Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 96
Filtrar
1.
Carbohydr Res ; 522: 108704, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36306549

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structure of the lipopolysaccharide O-chain (OPS) of three strains of F. nucleatum HM-994, HM-995, and HM-997, isolated from cancerous tissues: -3-ß-D-ManNAc4Lac-4-ß-D-Glc6OAc-3-ß-D-FucNAc4N- HM-994. -4-α-L-GalNHBuA-3-α-D-QuiNAc4NHBu-3-α-L-Rha-6-α-D-GalN- HM-995. -3-[α-L-GulNAcA-4-]-ß-D-Glc-4-ß-D-ManNAcAN-3-ß-D-FucNAc4N-3- HM-997. where HBu is 3-hydroxybutyryl, ManNAc4Lac is 4-O-(1-carboxyethyl)-2-acetamido-2-deoxy-mannose. All monosaccharides are in the pyranose form. The structures were determined using standard NMR (2D homo- and hetero-nuclear techniques), MS and chemical methods following gtypical LPS isolation and purification methods. In some cases polymeric material was further degraded in order to produce compounds that gave improved NMR spectra that were easier to be fully interpreted. Structure of the OPS from strain HM-994 was identical to the OPS from F. nucleatum strain MJR 7757 B. Structures of the OPS from HM-995 and HM-997 are novel and to our knowledge have not been previously reported and include the often observed 6-deoxy- sugars found in several F. nucleatum strains and butyrate rather than acetate modifications in the HM-995 strain. This structural knowledge adds to the ever increasing variation found in LPS O-antigen structures from F. nucleatum strain from both oral and cancerous origin and suggests that there may be a multitude of different LPS O-antigen structures elaborated by this organism that may present challenges to any serotyping efforts.


Assuntos
Fusobacterium nucleatum , Antígenos O , Gravidez , Feminino , Humanos , Antígenos O/química , Fusobacterium nucleatum/química , Lipopolissacarídeos , Composição de Bases , RNA Ribossômico 16S , Filogenia , Análise de Sequência de DNA , Monossacarídeos
2.
Carbohydr Res ; 521: 108648, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36030633

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report four new structures of the lipopolysaccharide O-chain (OPS) from five strains of F. nucleatum CTX47T, CC2_6JVN3, CC2_3FMU1, CC2_1JVN3, HM-996, isolated from cancerous tissues. Three of the four structures have a common sequence of hexose-diaminofucose-hexitol-phosphate in the main chain.


Assuntos
Fusobacterium nucleatum , Antígenos O , Animais , Anticorpos Monoclonais , Composição de Bases , Feminino , Fusobacterium nucleatum/química , Hexoses , Humanos , Lipopolissacarídeos , Camundongos , Antígenos O/química , Fosfatos , Filogenia , Gravidez , RNA Ribossômico 16S , Análise de Sequência de DNA , Álcoois Açúcares
3.
ACS Infect Dis ; 8(7): 1347-1355, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35674342

RESUMO

Pseudomonas aeruginosa was added to the World Health Organization's priority pathogen list for research and development of new antibiotics in 2017. Alongside the development of new antibiotics to fight antimicrobial-resistant P. aeruginosa, vaccines would be an appealing addition to the toolbox health professionals have against this bacteria, which causes life-threatening respiratory infections. Recently, the structure of a novel immunogenic terminal carbohydrate moiety on the cell surface of P. aeruginosa was elucidated, consisting of a 3-O-methyl (1→4)-α-d-rhamnan pentasaccharide. As isolating this oligosaccharide from P. aeruginosa in sufficient amounts for producing a conjugate vaccine is challenging, herein we describe the synthesis of 3-O-methyl d-rhamnose oligosaccharide. We also report the conjugation of the synthetic pentasaccharide to human serum albumin and its resulting immunogenicity.


Assuntos
Mananas , Pseudomonas aeruginosa , Antibacterianos , Desoxiaçúcares , Humanos , Oligossacarídeos
4.
ACS Infect Dis ; 8(7): 1336-1346, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35653593

RESUMO

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.


Assuntos
Desoxiaçúcares , Pseudomonas aeruginosa , Animais , Anticorpos Monoclonais , Desoxiaçúcares/química , Epitopos , Mananas , Camundongos , Polissacarídeos , Coelhos , Trissacarídeos
5.
Carbohydr Res ; 517: 108576, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35526359

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and stillbirths. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the structures of the lipopolysaccharide O-chain (OPS) of two strains of F. nucleatum, SB-106CP and HM-992, both isolated from cancerous tissues. These strains elaborate the same sugar chain, differing only by their N-acylation pattern: -6-α-D-GlcNAc-4-ß-D-GlcNHBu3NABuA-3-ß-D-QuiNAc4NABuAc- SB-106CP -6-α-D-GlcNAc-4-ß-D-GlcNHBu3NABuA-3-ß-D-QuiNAc4NAc- HM-992 ABu = (R)-3-amino-butyryl AbuAc = (R)-3-N-acetyl-3-aminobutyryl HBu = (R)-3-hydroxy-butyryl All monosaccharides are in the pyranose form. Previously we published the structure of the OPS from F. nucleatum 12230, a transtracheal isolate, which had similar sugar chain, differing by replacement of GlcNAc with Glc and a different acylation pattern: -6-α-d-Glc-4-ß-d-GlcNHBu3NHBuA-3-ß-d-QuiNAc4NABu- A mouse monoclonal antibody specific for the 12230 O-antigen did not cross react with the LPS of strains SB-106CP and HM-992 confirming the structural differentiation.


Assuntos
Fusobacterium nucleatum , Antígenos O , Animais , Composição de Bases , Fusobacterium nucleatum/química , Lipopolissacarídeos , Camundongos , Monossacarídeos , Antígenos O/química , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA
6.
Glycoconj J ; 38(6): 735-746, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34491462

RESUMO

Haemophilus influenzae is a leading cause of meningitis disease and mortality, particularly in young children. Since the introduction of a licensed conjugate vaccine (targeting the outer capsular polysaccharide) against the most prevalent serotype, Haemophilus influenzae serotype b, the epidemiology of the disease has changed and Haemophilus influenzae serotype a is on the rise, especially in Indigenous North American populations. Here we apply molecular modeling to explore the preferred conformations of the serotype a and b capsular polysaccharides as well as a modified hydrolysis resistant serotype b polysaccharide. Although both serotype b and the modified serotype b have similar random coil behavior, our simulations reveal some differences in the polysaccharide conformations and surfaces which may impact antibody cross-reactivity between these two antigens. Importantly, we find significant conformational differences between the serotype a and b polysaccharides, indicating a potential lack of cross-reactivity that is corroborated by immunological data showing little recognition or killing between heterologous serotypes. These findings support the current development of a serotype a conjugate vaccine.


Assuntos
Infecções por Haemophilus , Vacinas Anti-Haemophilus , Haemophilus influenzae tipo b , Anticorpos Antibacterianos , Criança , Pré-Escolar , Infecções por Haemophilus/epidemiologia , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae , Humanos , Lactente , Polissacarídeos , Polissacarídeos Bacterianos , Vacinas Conjugadas
7.
Glycobiology ; 31(3): 307-314, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32839812

RESUMO

We describe the structural characterization of the capsular polysaccharides (CPSs) of Pasteurella multocida serotypes B and E. CPS was isolated following organic solvent precipitation of the supernatant from flask grown cells. Structural analysis utilizing nuclear magnetic resonance spectroscopy enabled the determination of the CPS structures and revealed significant structural similarities between the two serotypes, but also provided an explanation for the serological distinction. This observation was extended by the development of polyclonal sera to the glycoconjugate of serotype B CPS that corroborated the structural likenesses and differences. Finally, identification of these structures enabled a more comprehensive interrogation of the genetic loci and prediction of roles for some of the encoded proteins in repeat unit biosynthesis.


Assuntos
Pasteurella multocida/química , Polissacarídeos , Configuração de Carboidratos , Pasteurella multocida/imunologia , Polissacarídeos/química , Polissacarídeos/genética , Polissacarídeos/imunologia , Sorotipagem
8.
Carbohydr Res ; 499: 108198, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33280822

RESUMO

Fusobacterium nucleatum is a gram-negative bacterium, part of the normal human microflora. It is associated with various health complications, including periodontitis and colorectal cancer. Its surface is covered with lipopolysaccharide, which interacts with the immune system and can be involved in various processes in health and disease conditions. Here we present the results of structural analysis of core oligosaccharides from the lipopolysaccharides of several strains of F. nucleatum. Pure compounds were isolated using mild acid hydrolysis or alkaline deacylation of the lipopolysaccharides and analyzed by NMR spectroscopy, mass-spectrometry and chemical methods. All cores analyzed had a common octasaccharide region, including five heptose residues and a non-phosphorylated 3-deoxy-d-manno-oct-2-ulosonic acid residue. The common region is substituted with different additional components specific for each strain. By structure type the F. nucleatum core is similar to that produced by Aeromonas.


Assuntos
Fusobacterium nucleatum/química , Lipopolissacarídeos/química , Oligossacarídeos/química , Configuração de Carboidratos , Espectroscopia de Ressonância Magnética , Oligossacarídeos/isolamento & purificação
9.
Can J Microbiol ; 66(9): 529-534, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32396022

RESUMO

Fusobacterium nucleatum is becoming increasingly recognised as an emerging pathogen, gaining attention as a potential factor for exacerbating colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about the virulence factors of this organism; thus, we have initiated studies to examine the bacterium's surface glycochemistry. In an effort to characterise the surface carbohydrates of F. nucleatum, the aims of this study were to investigate the structure of the lipopolysaccharide (LPS) O-antigen of the cancer-associated isolate F. nucleatum strain CC 7/3 JVN3 C1 (hereafter C1) and to develop monoclonal antibodies (mAbs) to the LPS O-antigen that may be beneficial to the growing field of F. nucleatum research. In this study, we combined several technologies, including nuclear magnetic resonance (NMR) spectroscopy, to elucidate the structure of the LPS O-antigen repeat unit as -[-4-ß-Gal-3-α-FucNAc4N-4-α-NeuNAc-]-. We have previously identified this structure as the LPS O-antigen repeat unit from strain 10953. In this present study, we developed a mAb to the C1 LPS O-antigen and confirmed the mAbs cross-reactivity to the 10953 strain, thus confirming the structural identity.


Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Fusobacterium nucleatum/imunologia , Antígenos O/química , Antígenos O/imunologia , Animais , Antígenos de Bactérias/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Sorotipagem , Fatores de Virulência
10.
ACS Chem Biol ; 15(4): 1050-1058, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32191024

RESUMO

Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA)of C. difficile. The mAbs were characterized in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen, and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerized with human constant region domains and was found to recognize the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (standalone, in conjunction with known antitoxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Clostridioides difficile/imunologia , Lipopolissacarídeos/imunologia , Ácidos Teicoicos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/química , Clostridioides difficile/química , Humanos , Camundongos , Estabilidade Proteica
11.
J Immunol ; 201(8): 2385-2391, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30224513

RESUMO

d-Glycero-ß-d-manno-heptose 1,7-biphosphate (ß-HBP) is a novel microbial-associated molecular pattern that triggers inflammation and thus has the potential to act as an immune modulator in many therapeutic contexts. To better understand the structure-activity relationship of this molecule, we chemically synthesized analogs of ß-HBP and tested their ability to induce canonical TIFA-dependent inflammation in human embryonic kidney cells (HEK 293T) and colonic epithelial cells (HCT 116). Of the analogs tested, only d-glycero-ß-d-manno-heptose 1-phosphate (ß-HMP) induced TIFA-dependent NF-κB activation and cytokine production in a manner similar to ß-HBP. This finding expands the spectrum of metabolites from the Gram-negative ADP-heptose biosynthesis pathway that can function as innate immune agonists and provides a more readily available agonist of the TIFA-dependent inflammatory pathway that can be easily produced by synthetic methods.


Assuntos
Bactérias Gram-Negativas/fisiologia , Heptoses/imunologia , Imunidade Inata , Fatores Imunológicos/imunologia , Inflamação/imunologia , Manose/imunologia , Moléculas com Motivos Associados a Patógenos/imunologia , Fosfatos/imunologia , Piranos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células HEK293 , Heptoses/síntese química , Humanos , Imunização , Fatores Imunológicos/síntese química , Inflamação/induzido quimicamente , Manose/síntese química , Fosfatos/síntese química , Piranos/síntese química , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato
12.
PLoS One ; 13(8): e0201282, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30110339

RESUMO

During the last two decades, Haemophilus influenzae serotype a (Hia) emerged as an important cause of invasive disease in Canadian First Nations and Inuit, and Alaskan Native populations, with the highest rates reported in young children. Immunocompetent adults, in contrast to children, do not typically develop invasive Hia disease. To clarify factors responsible for an increased burden of invasive Hia disease in certain population groups we studied serum bactericidal activity (SBA) against Hia and quantified IgG and IgM specific to Hia capsular polysaccharide in healthy adult members of two First Nations communities: 1) with reported cases of invasive Hia disease (Northern Ontario, NO), and 2) without reported cases (Southern Ontario, SO), in comparison to non-First Nations living in proximity to the NO First Nations community, and non-First Nations elderly non-frail Canadians from across the country (total of 110 First Nations and 76 non-First Nations). To elucidate the specificity of bactericidal antibodies, sera were absorbed with various Hia antigens. Naturally acquired SBA against Hia was detected at higher rates in First Nations (NO, 80%; SO, 96%) than non-First Nations elderly Canadians (64%); the SBA titres in First Nations were higher than in non-First Nations elderly Canadians (P<0.001) and NO non-First Nations adults (P>0.05). Among First Nations, SBA was mediated predominantly by IgM, and by both antibodies specific to Hia capsular polysaccharide and lipooligosaccharide. CONCLUSIONS: The SBA against Hia is frequently present in sera of First Nations adults regardless of the burden of Hia disease observed in their community; it may represent part of the natural antibody repertoire, which is potentially formed in this population under the influence of certain epigenetic factors. Although the nature of these antibodies deserves further studies to understand their origin, the data suggest that they may represent important protective mechanism against invasive Hia disease.


Assuntos
Anticorpos Antibacterianos/imunologia , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/administração & dosagem , Haemophilus influenzae/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/sangue , Atividade Bactericida do Sangue/efeitos dos fármacos , Atividade Bactericida do Sangue/imunologia , Canadá , Feminino , Infecções por Haemophilus/sangue , Vacinas Anti-Haemophilus/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade
13.
Carbohydr Res ; 468: 69-72, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30153554

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and still births. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the following structure of the lipopolysaccharide O-chain of a spontaneous streptomycin resistant (SmR) mutant of F. nucleatum strain ATCC 23726: -4-ß-Non5Am7Ac-4-ß-d-GlcNAcyl3NFoAN-3-ß-d-FucNAc4N- where GlcNAcyl3NFoAN indicates 2,3-diamino-2,3-dideoxyglucuronic acid amide with Fo at N-3 being formyl and Acyl at N-2 being propanoyl (∼70%) or butanoyl (∼30%); Non5Am7Ac indicates 7-acetamido-5-acetimidoylamino-3,5,7,9-tetradeoxy-l-gluco-non-2-ulosonic acid presumably having the d-glycero-l-gluco configuration. To our knowledge, no l-gluco isomer of higher sugars of this class as well as no N-propanoyl or N-butanoyl group have so far been found in bacterial polysaccharides.


Assuntos
Fusobacterium nucleatum/química , Glucose/química , Antígenos O/química , Isomerismo
14.
Infect Immun ; 86(8)2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29844237

RESUMO

Sialylation of lacto-N-neotetraose (LNnT) extending from heptose I (HepI) of gonococcal lipooligosaccharide (LOS) contributes to pathogenesis. Previously, gonococcal LOS sialyltransterase (Lst) was shown to sialylate LOS in Triton X-100 extracts of strain 15253, which expresses lactose from both HepI and HepII, the minimal structure required for monoclonal antibody (MAb) 2C7 binding. Ongoing work has shown that growth of 15253 in cytidine monophospho-N-acetylneuraminic acid (CMP-Neu5Ac)-containing medium enables binding to CD33/Siglec-3, a cell surface receptor that binds sialic acid, suggesting that lactose termini on LOSs of intact gonococci can be sialylated. Neu5Ac was detected on LOSs of strains 15253 and an MS11 mutant with lactose only from HepI and HepII by mass spectrometry; deleting HepII lactose rendered Neu5Ac undetectable. Resistance of HepII lactose Neu5Ac to desialylation by α2-3-specific neuraminidase suggested an α2-6 linkage. Although not associated with increased factor H binding, HepII lactose sialylation inhibited complement C3 deposition on gonococci. Strain 15253 mutants that lacked Lst or HepII lactose were significantly attenuated in mice, confirming the importance of HepII Neu5Ac in virulence. All 75 minimally passaged clinical isolates from Nanjing, China, expressed HepII lactose, evidenced by reactivity with MAb 2C7; MAb 2C7 was bactericidal against the first 62 (of 75) isolates that had been collected sequentially and were sialylated before testing. MAb 2C7 effectively attenuated 15253 vaginal colonization in mice. In conclusion, this novel sialylation site could explain the ubiquity of gonococcal HepII lactose in vivo Our findings reinforce the candidacy of the 2C7 epitope as a vaccine antigen and MAb 2C7 as an immunotherapeutic antibody.


Assuntos
Gonorreia/microbiologia , Heptoses/metabolismo , Lactose/metabolismo , Lipopolissacarídeos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neisseria gonorrhoeae/metabolismo , Neisseria gonorrhoeae/patogenicidade , Adulto , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , China , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Lipopolissacarídeos/química , Masculino , Espectrometria de Massas , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Ácido N-Acetilneuramínico/análise , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/isolamento & purificação
15.
Carbohydr Res ; 463: 37-39, 2018 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-29753950

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and still births. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the following structure of the lipopolysaccharide O-chain of F. nucleatum strain MJR 7757 B:where Lac is (R)-1-carboxyethyl (lactic acid residue); all monosaccharides are in the pyranose form. ManNAc4Lac, analogue of N-acetylmuramic acid, is found for the first time in natural sources.


Assuntos
Fusobacterium nucleatum/metabolismo , Antígenos O/química , Fusobacterium nucleatum/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular
16.
Glycoconj J ; 35(1): 53-64, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28971282

RESUMO

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.


Assuntos
Desoxiaçúcares/imunologia , Glicoconjugados/imunologia , Mananas/imunologia , Streptococcus mutans/imunologia , Animais , Linhagem Celular Tumoral , Desoxiaçúcares/química , Feminino , Glicoconjugados/química , Humanos , Mananas/química , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Sorogrupo , Streptococcus mutans/química , Streptococcus mutans/genética , Vacinas Conjugadas/imunologia
17.
Infect Immun ; 85(11)2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28874446

RESUMO

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.


Assuntos
Proteínas de Bactérias/genética , Proteínas Sanguíneas/toxicidade , Farmacorresistência Bacteriana/genética , Etanolaminofosfotransferase/genética , Regulação Bacteriana da Expressão Gênica , Pasteurella multocida/genética , Pasteurella multocida/patogenicidade , Precursores de Proteínas/toxicidade , Animais , Proteínas de Bactérias/metabolismo , Galinhas , Biologia Computacional , Etanolaminofosfotransferase/metabolismo , Etanolaminas/química , Etanolaminas/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Galactose/química , Galactose/metabolismo , Perfilação da Expressão Gênica , Heptoses/química , Heptoses/metabolismo , Isoenzimas , Lipídeo A/química , Lipídeo A/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Infecções por Pasteurella/microbiologia , Infecções por Pasteurella/patologia , Pasteurella multocida/classificação , Pasteurella multocida/efeitos dos fármacos , Filogenia , Açúcares Ácidos/química , Açúcares Ácidos/metabolismo , Transcriptoma
18.
Vaccine ; 35(45): 6129-6136, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28951087

RESUMO

After the introduction of the glycoconjugate vaccine based upon the capsular polysaccharide ofHaemophilus influenzaetype b in the mid 1980s there was a remarkable decrease in the number of invasive cases reported for this organism. Since the 1990s several groups have observed the emergence ofHaemophilus influenzaetype a (Hia), especially in indigenous communities in the northern regions of Canada and Alaska, to a stage where a solution is warranted to prevent further unnecessary deaths due to this pathogen. A glycoconjugate vaccine solution based upon the type a capsular polysaccharide (CPS) was investigated pre-clinically in an effort to illustrate the proof of concept for this approach. In this study we describe the growth of Hia and the isolation, purification and conjugation of the CPS to several carrier proteins. The resulting glycoconjugates were immunised in mice and rabbits provoking sera that facilitated bactericidal killing against all type a strains that we tested. This study has illustrated the pre-clinical proof of concept of a glycoconjugate vaccine based on the CPS of Hia asa solution to this emerging disease.


Assuntos
Cápsulas Bacterianas/imunologia , Glicoconjugados/imunologia , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae/imunologia , Polissacarídeos Bacterianos/imunologia , Alaska , Animais , Canadá , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Saúde Pública , Coelhos , Sorotipagem/métodos , Vacinação/métodos
19.
Carbohydr Res ; 448: 115-117, 2017 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-28651243

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. It was also found in colorectal cancer tissues and is linked with pregnancy complications, including pre-term and still births. Cell surface structures of the bacterium could be implicated in pathogenesis. Here we report the following structure of the lipopolysaccharide O-chain of F. nucleatum strain 12230: -6-α-d-Glc-4-ß-d-GlcNHBu3NHBuA-3-ß-d-QuiNAc4NABu- where ABu and HBu indicate (R)-3-aminobutanoyl and (R)-3-hydroxybutanoyl, respectively; all monosaccharides are in the pyranose form.


Assuntos
Fusobacterium nucleatum/química , Antígenos O/química , Sequência de Carboidratos
20.
Carbohydr Res ; 440-441: 38-42, 2017 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-28199859

RESUMO

Fusobacterium nucleatum is an anaerobic bacterium found in the human mouth where it causes periodontitis. Recently, it has been gaining attention as a potential causative agent for colorectal cancer and is strongly linked with pregnancy complications including pre-term and still births. Little is known about virulence factors of this organism and thus we have initiated studies to examine the bacterial surface glycochemistry. Consistent with a recent paper suggesting that F. nucleatum strain 10593 can synthesize sialic acid, a staining technique identified sialic acid on the bacterial surface. We isolated lipopolysaccharide from this F. nucleatum strain and performed structural analysis on the O-antigen. Our studies identified a trisaccharide repeating unit of the O-antigen with the following structure: -[→4)-α-Neup5Ac-(2 â†’ 4)-ß-d-Galp-(1 â†’ 3)-α-d-FucpNAc4NAc-(1-]- where Ac indicates 4-N-acetylation of ∼30% FucNAc4N residues. The presence of sialic acid as a constituent of the O-antigen is consistent with recent data identifying de novo sialic acid synthesis in this strain.


Assuntos
Fusobacterium nucleatum/química , Ácido N-Acetilneuramínico/química , Antígenos O/química , Trissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Ácido N-Acetilneuramínico/isolamento & purificação , Antígenos O/isolamento & purificação , Coloração e Rotulagem/métodos , Trissacarídeos/isolamento & purificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA