RESUMO
Pectobacterium brasiliense is a widely distributed phytopathogenic bacterium that causes diseases such as soft rot and blackleg, leading to significant yield losses in potatoes as well as other vegetables and ornamental plants. Lipopolysaccharide (LPS) is an important virulence factor that plays an essential role in colonisation of plant tissues and overcoming the host defence mechanisms. The O-polysaccharide from the LPS of P. brasiliense strain NCPPB 4609TS (=CFBP 6617TS = LMG 21371TS = IFB5390) was structurally characterised using spectroscopic techniques and chemical methods. The analyses revealed that the polysaccharide repeating unit consists of Gal, GlcN and an unusual 3-amino-3,6-dideoxyglucose decorated with (R)-3-hydroxybutyric acid according to the structure shown below: In addition, another polysaccharide was isolated from bacterial cells, analysis of which led to the identification of an enterobacterial common antigen, containing N-acetyl-d-glucosamine, N-acetyl-d-mannosaminouronic acid, and 4-acetamido-4,6-dideoxy-d-galactose.
Assuntos
Antígenos O , Pectobacterium , Antígenos O/química , Lipopolissacarídeos/químicaRESUMO
We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.
Assuntos
Apolipoproteínas A/metabolismo , Colesterol/metabolismo , Produtos Finais de Glicação Avançada , Lipopolissacarídeos/química , Macrófagos/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/química , Animais , Apolipoproteínas A/química , Células da Medula Óssea/citologia , Cromatografia Líquida , Perfilação da Expressão Gênica , Humanos , Inflamação , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Proteínas Recombinantes/químicaRESUMO
Pathogenic spirochetes from genus Leptospira are etiologic agents of leptospirosis. Cellular vaccines against Leptospira infection often elicit mainly response against the LPS antigen of the serovars present in the formulation. There is no suitable protein candidate capable of replacing whole-cell vaccines, thus requiring new approaches on vaccine development to improve leptospirosis prevention. Our goal was to develop a whole-cell vaccine sorovar-independent based on LPS removal and conservation of protein antigens exposure, to evaluate the protective capacity of monovalent or bivalent vaccines against homologous and heterologous virulent Leptospira in hamster. Leptospire were subjected to heat inactivation, or to LPS extraction with butanol and in some cases further inactivation with formaldehyde. Hamsters were immunized and challenged with homologous or heterologous virulent serovars, blood and organs were collected from the survivors for bacterial quantification, chemokine evaluation, and analysis of sera antibody reactivity and cross-reactivity by Western blot. Immunization with either heated or low LPS vaccines with serovar Copenhageni or Canicola resulted in 100% protection of the animals challenged with homologous virulent bacteria. Notably, different from the whole-cell vaccine, the low LPS vaccines produced with serovar Canicola provided only partial protection in heterologous challenge with the virulent Copenhageni serovar. Immunization with bivalent formulation results in 100% protection of immunized animals challenged with virulent serovar Canicola. All vaccines produced were able to eliminate bacteria from the kidney of challenged animals. All the vaccines raised antibodies capable to recognize antigens of serovars not present in the vaccine formulation. Transcripts of IFNγ, CXCL16, CCL5, CXCL10, CXCR6, and CCR5, increased in all immunized animals. Conclusion: Our results showed that bivalent vaccines with reduced LPS may be an interesting strategy for protection against heterologous virulent serovars. Besides the desirable multivalent protection, the low LPS vaccines are specially promising due to the expected lower reatogenicity.
Assuntos
Vacinas Bacterianas , Leptospira/imunologia , Leptospirose/imunologia , Lipopolissacarídeos/química , Vacinação , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Cricetinae , Leptospira/química , Leptospirose/prevenção & controleRESUMO
Gram-negative bacterial infections induce inflammation and pain. Lipopolysaccharide (LPS) is a pathogen-associated molecular pattern and the major constituent of Gram-negative bacterial cell walls. Diosmin is a citrus flavonoid with antioxidant and anti-inflammatory activities. Here we investigated the efficacy of diosmin in a nonsterile model of inflammatory pain and peritonitis induced by LPS. Diosmin reduced in a dose-dependent manner LPS-induced inflammatory mechanical hyperalgesia, thermal hyperalgesia, and neutrophil recruitment to the paw (myeloperoxidase activity). Diosmin also normalized changes in paw weight distribution assessed by static weight bearing as a nonreflexive method of pain measurement. Moreover, treatment with diosmin inhibited LPS-induced peritonitis as observed by a reduction of leukocyte recruitment and oxidative stress. Diosmin reduced LPS-induced total ROS production (DCFDA assay) and superoxide anion production (NBT assay and NBT-positive cells). We also observed a reduction of LPS-induced oxidative stress and cytokine production (IL-1ß, TNF-α, and IL-6) in the paw. Furthermore, we demonstrated that diosmin inhibited LPS-induced NF-κB activation in peritoneal exudate. Thus, we demonstrated, using a model of nonsterile inflammation induced by LPS, that diosmin is a promising molecule for the treatment of inflammation and pain.
Assuntos
Anti-Inflamatórios/farmacologia , Hiperalgesia/tratamento farmacológico , Lipopolissacarídeos/farmacologia , NF-kappa B/antagonistas & inibidores , Peritonite/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Antioxidantes/farmacologia , Diosmina/efeitos adversos , Inflamação , Interleucina-1beta , Lipopolissacarídeos/química , Macrófagos/química , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , NF-kappa B/química , Infiltração de Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewes ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.
Assuntos
Animais , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Ovinos/fisiologia , Ovinos/metabolismo , OócitosRESUMO
Although a considerable number of studies have investigated the effects of lipopolysaccharide (LPS) on the reproductive performance of dairy cows, the response of ovine oocytes to LPS during their in vitro maturation and development is not well defined yet. Ewes ovaries were obtained from a slaughterhouse, the oocytes were collected and matured in the presence of increasing concentrations (0, 0.01, 0.1, 1 and 10 µg/mL) of LPS in order to evaluate the meiotic maturation by measuring the proportion of oocytes reaching the MII stage. The concentration of intracellular glutathione (GSH) was measured in oocytes following maturation in vitro. In addition, concentrations of selected metabolites including glucose, pyruvate, lactate and glutamine were quantified in the medium following maturation. A number of treated matured oocytes along with the control group were subsequently fertilized using frozen semen and assessed for the rate of cleavage and for the proportion reaching the blastocyst stage. The number of oocytes in MII stage was significantly reduced in response to the increasing concentrations of LPS (77.83%, 70.64%, 68.86%, 66.32%, respectively, in case of 0.01, 0.1, 1 and 10 µg/mL LPS when compared to the control group, 76.34%; P<0.05). There were no differences neither in the intracellular concentration of GSH in the oocytes nor in case of the metabolites in the maturation medium. Although the rate of cleaved oocytes was not affected by increasing levels of LPS, the blastocyst rate was reduced in a dose dependent manner (36.69%, 34.21%, 30.35%, 17.27% and 14.03% for the control, 0.01, 0.1, 1 and 10 µg/mL LPS, respectively (P<0.05). These results demonstrate that the developmental competence of ovine oocytes may be affected detrimentally by LPS and such deleterious effects could be related to the maturation process.(AU)
Assuntos
Animais , Ovinos/metabolismo , Ovinos/fisiologia , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , OócitosRESUMO
Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.
Assuntos
Azospirillum brasilense/química , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Antígenos O/química , Triticum/fisiologia , Adsorção , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Quimiotaxia/efeitos dos fármacos , Lipopolissacarídeos/isolamento & purificação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Espectroscopia de Prótons por Ressonância Magnética , Plântula/efeitos dos fármacos , Plântula/fisiologia , Triticum/efeitos dos fármacosRESUMO
Understanding the interactions between nanoparticles and biological surfaces is of great importance for many areas of nanomedicine and calls for detailed studies at the molecular level using simplified models of cellular membranes. In this paper, water-dispersed polyvinylpyrrolidonestabilized gold nanoparticles (AuNPs) were incorporated in floating monolayers of selected lipids at the air-water interface as cell membrane models. Surface pressure-area isotherms showed the condensation of glycoside-free lipid monolayers, suggesting their adsorption on the nanoparticle surface through the hydrophilic head groups. On the other hand, monolayers containing glycoside derivatives expanded upon AuNPs incorporation, pointing that the supramolecular structure formed should facilitate the incorporation of these nanoparticles in cellular membranes. These findings can be therefore correlated with the possible toxicity, microbicide and antitumorigenic effects of these nanoparticles in lipidic surfaces of erythrocyte and microbial membranes.
Assuntos
Ar , Membrana Celular/química , Ouro/química , Lipopolissacarídeos/química , Nanopartículas Metálicas/química , Peptidoglicano/química , Água/química , Modelos Químicos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
AIM: To investigate the influence of auxiliary chemical substances (ACSs) and calcium hydroxide [Ca(OH)2 ] dressings on lipopolysaccharides (LPS)/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4). METHODOLOGY: Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); (ii) intracanal medicament: Ca(OH)2 paste for various periods (1 h, 24 h, 7 days, 14 days and 30 days); (iii) combination of substances: (a) 2.5% NaOCl (1 h), followed by 17% EDTA (3 min) and Ca(OH)2 (7 days); (b) 2% CHX (1 h), afterwards, 17% EDTA (3 min) followed by Ca(OH)2 (7 days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrom (MALDI-TOF MS) whilst LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way anova with Tukey-Kramer post-hoc tests at the 5% significance level. RESULTS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (P ≤ .001). 5.25% NaOCl treatment led to the absence of lipid A peaks and LPS bands, whilst no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl-treated LPS did not activate TLR4 (P < .0001). As for Ca(OH)2 , lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24 h. LPS treated with Ca(OH)2 was a weak TLR4 activator (P < .0001). From 24 h onwards, no significant differences were found amongst the time periods tested (P > 0.05). The addition of Ca(OH)2 for 7 days to cells treated either with 2.5% NaOCl or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR4. CONCLUSION: 5.25% NaOCl and Ca(OH)2 dressings from 24 h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4.
Assuntos
Hidróxido de Cálcio/farmacologia , Fusobacterium nucleatum/efeitos dos fármacos , Lipídeo A/metabolismo , Lipopolissacarídeos/metabolismo , Irrigantes do Canal Radicular/farmacologia , Receptor 4 Toll-Like/metabolismo , Análise de Variância , Clorexidina/farmacologia , Ácido Edético/farmacologia , Fusobacterium nucleatum/química , Fusobacterium nucleatum/metabolismo , Lipídeo A/química , Lipídeo A/isolamento & purificação , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Tratamento do Canal Radicular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA - ∆lytS and ∆lytT; LTA - ∆dltA and ∆dltD; and insoluble exopolysaccharide - ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.