and uropathogenic pathogen. Despite its relevance as a disease-causing serotype, the associated capsular polysaccharide remains poorly characterized. We describe in this report the chemical structure of the K15 polysaccharide, based on chemical analysis and nuclear magnetic resonance (NMR) data. The repeating structure of the K15 polysaccharide consists of 4)-α-GlcpNAc-(1 â 5)-α-KDOp-(2 â partially O-acetylated at 3-hydroxyl of GlcNAc. We also report, the organization of the gene cluster responsible for capsule biosynthesis. We identify genes in this cluster that potentially encode an O-acetyltransferase, an N-acetylglucosamine transferase, and a KDO transferase consistent with the structure we report.
Asunto(s)
Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Escherichia coli/genética , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/genética , Espectroscopía de Resonancia Magnética con Carbono-13 , Disacáridos/química , Familia de Multigenes , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.
Asunto(s)
Vacunas contra el SIDA/inmunología , VIH-1/inmunología , Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas/inmunología , Femenino , Inmunización , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Neutralización , Péptidos/químicaRESUMEN
Polysialic acids (PSA) are important extracellular virulence factors of the human pathogens Neisseria meningitidis and Escherichia coli. The importance of these polysaccharides in virulence make the polysialyltransferases (PST) targets for therapeutic drugs and protein engineering to facilitate efficient vaccine production. Here, we have generated recombinant bovine nucleotide monophosphate kinase to facilitate steady state kinetic assays of the PST. We have characterized the N. meningitidis group C (NmC) PST kinetically, using substrate analogues to describe the polymerization reaction. We observed a decrease in Km as the length of the oligo-sialic acid acceptor was increased, indicating a tighter binding of longer oligomers. In addition, we observed a biphasic relationship between kcat and chain length, which can be attributed to a switch in the mechanism of transfer of sialic acid from distributive to processive as the chain length increased above six sialic acid units. Substitution of donor substrate with the analogue CMP-9-F-sialic acid had minimal effect on acceptor Km, but it decreased kcat 6-fold. We propose that this decrease in kcat is caused by a destabilization of the transition state and/or an increase affinity of the product due to presence of the fluoro substituent. The acceptor's hydrophobicity also plays a role in catalysis. The kinetic analysis of the NmC PST with hydrophobic aglycon acceptor substrates indicated that they bind tighter and are turned over at a faster rate than the α-2,9 polysialic acid substrates lacking the hydrophobic end. This finding suggests the presence of a secondary ligand binding site that tethers the acceptor substrate to the enzyme active site.
Asunto(s)
Proteínas Bacterianas/química , Ácido N-Acetilneuramínico Citidina Monofosfato/análogos & derivados , Neisseria meningitidis/enzimología , Sialiltransferasas/química , Animales , Proteínas Bacterianas/aislamiento & purificación , Bovinos , Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Estructura Molecular , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Polimerizacion , Sialiltransferasas/aislamiento & purificación , Especificidad por SustratoRESUMEN
Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.
Asunto(s)
Proteínas Bacterianas/metabolismo , Neisseria meningitidis/enzimología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/química , Polisacáridos Bacterianos/metabolismo , Unión Proteica , Transferasas (Grupos de Otros Fosfatos Sustitutos)/químicaRESUMEN
Neutrophil extracellular traps (NET) are formed against pathogens. However, various diseases are directly linked to this meshwork of DNA. The cytotoxic properties of extracellular histones especially seem to be an important trigger during these diseases. Furthermore, NET accumulation on implants is discussed to result in an impaired efficiency or failure, depending on the category of implant. Interestingly, mucins have been investigated as surface coatings potentially capable of reducing neutrophil adhesion. Similarly, polysialic acid was shown to inactivate the cytotoxic properties of extracellular histones. We wanted to combine the probability to decrease the adhesion of neutrophils using mucins with the capability of sialic acid polymers to counteract histone-mediated cytotoxicity. To this end, we elongate cervical mucins using bacterial polysialyltransferases. Subsequent cell-based experiments demonstrated the activity of elongated mucins against histone-mediated cytotoxicity. Thus, polysialylated mucins may represent a novel component to coat implants or to combat diseases with exaggerated NET formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Moco del Cuello Uterino/química , Trampas Extracelulares/fisiología , Histonas/antagonistas & inhibidores , Mucinas/metabolismo , Neisseria meningitidis/enzimología , Ácidos Siálicos/metabolismo , Sialiltransferasas/metabolismo , Animales , Bovinos , Adhesión Celular , Línea Celular , Pollos , Estro , Femenino , Histonas/fisiología , Histonas/toxicidad , Técnicas In Vitro , Neutrófilos/citología , PorcinosRESUMEN
By using O-SP-core (O-SPcNH2 ) polysaccharide, isolated from Vibrio cholera O1 lipopolysaccharide (LPS) and related synthetic substances, a detailed study of factors that affect conjugation of bacterial polysaccharides to protein carriers through squaric acid chemistry to form conjugate vaccines has been carried out. Several previously unrecognized processes that take place during the squarate labeling of the O-SPcNH2 and subsequent conjugation of the formed squarate (O-SPcNH-SqOMe) have been identified. The efficiency of conjugation at pHâ 8.5, 9.0, and 9.5 to bovine serum albumin (BSA) and to the recombinant tetanus toxin fragment C (rTT-Hc) has been determined. The study led to a protocol for more efficient labeling of O-SPcNH2 antigen with the methyl squarate group, to yield a higher-quality, more potent squarate conjugation reagent. Its use resulted in about twofold increases in conjugation efficiency (from 23-26 % on BSA to 51 % on BSA and 55 % on rTT-Hc). The spent conjugation reagent could be recovered and regenerated by treatment with MeI in the absence of additional base. The immunological properties of the experimental vaccine made from the regenerated conjugation reagent were comparable with those of the immunogen made from the parent O-SPcNH-SqOMe.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas contra el Cólera/inmunología , Ciclobutanos/inmunología , Glicoconjugados/inmunología , Animales , Antígenos Bacterianos/química , Bovinos , Cólera/inmunología , Vacunas contra el Cólera/química , Ciclobutanos/síntesis química , Ciclobutanos/química , Glicoconjugados/síntesis química , Glicoconjugados/química , Humanos , Concentración de Iones de Hidrógeno , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/inmunología , Toxina Tetánica/química , Toxina Tetánica/inmunología , Fiebre Tifoidea/inmunología , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunología , Vibrio choleraeRESUMEN
Neisseria meningitidis serogroups A, B, C, Y, W135 and X are responsible for most cases of meningococcal meningitis. Neisseria meningitidis serogroup X has recently emerged as a contributor to outbreaks of disease in Africa, but there is currently no vaccine against serogroup X. Understanding of the biosynthesis of the serogroup X capsular polysaccharide would provide useful tools for vaccine production. The serogroup X polysaccharide is a homopolymer of (α1â4)-linked N-acetylglucosamine (GlcNAc)-1-phosphate. It has been shown that the gene cluster xcbABC encodes synthesis of this polysaccharide. The xcbA gene product has significant homology with sacB, which is responsible for synthesis of the Neisseria serogroup A capsular polysaccharide, an (α1â6)-N-acetylmannosamine-1-phosphate homopolymer. The xcbA protein also shares homology with the catalytic domain of human N-acetylglucosamine-1-phosphoryltransferase, a key enzyme in the mannose-6-phosphate receptor pathway. In this study, we show that xcbA in the appropriate background is sufficient for the synthesis of N. meningitidis serogroup X polysaccharide. By ELISA we detected polysaccharide in fractions of Escherichia coli expressing the xcbA gene. We isolated polysaccharide from an E. coli strain expressing XcbA and demonstrated that this polysaccharide has a (13)C-NMR spectrum identical to that of polysaccharide isolated from N. meningitidis Group X. We also demonstrate that the purified XcbA protein is an N-acetylglucosamine-1-phosphotransferase that transfers N-acetylglucosamine-1-phosphate from UDP-GlcNAc to the 4-hydroxyl of an N-acetylglucosamine-1-phosphate oligosaccharide. Oligosaccharides fluorescently labeled at the aglycon are extended by XcbA only after the 4-phosphate occupying the non-reducing GlcNAc has been removed. The minimum size of fluorescent acceptors is a trisaccharide.
Asunto(s)
Meningitis Meningocócica , Neisseria meningitidis/clasificación , Neisseria meningitidis/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/inmunología , Cápsulas Bacterianas/metabolismo , Clonación Molecular , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Meningitis Meningocócica/sangre , Meningitis Meningocócica/inmunología , Datos de Secuencia Molecular , Neisseria meningitidis/inmunología , Neisseria meningitidis/metabolismo , Serotipificación , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
We report herein the glycation sites in a vaccine candidate for cholera formed by conjugation of the synthetic hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae, serotype Ogawa, to the recombinant tetanus toxin C-fragment (rTT-Hc) carrier. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the vaccine revealed that it is composed of a mixture of neoglycoconjugates with carbohydrate : protein ratios of 1.9 : 1, 3.0 : 1, 4.0 : 1, 4.9 : 1, 5.9 : 1, 6.9 : 1, 7.9 : 1 and 9.1 : 1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tryptic and GluC V8 digests allowed identification of 12 glycation sites in the carbohydrate-protein neoglycoconjugate vaccine. The glycation sites are located exclusively on lysine (Lys) residues and are listed as follows: Lys 22, Lys 61, Lys 145, Lys 239, Lys 278, Lys 318, Lys 331, Lys 353, Lys 378, Lys 389, Lys 396 and Lys 437. Based on the 3-D representation of the rTT-Hc protein, all the glycation sites correspond to lysines located at the outer surface of the protein.
Asunto(s)
Vacunas contra el Cólera/química , Glicoconjugados/química , Fragmentos de Péptidos/química , Polisacáridos Bacterianos/química , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Toxina Tetánica/química , Vibrio cholerae O1/química , Secuencia de Aminoácidos , Cólera/microbiología , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP < 47 (median DP 25) and DP < 100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS(E) analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP < 47 and DP < 100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.
Asunto(s)
Vacunas Meningococicas/química , Fragmentos de Péptidos/química , Ácidos Siálicos/química , Toxina Tetánica/química , Vacunas Conjugadas/química , Secuencia de Aminoácidos , Animales , Campylobacter jejuni/inmunología , Vacunas Meningococicas/inmunología , Ratones , Datos de Secuencia Molecular , Neisseria meningitidis/inmunología , Fragmentos de Péptidos/inmunología , Ácidos Siálicos/inmunología , Toxina Tetánica/inmunología , Vacunas Conjugadas/inmunologíaRESUMEN
Vaccines against Neisseria meningitidis group C are based on its α-2,9-linked polysialic acid capsular polysaccharide. This polysialic acid expressed on the surface of N. meningitidis and in the absence of specific antibody serves to evade host defense mechanisms. The polysialyltransferase (PST) that forms the group C polysialic acid (NmC PST) is located in the cytoplasmic membrane. Until recently, detailed characterization of bacterial polysialyltransferases has been hampered by a lack of availability of soluble enzyme preparations. We have constructed chimeras of the group C polysialyltransferase that catalyzes the formation α-2,9-polysialic acid as a soluble enzyme. We used site-directed mutagenesis to determine the region of the enzyme necessary for synthesis of the α-2,9 linkage. A chimera of NmB and NmC PSTs containing only amino acids 1 to 107 of the NmB polysialyltransferase catalyzed the synthesis of α-2,8-polysialic acid. The NmC polysialyltransferase requires an exogenous acceptor for catalytic activity. While it requires a minimum of a disialylated oligosaccharide to catalyze transfer, it can form high-molecular-weight α-2,9-polysialic acid in a nonprocessive fashion when initiated with an α-2,8-polysialic acid acceptor. De novo synthesis in vivo requires an endogenous acceptor. We attempted to reconstitute de novo activity of the soluble group C polysialyltransferase with membrane components. We found that an acapsular mutant with a defect in the polysialyltransferase produces outer membrane vesicles containing an acceptor for the α-2,9-polysialyltransferase. This acceptor is an amphipathic molecule and can be elongated to produce polysialic acid that is reactive with group C-specific antibody.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Neisseria meningitidis Serogrupo C/metabolismo , Receptores de Superficie Celular/metabolismo , Sialiltransferasas/metabolismo , Anticuerpos Antibacterianos/inmunología , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Mutación , Neisseria meningitidis Serogrupo C/enzimología , Neisseria meningitidis Serogrupo C/genética , Receptores de Superficie Celular/genética , Proteínas Recombinantes , Ácidos Siálicos/metabolismo , Sialiltransferasas/genéticaRESUMEN
Using recombinant tetanus toxin H(C) fragment (rTT-H(C)) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate-carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.
Asunto(s)
Adyuvantes Inmunológicos/síntesis química , Glicopéptidos/inmunología , Oligosacáridos/inmunología , Fragmentos de Péptidos/inmunología , Toxina Tetánica/inmunología , Vacunas Sintéticas/inmunología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicoconjugados/síntesis química , Glicoconjugados/química , Glicopéptidos/química , Datos de Secuencia Molecular , Oligosacáridos/química , Fragmentos de Péptidos/química , Proteínas Recombinantes/química , Toxina Tetánica/químicaRESUMEN
Polysaccharide (PS) and tetanus toxoid (TT) protein conjugate vaccines were prepared using O-acetylated (OAc+), O-acetyl negative (OAc(-)) and chemically de-O-acetylated (de-OAc) meningococcal W135 PS. The PSs were activated by periodate oxidation and coupled to hydrazine derivatized TT. High performance anion exchange chromatography of acid hydrolysates of periodate activated W135 PSs, showed that galactose residues in OAc+ PS were more sensitive to the periodate oxidation step than they were in the OAc(-) PS or de-OAc PS. Mouse antisera against OAc(-)-TT conjugate vaccines recognized both OAc(-) and OAc+ PS by ELISAs and had high bactericidal titers against both OAc+ and OAc(-) W135 strains. Purified high molecular weight (HMW) conjugates showed higher PS to protein ratios in OAc(-)-TT(HMW) and de-OAc-TT(HMW) indicating better conjugation efficiency than OAc+-TT(HMW) conjugate. Antisera against the HMW fractions gave higher bactericidal titers than antisera against unfractionated conjugates. Inhibition ELISAs indicated that OAc(-) and OAc+ HMW conjugates induced antibodies that bound both OAc+ and OAc(-) PS. Thus, for W135, PS O-acetylation does not contribute a dominant immunogenic epitope. The OAc(-) PS may be a good starting material for preparing W135 PS-TT conjugate vaccines using periodate oxidation.
Asunto(s)
Vacunas Bacterianas/inmunología , Neisseria meningitidis Serogrupo W-135/inmunología , Polisacáridos Bacterianos/inmunología , Toxoide Tetánico/inmunología , Acetilación , Animales , Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/química , Actividad Bactericida de la Sangre/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/inmunología , Femenino , Hidrólisis , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/prevención & control , Ratones , Neisseria meningitidis Serogrupo W-135/química , Oxidación-Reducción , Ácido Peryódico , Polisacáridos Bacterianos/química , Toxoide Tetánico/química , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
The extracellular polysaccharide capsule is an essential virulence factor of Neisseria meningitidis, a leading cause of severe bacterial meningitis and sepsis. Serogroup B strains, the primary disease causing isolates in Europe and America, are encapsulated in alpha-2,8 polysialic acid (polySia). The capsular polymer is synthesized from activated sialic acid by action of a membrane-associated polysialyltransferase (NmB-polyST). Here we present a comprehensive characterization of NmB-polyST. Different from earlier studies, we show that membrane association is not essential for enzyme functionality. Recombinant NmB-polyST was expressed, purified and shown to synthesize long polySia chains in a non-processive manner in vitro. Subsequent structure-function analyses of NmB-polyST based on refined sequence alignments allowed the identification of two functional motifs in bacterial sialyltransferases. Both (D/E-D/E-G and HP motif) are highly conserved among different sialyltransferase families with otherwise little or no sequence identity. Their functional importance for enzyme catalysis and CMP-Neu5Ac binding was demonstrated by mutational analysis of NmB-polyST and is emphasized by structural data available for the Pasteurella multocida sialyltransferase PmST1. Together our data are the first description of conserved functional elements in the highly diverse families of bacterial (poly)sialyltransferases and thus provide an advanced basis for understanding structure-function relations and for phylogenetic sorting of these important enzymes.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Neisseria meningitidis Serogrupo B/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Sialiltransferasas/química , Sialiltransferasas/metabolismo , Adolescente , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Niño , Humanos , Infecciones Meningocócicas/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Ácidos Siálicos/metabolismo , Sialiltransferasas/genéticaAsunto(s)
Alternativas a las Pruebas en Animales , Toxoide Diftérico , Directrices para la Planificación en Salud , Toxoide Tetánico , Pruebas de Toxicidad/métodos , Animales , Química Física , Toxoide Diftérico/química , Toxoide Diftérico/inmunología , Toxoide Diftérico/normas , Educación , Europa (Continente) , Inmunoquímica , Control de Calidad , Toxoide Tetánico/química , Toxoide Tetánico/inmunología , Toxoide Tetánico/normas , Pruebas de Toxicidad/economíaRESUMEN
Escherichia coli K92 produces a capsular polysialic acid with alternating alpha2,8 alpha2,9 NeuNAc linkages. This polysaccharide is cross-reactive with the neuroinvasive pathogen Neisseria meningitidis Group C. The K92 polysialyltransferase (PST) catalyzes the synthesis of the polysialic acid with alternating linkages by the transfer of NeuNAc from CMP-NeuNAc to the nonreducing end of the growing polymer. We used a fluorescent-based high-performance liquid chromatography assay to characterize the process of chain extension. The PST elongates the acceptor GT3-FCHASE in a biphasic fashion. The initial phase polymers are characterized by accumulation of product containing 1-8 additional sialic acid residues. This phase is followed by a very rapid formation of high-molecular weight (MW) polymer as the accumulated oligosaccharides containing 8-10 sialic acids are consumed. The high-MW polymer contains 90-100 sialic acids and is sensitive to degradation by periodate and K1-5 endoneuraminidase, suggesting that the polymer contains the alternating structure. The polymerization reaction does not appear to be strictly processive, since oligosaccharides of each intermediate size were detected before accumulation of high-molecular weight polymer. Synthesis can be blocked by CMP-9-azido-NeuNAc. These results suggest that the K92 PST forms both alpha2,8 and alpha2,9 linkages in a successive and nonprocessive fashion.
Asunto(s)
Escherichia coli/enzimología , Ácido N-Acetilneuramínico/química , Ácidos Siálicos/química , Sialiltransferasas/fisiología , Membrana Celular/química , Cromatografía Líquida de Alta Presión , Proteínas de Escherichia coli/química , Colorantes Fluorescentes/farmacología , Glicósido Hidrolasas/química , Modelos Químicos , Oligosacáridos/química , Polímeros/química , Polisacáridos Bacterianos/química , Sialiltransferasas/química , Factores de TiempoRESUMEN
The polysialyltransferase of Escherichia coli K92 catalyzes the transfer of sialic acid from CMP-sialic acid to a growing chain of polysialic acid at the nonreducing end. The enzyme encoded by the neuS gene is membrane-associated and has been suggested to be organized within a complex of several proteins encoded by the K92 gene cluster. Attempts to prepare a soluble active NeuS enzyme have been unsuccessful. Recent results suggest that de novo synthesis of polysialic acid requires coexpression of four genes from the cluster: neuS, neuE, kpsC, and kpsS. However, elongation of preexisting polysialic acid chains only requires expression of neuS. The molecular organization of the catalytic unit of bacterial polysialyltransferases has not been described. We used radiation inactivation to measure the size of the minimum functional unit catalyzing the polysialyltransferase chain extension and de novo reactions. Membranes harboring NeuS in the presence and absence of other products of the K92 gene cluster were exposed to high-energy electrons. The rate of loss of polysialyltransferase activity reveals the mass of the molecules essential for catalytic activity. We observed that the transfer of neuNAc from CMP-neuNAc to a polysialic acid acceptor is catalyzed by a complex with a target size larger than that of monomeric NeuS. The target size of the unit catalyzing the extension of existing polysialic acid chains does not differ significantly from the size of the unit catalyzing transfer of sialic acid to the endogenous acceptor. Parallel samples of membranes containing NeuS and a green fluorescent protein (GFP) chimera were compared by target analysis. The target size of this structural unit was estimated by analysis of the rate of decay of the GFP-NeuS chimera band migrating in the immunoblots. The target size of the structural unit is larger than expected for a monomer. The results of these experiments show that while the target size of the catalytic activity for K92 polysialyltransferase is larger than a monomer of NeuS, a large complex is not required for catalysis.