RESUMEN
The capsular material from Lactobacillus plantarum IMB19, an isolate from fermented vegetables, has been analyzed and our results demonstrate that most of the coat of this bacterium consists of glycerol- and ribitol-type teichoic acids, further decorated with other substituents (α-glucose and alanine), and of a capsular polysaccharide (CPS) with a linear nonasaccharide repeating unit, rich in rhamnose, interconnected to the next via a phosphodiester bridge. Stimulation of immune cells with the total capsular material resulted in the enhancement of immunostimulatory (IFNγ, TNF-α, IL-6 and IL-12) or immuno-regulatory (IL-10) cytokines in an in vitro splenocyte culture system. The capsular polysaccharide, and not the teichoic acids mixture, was responsible for the IFNγ production. Indeed, a significant increase of IFNγ along with other inflammatory cytokines, and a dose response in IFNγ expression with an EC50 of 3.16 µM was found for CPS, disclosing that this polysaccharide is a potent immunostimulatory molecule.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Lactobacillus plantarum/química , Polisacáridos Bacterianos/farmacología , Ácidos Teicoicos/química , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Cápsulas Bacterianas/química , Glicocálix/química , Ratones Endogámicos C57BL , Estructura Molecular , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/aislamiento & purificación , Ramnosa/química , Bazo/citología , Bazo/efectos de los fármacos , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
The innate immune complement system helps clear invading pathogens by forming membrane attack complexes (MACs) on their surface. Abnormal activation of the complement system may aggravate atopic dermatitis (AD) symptoms in AD patients. Here, we investigated the anti-AD effects of LTAs isolated from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) by examination of complement regulatory proteins (CRPs). Combination treatment with pLTA and aLTA increased CD55 and CD59 production in HaCaT cells. The regulation of CD55 and CD59 was mediated by p38 mitogen-activated protein kinase (p38) signaling pathways in pLTA- and aLTA-treated cells. Complement-dependent cytotoxicity (CDC) and bactericidal assays revealed that combination treatment with pLTA and aLTA down-regulated the complement system. In experiments using an irritant contact dermatitis (ICD)-induced mouse model, the levels of MAC and C3 convertase (C3C) were lower in serum collected from pLTA- and aLTA-injected mice than in serum from mice who were untreated or received pLTA or aLTA alone. Combination treatment also inhibited IgE and CCL2 levels in ICD mice. On the other hand, IFN-γ level was significantly increased, indicating that combination treatment switches the Th2 response to a Th1 response. Our results suggest that combination treatment with LTAs could be a good therapeutic approach to alleviate AD by reducing formation of MACs and inducing a Th1 response.
Asunto(s)
Antígenos CD55/inmunología , Antígenos CD59/inmunología , Dermatitis Atópica , Lactobacillus plantarum/química , Lipopolisacáridos , Staphylococcus aureus/química , Ácidos Teicoicos , Regulación hacia Arriba/efectos de los fármacos , Animales , Línea Celular , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Ácidos Teicoicos/química , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/farmacologíaRESUMEN
There is increasing evidence of the relevant connection and regulation between the gut and skin immune axis. In fact, oral administration of lipoteichoic acid (LTA) from Lactobacillus rhamnosus GG (LGG) prevents the development of UV-induced skin tumors in chronically exposed mice. Here we aim to evaluate whether this LTA is able to revert UV-induced immunosuppression as a mechanism involved in its anti-tumor effect and whether it has an immunotherapeutic effect against cutaneous squamous cell carcinoma. Using a mouse model of contact hypersensitivity, we demonstrate that LTA overcomes UV-induced skin immunosuppression. This effect was in part achieved by modulating the phenotype of lymph node resident dendritic cells (DC) and the homing of skin migratory DC. Importantly, oral LTA reduced significantly the growth of established skin tumors once UV radiation was discontinued, demonstrating that it has a therapeutic, besides the already demonstrated preventive antitumor effect. The data presented here strongly indicates that oral administration of LTA represents a promising immunotherapeutic approach for different conditions in which the skin immune system is compromised.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Lacticaseibacillus rhamnosus/química , Lipopolisacáridos/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Ácidos Teicoicos/farmacología , Rayos Ultravioleta/efectos adversos , Administración Oral , Animales , Antineoplásicos/aislamiento & purificación , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Movimiento Celular/efectos de la radiación , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Dendríticas/efectos de la radiación , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Femenino , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/patología , Tracto Gastrointestinal/efectos de la radiación , Lipopolisacáridos/aislamiento & purificación , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ganglios Linfáticos/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Piel/efectos de los fármacos , Piel/inmunología , Piel/patología , Piel/efectos de la radiación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
Enterococcus faecium (E. faecium) has emerged as one of today's leading causes of health care-associated infections that is difficult to treat with the available antibiotics. These pathogens produce capsular polysaccharides on the cell surface which play a significant role in adhesion, virulence and evasion. Therefore, we aimed at the identification and characterization of bacterial polysaccharide antigens which are central for the development of vaccine-based prophylactic approaches. The crude cell wall-associated polysaccharides from E. faecium, its mutant and complemented strains were purified and analyzed by a primary antibody raised against lipoteichoic acid (LTA) and diheteroglycan (DHG). The resistant E. faecium strains presumably possess novel capsular polysaccharides that allow them to avoid the evasion from opsonic killing. The E. faecium U0317 strain was very well opsonized by anti-U0317 (~95%), an antibody against the whole bacterial cell. The deletion mutant showed a significantly increased susceptibility to opsonophagocytic killing (90-95%) against the penicillin binding protein (anti-PBP-5). By comparison, in a mouse urinary tract and rat endocarditis infection model, respectively, there were no significant differences in virulence. In this study we explored the biological role of the capsule of E. faecium. Our findings showed that the U0317 strain is not only sensitive to anti-LTA but also to antibodies against other enterococcal surface proteins. Our findings demonstrate that polysaccharides capsule mediated-resistance to opsonophagocytosis. We also found that the capsular polysaccharides do not play an important role in bacterial virulence in urinary tract and infective endocarditis in vivo models.
Asunto(s)
Anticuerpos Antibacterianos/farmacología , Antígenos Bacterianos/aislamiento & purificación , Pared Celular/química , Enterococcus faecium/química , Lipopolisacáridos/aislamiento & purificación , Polisacáridos Bacterianos/aislamiento & purificación , Ácidos Teicoicos/aislamiento & purificación , Animales , Antibacterianos/farmacología , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Cápsulas Bacterianas/química , Cápsulas Bacterianas/inmunología , Pared Celular/inmunología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Endocarditis Bacteriana/tratamiento farmacológico , Endocarditis Bacteriana/microbiología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/inmunología , Enterococcus faecium/patogenicidad , Femenino , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Proteínas Opsoninas/farmacología , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/inmunología , Proteínas de Unión a las Penicilinas/aislamiento & purificación , Proteínas de Unión a las Penicilinas/farmacología , Fagocitosis/efectos de los fármacos , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Cultivo Primario de Células , Ratas , Ratas Wistar , Ácidos Teicoicos/química , Ácidos Teicoicos/inmunología , Ácidos Teicoicos/farmacología , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/microbiologíaRESUMEN
The envelope of gram-positive bacteria encompasses the cell wall, a rigid exoskeleton comprised of peptidoglycan that provides protection against lysis and governs bacterial cell shapes. Peptidoglycan also serves as the site of attachment for proteins and nonproteinaceous polymers that interact with the bacterial environment. Nonproteinaceous molecules include teichoic acids, capsular polysaccharides, and secondary cell wall polysaccharides (SCWP). Treatment of gram-positive bacterial cells with proteases, nucleases, and detergents results in the isolation of "murein sacculi" (i.e., peptidoglycan with bound carbohydrate polymers). Incubation of sacculi with acid or base releases carbohydrate polymers that can be purified for further biochemical characterization. This protocol describes the hydrofluoric acid extraction and purification of the secondary cell wall polymer of Bacillus anthracis that is also found in the envelope of the other members of the Bacillus cereus sensu lato group of bacteria.
Asunto(s)
Pared Celular/química , Cromatografía en Gel/métodos , Cromatografía de Fase Inversa/métodos , Bacterias Grampositivas/química , Ácido Fluorhídrico/química , Peptidoglicano/aislamiento & purificación , Polisacáridos Bacterianos/aislamiento & purificación , Bacillus anthracis/química , Fraccionamiento Químico/métodos , Cromatografía Líquida de Alta Presión/métodos , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Peptidoglicano/química , Polisacáridos Bacterianos/química , Ácidos Teicoicos/química , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
INTRODUCTION: Apical periodontitis is an inflammatory disease in the periradicular region of teeth that results from infection by multispecies bacterial biofilm residing in the root canal system. In this study, we investigated whether Lactobacillus plantarum lipoteichoic acid (Lp.LTA) could inhibit multispecies oral pathogenic bacterial biofilm formation. METHODS: Highly pure and structurally intact Lp.LTA was purified from L. plantarum. Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were co-cultured to form oral multispecies biofilm in the presence or absence of Lp.LTA on culture plates or human dentin slices. Preformed biofilm was treated with or without Lp.LTA, followed by additional treatment with intracanal medicaments such as calcium hydroxide or chlorhexidine digluconate. Confocal microscopy and crystal violet assay were performed to determine biofilm formation. Biofilm on human dentin slices was visualized with a scanning electron microscope. RESULTS: Biofilm formation of multispecies bacteria on the culture dishes was dose-dependently reduced by Lp.LTA compared with the nontreatment control group. Lp.LTA also inhibited multispecies biofilm formation on the dentin slices in a dose-dependent manner. Interestingly, Lp.LTA was shown to reduce preformed multispecies biofilm compared with the nontreatment group. Moreover, Lp.LTA potentiated the effectiveness of the intracanal medicaments in the removal of preformed multispecies biofilm. CONCLUSIONS: These results suggest that Lp.LTA is a potential anti-biofilm agent for treatment or prevention of oral infectious disease, including apical periodontitis, which is mainly caused by multispecies bacterial biofilm.
Asunto(s)
Actinomyces/fisiología , Biopelículas/efectos de los fármacos , Enterococcus faecalis/fisiología , Lactobacillus plantarum/química , Ligilactobacillus salivarius/fisiología , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/farmacología , Actinomyces/patogenicidad , Hidróxido de Calcio/farmacología , Clorhexidina/análogos & derivados , Clorhexidina/farmacología , Dentina/microbiología , Depresión Química , Relación Dosis-Respuesta a Droga , Enterococcus faecalis/patogenicidad , Humanos , Ligilactobacillus salivarius/patogenicidad , Lipopolisacáridos/uso terapéutico , Periodontitis Periapical/tratamiento farmacológico , Periodontitis Periapical/microbiología , Periodontitis Periapical/prevención & control , Irrigantes del Conducto Radicular/farmacología , Ácidos Teicoicos/uso terapéuticoRESUMEN
Streptococcus suis serotype 2 is an important porcine and human pathogen. Lipoteichoic acid (LTA) from S. suis has been suggested to contribute to its virulence, and absence of d-alanylation from the S. suis LTA is associated with increased susceptibility to cationic antimicrobial peptides. Here, using high-resolution NMR spectroscopy and MS analyses, we characterized the LTA structures from three S. suis serotype 2 strains differing in virulence, sequence type (ST), and geographical origin. Our analyses revealed that these strains possess-in addition to the typical type I LTA present in other streptococci-a second, mixed-type series of LTA molecules of high complexity. We observed a ST-specific difference in the incorporation of glycosyl residues into these mixed-type LTAs. We found that strains P1/7 (ST1, high virulence) and SC84 (ST7, very high virulence) can attach a 1,2-linked α-d-Glcp residue as branching substituent to an α-d-Glcp that is 1,3-linked to glycerol phosphate moieties and that is not present in strain 89-1591 (ST25, intermediate virulence). In contrast, the latter strain could glycosylate its LTA at the glycerol O-2 position, which was not observed in the other two strains. Using LTA preparations from WT strains and from mutants with an inactivated prolipoprotein diacylglyceryl transferase, resulting in deficient lipoprotein acylation, we show that S. suis LTAs alone do not induce Toll-like receptor 2-dependent pro-inflammatory mediator production from dendritic cells. In summary, our study reveals an unexpected complexity of LTAs present in three S. suis serotype 2 strains differing in genetic background and virulence.
Asunto(s)
Adyuvantes Inmunológicos/química , Células Dendríticas/efectos de los fármacos , Lipopolisacáridos/química , Streptococcus suis/química , Ácidos Teicoicos/química , Transferasas/deficiencia , Adyuvantes Inmunológicos/aislamiento & purificación , Adyuvantes Inmunológicos/farmacología , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Quimiocina CCL3/genética , Quimiocina CCL3/inmunología , Quimiocina CXCL1/genética , Quimiocina CXCL1/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Expresión Génica , Glicosilación , Interleucina-6/genética , Interleucina-6/inmunología , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Éteres Fosfolípidos/química , Cultivo Primario de Células , Serogrupo , Streptococcus suis/clasificación , Streptococcus suis/patogenicidad , Relación Estructura-Actividad , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/farmacología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Transferasas/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , VirulenciaRESUMEN
Teichoic acid (TA), a crucial cell wall constituent of the pathobiont Streptococcus pneumoniae, is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, LTA). Both TA polymers share a common precursor synthesis pathway, but differ in the final transfer of the TA chain to either peptidoglycan or a glycolipid. Here, we show that LTA exhibits a different linkage conformation compared to WTA, and identify TacL (previously known as RafX) as a putative lipoteichoic acid ligase required for LTA assembly. Pneumococcal mutants deficient in TacL lack LTA and show attenuated virulence in mouse models of acute pneumonia and systemic infections, although they grow normally in culture. Hence, LTA is important for S. pneumoniae to establish systemic infections, and TacL represents a potential target for antimicrobial drug development.
Asunto(s)
Pared Celular/metabolismo , Lipopolisacáridos/deficiencia , Neumonía Neumocócica/microbiología , Sepsis/microbiología , Streptococcus pneumoniae/patogenicidad , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Ligasas/genética , Ligasas/metabolismo , Lipopolisacáridos/aislamiento & purificación , Masculino , Ratones , Microscopía Electrónica , Mutagénesis , Peptidoglicano/metabolismo , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/ultraestructura , Ácidos Teicoicos/aislamiento & purificación , VirulenciaRESUMEN
Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is recognized by Toll-like receptor 2, expressed on certain mammalian cell surfaces, initiating signaling cascades that include nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase. There are many structural and functional varieties of LTA, which vary according to the different species of gram-positive bacteria that produce them. In this study, we examined whether LTA isolated from Staphylococcus aureus (aLTA) affects the expression of junction proteins in keratinocytes. In HaCaT cells, tight junction-related gene expression was not affected by aLTA, whereas adherens junction-related gene expression was modified. High doses of aLTA induced the phosphorylation of extracellular signal-regulated protein kinases 1 and 2, which in turn induced the epithelial-mesenchymal transition (EMT) of HaCaT cells. When cells were given a low dose of aLTA, however, NF-κB was activated and the total cell population increased. Taken together, our study suggests that LTA from S. aureus infections in the skin may contribute both to the outbreak of EMT-mediated carcinogenesis and to the genesis of wound healing in a dose-dependent manner.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/farmacología , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Línea Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos , Lipopolisacáridos/administración & dosificación , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosforilación , ARN Mensajero/biosíntesis , Transducción de Señal , Enfermedades de la Piel/microbiología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/patogenicidad , Ácidos Teicoicos/administración & dosificación , Receptor Toll-Like 2/metabolismoRESUMEN
Staphylococcus aureus (S. aureus) causes severe inflammation in various infectious diseases, leading to high mortality. The clinical application of antibiotics has gained a significant curative effect. However, it has led to the emergence of various resistant bacteria. Therefore, in this study, we investigated the protective effect of polydatin (PD), a traditional Chinese medicine extract, on S. aureus lipoteichoic acid (LTA)-induced injury in vitro and in vivo. First, a significant improvement in the pathological conditions of PD in vivo was observed, suggesting that PD had a certain protective effect on LTA-induced injury in a mouse model. To further explore the underlying mechanisms of this protective effect of PD, LTA-induced murine macrophages were used in this study. The results have shown that PD could reduce the NF-κB p65, and IκBα phosphorylation levels increased by LTA, resulting in a decrease in the transcription of pro-inflammatory factors, such as TNF-α, IL-1ß and IL-6. However, LTA can not only activate NF-κB through the recognition of TLR2 but also increase the level of intracellular reactive oxygen species (ROS), thereby activating NF-κB signalling. We also detected high levels of ROS that activate caspases 9 and 3 to induce apoptosis. In addition, using a specific NF-κB inhibitor that could attenuate apoptosis, namely NF-κB p65, acted as a pro-apoptotic transcription factor in LTA-induced murine macrophages. However, PD could inhibit the generation of ROS and NF-κB p65 activation, suggesting that PD suppressed LTA-induced injury by attenuating ROS generation and TLR2-NFκB signalling.
Asunto(s)
Antioxidantes/farmacología , Endometriosis/tratamiento farmacológico , Glucósidos/farmacología , Sustancias Protectoras/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Estilbenos/farmacología , Receptor Toll-Like 2/genética , Factor de Transcripción ReIA/genética , Animales , Supervivencia Celular/efectos de los fármacos , Endometriosis/inducido químicamente , Endometriosis/genética , Endometriosis/inmunología , Femenino , Regulación de la Expresión Génica , Proteínas I-kappa B/genética , Proteínas I-kappa B/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo/efectos de los fármacos , Células RAW 264.7 , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Staphylococcus aureus/química , Ácidos Teicoicos/antagonistas & inhibidores , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/toxicidad , Receptor Toll-Like 2/inmunología , Factor de Transcripción ReIA/inmunología , Útero/efectos de los fármacos , Útero/inmunología , Útero/patologíaRESUMEN
The cell wall glycopolymers of three strains of Streptomyces albus and the type strain of Streptomyces pathocidini were investigated. The structures of the glycopolymers were established using a combination of chemical and NMR spectroscopic methods. The cell wall of S. albus subsp. albus VKM Ac-35(T) was found to be comprised of three glycopolymers, viz. unsubstituted 1,5-poly(ribitol phosphate), 1,3-poly(glycerol phosphate) substituted with ß-D-glucopyranose, and the major polymer, a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid (Kdn)-teichulosonic acid: ß-D-Glcp-(1 â 8)-α-Kdnp-(2[(â6)-ß-D-Glcp-(1 â 8)-α-Kdnp-(2 â] n 6)-ß-D-Glcp-(1 â 8)-ß-Kdnp-(2-OH, where n ≥ 3. The cell walls of 'S. albus' J1074 and 'S. albus' R1-100 were found to contain three glycopolymers of identical structures, viz. unsubstituted 1,3- and 2,3-poly(glycerol phosphates), and the major polymer, a Kdn-teichulosonic acid with an unusual structure that has not been previously described: ß-D-Galp-(1 â 9)-α-Kdnp-(2[(â3)-ß-D-Galp-(1 â 9)-α-Kdnp-(2 â] n 3)-ß-D-Galp-(1 â 9)-ß-Kdnp-(2-OH, where n ~ 7-8. The cell wall of S. pathocidini (formerly S. albus subsp. pathocidicus) VKM Ac-598(T) was found to contain two glycopolymers, viz. 1,3-poly(glycerol phosphate) partially O-glycosylated with 2-acetamido-2-deoxy-α-D-glucopyranose and/or O-acylated with L-lysine, and a poly(diglycosyl 1-phosphate) of hitherto unknown structure: -6)-α-D-Glcp-(1 â 6)-α-D-GlcpNAc-(1-P-.
Asunto(s)
Polisacáridos Bacterianos/metabolismo , Streptomyces/metabolismo , Pared Celular/metabolismo , Lisina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Fosfatos/aislamiento & purificación , Fosfatos/metabolismo , Polisacáridos Bacterianos/aislamiento & purificación , Streptomyces/química , Azúcares Ácidos/aislamiento & purificación , Azúcares Ácidos/metabolismo , Ácidos Teicoicos/aislamiento & purificación , Ácidos Teicoicos/metabolismo , Ácidos Urónicos/aislamiento & purificación , Ácidos Urónicos/metabolismoRESUMEN
Lipoteichoic acid (LTA) is one of microbe-associated molecular pattern (MAMP) molecules of gram-positive bacteria. In this study, we demonstrated that Clostridium butyricum LTA (bLTA) significantly inhibited the inflammatory response and apoptosis induced by Staphylococcus aureus LTA (aLTA) in HT-29 cells. aLTA stimulated the inflammatory responses by activating a strong signal transduction cascade through NF-κB and ERK, but bLTA did not activate the signaling pathway. bLTA pretreatment inhibited the activation of the NF-κB and ERK signaling pathway induced by aLTA. The expression and release of cytokines such as IL-8 and TNF-α were also suppressed by bLTA pretreatment. aLTA treatment induced apoptosis in HT-29 cells, but bLTA did not affect the viability of the cells. Further study indicated that bLTA inhibited apoptosis in HT-29 cells induced by aLTA. These results suggest that bLTA may act as an aLTA antagonist and that an antagonistic bLTA may be a useful agent for suppressing the pro-inflammatory activities of gram-positive pathogenic bacteria.
Asunto(s)
Clostridium butyricum/química , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Staphylococcus aureus/química , Ácidos Teicoicos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Clostridium butyricum/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/inmunología , Células HT29 , Humanos , Inflamación/prevención & control , Interleucina-8/genética , Interleucina-8/inmunología , Lipopolisacáridos/aislamiento & purificación , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Transducción de Señal , Especificidad de la Especie , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Ácidos Teicoicos/aislamiento & purificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Lactic acid bacteria (LAB) have beneficial effects on intestinal health and skin diseases. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is known to induce the production of several cytokines such as TNF-α, IL-1ß, and IL-8 and affect the intestinal microflora, anti-aging, sepsis, and cholesterol level. In this study, Weissella cibaria was isolated from Indian dairy products, and we examined its immune-enhancing effects. Live and heatkilled W. cibaria did not induce the secretion of immune-related cytokines, whereas LTA isolated from W. cibaria (cLTA) significantly increased the secretion of TNF-α, IL-1ß, and IL-6 in a dose-dependent manner. cLTA increased the phosphorylation of nuclear factor kappalight-chain-enhancer of activated B cells, p38 mitogen-activated protein kinases, and c-Jun N-terminal kinases in THP-1 cells. The secretion of TNF-α and IL-6 was also increased in the cLTA-treated mouse splenocytes. These results suggest that cLTA, but not W. cibaria whole cells, has immune-boosting potential and can be used to treat immunosuppression diseases.
Asunto(s)
Citocinas/biosíntesis , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Bazo/citología , Ácidos Teicoicos/farmacología , Weissella/química , Animales , Línea Celular , Humanos , Inmunomodulación/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/aislamiento & purificación , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/inmunología , FN-kappa B/metabolismo , Fosforilación , Filogenia , ARN Ribosómico 16S/genética , Ácidos Teicoicos/aislamiento & purificación , Weissella/clasificación , Weissella/genéticaRESUMEN
Two different teichoic acids, along with a teichulosonic and a teichuronic acids, were identified in the cell wall of Brevibacterium aurantiacum VKM Ac-2111(Т). One teichoic acid is 1,3-poly(glycerol phosphate) with 2-acetamido-2-deoxy-α-D-galactopyranose and L-glutamic acid as non-stoichiometric substituents at O-2 of the glycerol residue. The second one is a poly(glycosylglycerol phosphate) with -4)-α-D-Galp-(1 â 2)-sn-Gro-(3-P- and/or -6)-α-D-Galp-(1 â 2)-sn-Gro-(3-P- units in the main chain. The structure of the first has not been reported so far, while the latter one is new for actinobacteria. The teichulosonic acid with α-3-deoxy-ß-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn) and ß-D-glucopyranose residues in the backbone represents a novel polymer: â 8)-α-Kdn-(2 â 6)-ß-D-Glcp-(1 â. The teichuronic acid has also hitherto unknown structure: â 3)-ß-D-Galf(2OAc)0.3-(1 â 3)-ß-D-GlcpÐ-(1 â and is found in members of the genus Brevibacterium for the first time. The polymer structures were elucidated using 1D- and 2D-NMR spectroscopy: (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, HSQC-TOCSY, and (1)H,(13)C and (1)H,(31)P HMBC.
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Brevibacterium/química , Pared Celular/química , Ácidos Teicoicos/aislamiento & purificación , Ácidos Urónicos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Estructura Molecular , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/aislamiento & purificación , Ácidos Teicoicos/química , Ácidos Urónicos/químicaRESUMEN
Bifidobacteria are colonizers of the human gut, where they are interacting with their host as well as with other members of the intestinal microbiota. Teichoic acids (TAs) have previously been shown to play an important role in modulating microbe-host interactions in the human gut. However, so far, there is a paucity of information regarding the presence of TAs in the cell envelope of bifidobacteria. In silico analyses targeting the chromosomes of all 48 (sub)species that currently represent the genus Bifidobacterium revealed the presence of genes responsible for TA biosynthesis, suggesting that bifidobacteria contain both wall TAs and lipoteichoic acids. Transcriptome analyses of the infant gut commensal Bifidobacterium bifidum PRL2010 highlighted that the transcription of the presumptive TA biosynthetic loci is modulated in response to environmental conditions reflecting those of the human gut. Furthermore, chemical characterization of TAs produced by PRL2010 indicates the presence of lipoteichoic acids.
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Bifidobacterium/enzimología , Bifidobacterium/genética , Vías Biosintéticas/genética , Genoma Bacteriano , Ácidos Teicoicos/biosíntesis , Animales , Simulación por Computador , Microbioma Gastrointestinal/fisiología , Tracto Gastrointestinal/química , Tracto Gastrointestinal/fisiología , Perfilación de la Expresión Génica , Humanos , Lactante , Lipopolisacáridos/genética , Lipopolisacáridos/aislamiento & purificación , Ratones , Probióticos , Ácidos Teicoicos/química , Ácidos Teicoicos/genética , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
BACKGROUND: Ultraviolet (UV) irradiation from the sun is the primary environmental factor that causes human skin aging. UV irradiation induces the expressions of matrix metalloproteinases (MMPs) and extracellular matrix degrading enzymes. Among the members of MMP family, MMP-1 is an interstitial collagenase that degrades the collagen triple helix. We investigated the effect of Lactobacillus plantarum, well known as useful microorganism, on UV-induced-MMP-1 expression in human dermal fibroblasts. METHODS: Human dermal fibroblasts (HDF) was pre-stimulated with lipoteichoic acid isolated from L. plantarum followed by UV irradiation. Secreted protein level of MMP-1 was evaluated by Western blot analysis. The phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) from the cell lysates was also examined by western blotting. Electrophoretic mobility-shift assay (EMSA) was used to detect the activated transcription factor, AP-1 and NF-κB. The detection of type 1 procollagen was carried with Procollagen type 1 C-peptide (PIP) EIA kit. The generation of reactive oxygen species (ROS) by LTA and UV irradiation was examined by Griess reagent assay and fluorescence microscope. RESULTS: We found that lipoteichoic acid (LTA), a cell-wall component of Gram-positive bacteria, isolated from L. plantarum, inhibited MMP-1 expression. Pretreatment with LTA from L. plantarum (pLTA) reduced MMP-1 expression in a dose-dependent manner and inhibited activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). It also led to the inhibition of DNA binding activity of activator protein-1 (AP-1) and of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB). Furthermore, LTA promoted type 1 procollagen synthesis and reduced the generation of ROS induced by UV irradiation. CONCLUSION: Our study demonstrates that pLTA inhibits degradation of collagen and promotes its synthesis and that pLTA contributes to a decrease in ROS production. Therefore, pLTA from L. plantarum has potential abilities to prevent and treat skin photo-aging.
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Colágeno Tipo I/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Lactobacillus plantarum/química , Lipopolisacáridos/farmacología , Metaloproteinasa 1 de la Matriz/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácidos Teicoicos/farmacología , Rayos Ultravioleta , Regulación hacia Arriba/efectos de los fármacos , Acetilcisteína/farmacología , Regulación hacia Abajo/efectos de la radiación , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/efectos de la radiación , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/aislamiento & purificación , FN-kappa B/metabolismo , Ácidos Teicoicos/aislamiento & purificación , Factor de Transcripción AP-1/metabolismoRESUMEN
Heat-killed Lactobacillus plantarum strain L-137 (HK L-137) is a more potent inducer of interleukin (IL)-12 than other heat-killed Lactobacillus strains. To elucidate the mechanism involved in this IL-12p40 induction, we compared HK L-137 with heat-killed L. plantarum strain JCM1149 (HK JCM1149) by nuclear magnetic resonance and mass spectrometry. Results showed that HK L-137 contained lipoteichoic acid (LTA) with a chemical structure similar to that of JCM1149, except for a lower degree of glucosyl substitution in the poly(glycerol phosphate) backbone. Lysozyme sensitivity and electrophoretic moiety analysis revealed that HK L-137 exposed more LTA on its cell surface than HK JCM1149. Phagocytosis of HK L-137 by splenic adherent cells was significantly greater than that of HK JCM1149. Anti-LTA antibody and anti-scavenger receptor-A (SR-A) antibody selectively inhibited phagocytosis of HK L-137, as well as IL-12p40 production, by splenic adherent cells. Thus, a higher efficiency of phagocytosis of HK L-137 via SR-A for LTA is responsible for the potent IL-12p40 induction.
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Subunidad p40 de la Interleucina-12/biosíntesis , Lactobacillus plantarum/metabolismo , Lipopolisacáridos/metabolismo , Receptores Depuradores/metabolismo , Ácidos Teicoicos/metabolismo , Animales , Anticuerpos/farmacología , Citocinas , Femenino , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Lipopolisacáridos/aislamiento & purificación , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Receptores de IgG/genética , Receptores Depuradores/inmunología , Ácidos Teicoicos/química , Ácidos Teicoicos/inmunología , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
The structures of the cell wall teichoic acids (TA) from some species of the genus Nocardiopsis were established by chemical and NMR spectroscopic methods. The cell walls of Nocardiopsis synnemataformans VKM Ac-2518(T) and Nocardiopsis halotolerans VKM Ac-2519(T) both contain two TA with unique structures-poly(polyol phosphate-glycosylpolyol phosphate)-belonging to the type IV TA. In both organisms, the minor TA have identical structures: poly(glycerol phosphate-N-acetyl-ß-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-4 of the amino sugar. This structure is found for the first time. The major TA of N. halotolerans has a hitherto unknown structure: poly(glycerol phosphate-N-acetyl-ß-galactosaminylglycerol phosphate), the N-acetyl-ß-galactosamine being acetalated with pyruvic acid at positions 4 and 6. The major TA of N. synnemataformans is a poly(glycerol phosphate-N-acetyl-ß-galactosaminylglycerol phosphate) with the phosphodiester bond between C-3 of glycerol and C-3 of the amino sugar. The cell walls of Nocardiopsis composta VKM Ac-2520 and N. composta VKM Ac-2521(T) contain only one TA, namely 1,3-poly(glycerol phosphate) partially substituted with N-acetyl-α-glucosamine. The cell wall of Nocardiopsis metallicus VKM Ac-2522(T) contains two TA. The major TA is 1,5-poly(ribitol phosphate), each ribitol unit carrying a pyruvate ketal group at positions 2 and 4. The structure of the minor TA is the same as that of N. composta. The results presented correlate well with the phylogenetic grouping of strains and confirm the species and strain specific features of cell wall TA in members of the genus Nocardiopsis.
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Actinobacteria/química , Pared Celular/química , Ácidos Teicoicos/química , Ácidos Teicoicos/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
It is known that lactic acid bacteria (LAB) have many beneficial health effects, including antioxidative activity and immune regulation. In this study, the immune regulatory effects of Lactobacillus sakei and Lactobacillus plantarum, which are found in different types of kimchi, were evaluated. L. sakei and its lipoteichoic acid (LTA) have greater immune stimulating potential in IL-12, IFN-γ, and TNF-α production as compared with L. plantarum in an in vitro condition. On the other hand, L. plantarum is assumed to repress the Th1 immune response in murine experiments. After being injected with LPS, L. plantarum-fed mice maintained a healthier state, and the level of TNF-α in their blood was lower than in other bacterial strainfed mice and in the LPS-only control mice. Additionally, IL-12 production was significantly decreased and the production of IL-4 was greatly increased in the splenocytes from L. plantarum-fed mice. Further experiments revealed that the pre-injection of purified LTA from L. plantarum (pLTA), L. sakei (sLTA), and S. aureus (aLTA) decreased TNF-α and IL-4 production in LPS-injected mice. Mouse IL-12, however, was significantly increased by aLTA pre-injection. In conclusion, the L. sakei and L. plantarum strains have immune regulation effects, but the effects differ in cytokine production and the regulatory effects of the Th1/Th2 immune response.
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Microbiología de Alimentos , Lactobacillus/crecimiento & desarrollo , Lactobacillus/inmunología , Animales , Células Cultivadas , Citocinas/sangre , Citocinas/metabolismo , Lactobacillus/aislamiento & purificación , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Ácidos Teicoicos/inmunología , Ácidos Teicoicos/aislamiento & purificaciónRESUMEN
Understanding molecular interactions of bacteria with their environment requires the purification and characterization of cell wall components. Here, we describe detailed experimental methods for the extraction, purification, and analysis of wall teichoic acids (WTA), which assume important roles as major constituents of Gram-positive cell walls, such as mediating interaction with cell wall-associated proteins, eukaryotic host cells, and bacteriophages. Specifically, we present a procedure for compositional WTA characterization to study large diversity of carbohydrate substitution on Listeria monocytogenes WTA. This protocol may also be used and adapted to analyze WTA from other bacteria.