RESUMEN
Teichoic acids (TAs) are key components of the Gram-positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well-defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We present here a streamlined automated solid-phase synthesis approach for the rapid generation of well-defined glycosylated, glycerol-based TA oligomers. Building on the use of a "universal" linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated. These are used to create a structurally diverse TA-microarray, which is used to reveal, for the first time, the binding preferences of anti-LTA (lipoteichoic acids) antibodies at the molecular level.
Asunto(s)
Ácidos Teicoicos/síntesis química , Alanina/metabolismo , Pared Celular/química , Glicosilación , Bacterias Grampositivas/química , Bacterias Grampositivas/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/inmunología , Estructura Molecular , Técnicas de Síntesis en Fase Sólida , Alcoholes del Azúcar/química , Ácidos Teicoicos/química , Ácidos Teicoicos/inmunologíaRESUMEN
Infections with Clostridium difficile increasingly cause morbidity and mortality worldwide. Bacterial surface glycans including lipoteichoic acid (LTA) were identified as auspicious vaccine antigens to prevent colonization. Here, we report on the potential of synthetic LTA glycans as vaccine candidates. We identified LTA-specific antibodies in the blood of C. difficile patients. Therefore, we evaluated the immunogenicity of a semi-synthetic LTA-CRM197 glycoconjugate. The conjugate elicited LTA-specific antibodies in mice that recognized natural LTA epitopes on the surface of C. difficile bacteria and inhibited intestinal colonization of C. difficile in mice in vivo. Our findings underscore the promise of synthetic LTA glycans as C. difficile vaccine candidates.
Asunto(s)
Antibacterianos/farmacología , Vacunas Bacterianas/farmacología , Clostridioides difficile/efectos de los fármacos , Infecciones por Clostridium/tratamiento farmacológico , Lipopolisacáridos/farmacología , Polisacáridos/farmacología , Ácidos Teicoicos/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Vacunas Bacterianas/síntesis química , Vacunas Bacterianas/química , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Lipopolisacáridos/síntesis química , Lipopolisacáridos/química , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Polisacáridos/síntesis química , Polisacáridos/química , Ácidos Teicoicos/síntesis química , Ácidos Teicoicos/químicaRESUMEN
The first synthesis of different Enterococcus faecium wall teichoic acid (WTA) fragments is presented. The structure of these major cell wall components was elucidated recently and it was shown that these glycerolphosphate (GroP) based polymers are built up from -6-(GalNAc-α(1-3)-GalNAc-ß(1-2)-GroP)- repeating units. We assembled WTA fragments up to three repeating units in length, in two series that differ in the stereochemistry of the glycerolphosphate moiety. The key GalNAc-GalNAc-GroP synthons, required for the synthesis, were generated from galactosazide building blocks that were employed in highly stereoselective glycosylation reactions to furnish both the α- and ß-configured linkages. By comparing the NMR spectra of the synthesized fragments with the isolated material it appears that the hereto undefined stereochemistry of the glycerol phosphate moiety is sn-glycerol-3-phosphate. The generated fragments will be valuable tools to study their immunological activity at the molecular level.
Asunto(s)
Pared Celular/química , Enterococcus faecium/química , Ácidos Teicoicos/síntesis química , Secuencia de Carbohidratos , EstereoisomerismoRESUMEN
The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure-activity relationship studies. To ensure reliable biological results, pure and well-defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.
Asunto(s)
Clostridioides difficile/química , Enterocolitis Seudomembranosa/microbiología , Lipopolisacáridos/síntesis química , Ácidos Teicoicos/síntesis química , Humanos , Interleucina-6/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Espectroscopía de Resonancia Magnética , Ácidos Teicoicos/química , Ácidos Teicoicos/farmacologíaRESUMEN
Clostridium difficile is a leading cause of severe nosocomial infections. Cell-surface carbohydrate antigens are promising vaccine candidates. Here we report the first total synthesis of oligomers of the lipoteichoic acid antigen repeating unit. Synthetic glycan microarrays revealed anti-glycan antibodies in the blood of patients that help to define epitopes for vaccine development.
Asunto(s)
Vacunas Bacterianas/síntesis química , Clostridioides difficile , Lipopolisacáridos/química , Polisacáridos/química , Ácidos Teicoicos/química , Vacunas Bacterianas/química , Vacunas Bacterianas/farmacología , Clostridioides difficile/efectos de los fármacos , Humanos , Lipopolisacáridos/síntesis química , Análisis por Micromatrices , Polisacáridos/farmacología , Polisacáridos/uso terapéutico , Ácidos Teicoicos/síntesis químicaRESUMEN
In this study, the role of lipoteichoic acid (LTA) anchors in the activation of the innate immune response was investigated through the chemical synthesis of a series of LTA derivatives and the determination of their ability to induce NO production in bone marrow-derived macrophages (BMM). To this end, an efficient synthesis of the sn-3-O-(α-D-galactofuranosyl)-1,2-di-O-acylglycerol LTA core was developed, which was then used as a key structure to produce both phosphate and glycerylphosphate-funtionalised LTA anchors, as well as galactofuranosyldiglycerides with different fatty acid chain lengths. With a series of LTA anchors in hand, we then determined the effect of these glycolipids on the innate immune response by exploring their capacity to activate macrophages. Here, we report that several of the LTA-derivatives were able to induce NO production by BMMs. In general, the unnatural (sn-1) core glycolipid anchors showed lower levels of activity than the corresponding natural (sn-3) analogues, and the activity of the glycolipids also appears to be dependent on the length of lipid present, with an optimum lipid length of C20 for the sn-3 derivatives. Interestingly, a triacylated anchor and the 6-O-phosphorylated anchor, showed only modest activity, while the 6-O-glycerophosphorylated derivative was unable to induce NO production. Taken as a whole, our results highlight the subtle effects that glycolipid length can have on the ability to activate BMMs.
Asunto(s)
Glucolípidos/química , Lipopolisacáridos/farmacología , Streptococcus/química , Ácidos Teicoicos/farmacología , Animales , Glucolípidos/metabolismo , Lipopolisacáridos/síntesis química , Lipopolisacáridos/química , Macrófagos/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Ácidos Teicoicos/síntesis química , Ácidos Teicoicos/químicaRESUMEN
We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a terminal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers featuring a terminal alcohol functionality. These complementary linker systems have been developed because the nature of the TA chain terminus can play a role in the biological activity of the synthetic TAs. A novel α-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust light fluorous synthetic protocol.
Asunto(s)
Enterococcus faecalis/química , Hidrocarburos Fluorados/química , Ácidos Teicoicos/síntesis química , Pared Celular/química , Glicosilación , Estructura Molecular , Compuestos Organofosforados/químicaRESUMEN
The use of perfluorooctylpropylsulfonylethanol as a new phosphate protecting group and fluorous linker is evaluated in the stepwise solution phase synthesis of a number of biologically relevant (carbohydrate substituted) glycerol teichoic acid fragments. Teichoic acid fragments, up to the dodecamer level, were assembled by means of phosphoramidite chemistry, using a relatively small excess of the building blocks and a repetitive efficient purification procedure of the protected intermediates by fluorous solid phase extraction (F-SPE).
Asunto(s)
Flúor/química , Ácidos Teicoicos/síntesis química , Glucosamina/química , Estructura MolecularRESUMEN
This communication describes the first automated solid phase synthesis of teichoic acids (TAs) and the preparation by this method of a number of well-defined TA structures, which were probed for their antigenicity. An opsonophagocytic killing assay revealed a clear TA-length-activity relationship and indicated a promising candidate for future vaccine development.
Asunto(s)
Ácidos Teicoicos/química , Animales , Anticuerpos/inmunología , Enterococcus faecalis/efectos de los fármacos , Glucosa/química , Glicerol/química , Conejos , Ácidos Teicoicos/síntesis química , Ácidos Teicoicos/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunologíaRESUMEN
During infections caused by Gram-negative bacteria, lipopolysaccharide (LPS, endotoxin) has a dominant role leading to fulminant pro-inflammatory reactions in the host. As there is no LPS in Gram-positive bacteria, other microbial cell wall components have been identified to be the causative agent for the pro-inflammatory activity since also Gram-positive bacterial infections lead to comparable clinical symptoms and reactions. On search for the "Gram-positive endotoxin" a widely accepted hypothesis has been raised in that the lipoteichoic acids (LTAs) serve as pathogen-associated molecular patterns (PAMPs) during Gram-positive sepsis, although the amount necessary for a pro-inflammatory in vitro response is several orders of magnitude higher than that for LPS. Therefore, LTA cannot be considered to be "the (endo)toxin of Gram-positive bacteria". Although LPS and LTA show structural relatedness (amphiphilic, negatively charged glycophospholipids), they are structurally quite different from each other and one might expect that they are also recognized by different receptors of the innate immune system, the so called toll-like receptors 4 and 2 (TLR4 and TLR2), respectively. Based on their chemical structure, the LTAs were classified into four types (type I-IV) of which we have carefully investigated the LTA of Staphylococcus aureus (type I), Lactococcus garvieae (type II) and Streptococcus pneumoniae (type IV). Hence, these LTAs have been synthesized in our group and biologically evaluated with respect to their potency to activate cytokines in transiently TLR2/CD14-transfected human endothelial kidney cells (HEK 293) or human macrophages and whole blood cells. Although LTA of type I and IV are structurally quite different, especially in their hydrophilic moiety, they originally were believed to interact with the same receptor (TLR2). Hence, the chemical syntheses leading to structurally defined, non-contaminated stimuli have a major impact on the outcome and interpretation of these biological studies of the innate immune system. With this material, it became evident that synthetic LTA from S. aureus and S. pneumoniae are not recognized by TLR2. Instead, another receptor of the innate immune system, the lectin pathway of the complement, known since many years to interact with LTA in quite a specific way, has gained increasing attractivity. With the help of synthetic LTA we obtained first evidences that this receptor is indeed the pathogen recognition receptor (PRR) for LTA.
Asunto(s)
Lactococcus/química , Lipopolisacáridos/síntesis química , Staphylococcus aureus/química , Streptococcus pneumoniae/química , Ácidos Teicoicos/síntesis química , Humanos , Lipopolisacáridos/inmunología , Ácidos Teicoicos/inmunologíaRESUMEN
Streptococcus pneumoniae LTA is a highly complex glycophospholipid that consists of nine carbohydrate residues: three glucose, two galactosamine and two 2-acetamino-4-amino-2,4,6-trideoxygalactose (AATDgal) residues that are each differently linked, one ribitol and one diacylated glycerol (DAG) residue. Suitable building blocks for the glucose and the AATDgal residues were designed and their synthesis is described in this paper. These building blocks permitted the successful synthesis of the core structure Glcß(1-3)AATDgalß(1-3)Glcα(1-O)DAG in a suitably protected form for further chain extension (1 b, 1 c) and as unprotected glycolipid (1 a) that was employed in biological studies. These studies revealed that 1 a as well as 1 lead to interleukin-8 release, however not via TLR2 or TLR4 as receptor.
Asunto(s)
Lipopolisacáridos/síntesis química , Streptococcus pneumoniae/inmunología , Ácidos Teicoicos/síntesis química , Glucolípidos/química , Células HEK293 , Humanos , Interleucina-8/sangre , Interleucina-8/efectos de los fármacos , Lipopolisacáridos/sangre , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Estructura Molecular , Streptococcus pneumoniae/química , Ácidos Teicoicos/sangre , Ácidos Teicoicos/química , Ácidos Teicoicos/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
The lipoteichoic acid (LTA) of the Streptococcus species DSM 8747 consists of a beta-d-galactofuranosyl diacylglycerol moiety (with different acyl groups) that is linked via 6-O to a poly(glycerophosphate) backbone; about 30% of the glycerophosphate moieties carry at 2-O hydrolytically labile d-alanyl residues. As typical LTA for this array of compounds LTA 1a was synthesized. To this end, from d-galactose the required galactofuranosyl building block 5 was obtained. The anomeric stereocontrol in the glycosylation step with 1,2-O-cyclohexylidene-sn-glycerol (4) was based on anchimeric assistance, thus finally leading to the unprotected core glycolipid 16. Regioselective protection and deprotection procedures permitted the defined attachment of the pentameric glycerophosphate 3 to the 6-hydroxy group of the galactose residue. Introduction of four d-alanyl residues led after global deprotection and purification to target molecule 1a possessing on average about two d-alanyl residues at 2-O of the pentameric glycerophosphate backbone, thus being in close accordance with the structure of the natural material.
Asunto(s)
Lipopolisacáridos/síntesis química , Streptococcus/química , Ácidos Teicoicos/síntesis química , Productos Biológicos/síntesis química , Galactosa/química , Glicerofosfatos/química , GlicosilaciónRESUMEN
In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors, provided with various protecting groups at the C2 hydroxyl function with an orthogonally protected glycerol acceptor. After selective deprotection, the resulting 1,2-cis-linked pseudodisaccharide acceptor was coupled to an alpha-directing thioglucose donor, giving the corresponding pseudotrisaccharide, which is then transformed to a phosphoramidite synthon. The kojibiosyl-glycerol phosphoramidite in combination with a glycerolphosphoramidite, an aminohexylphosphoramidite and dibenzylglycerol were coupled to a fully protected glycerol TA hexamer, using chemistry that can be amended for future automated synthesis. Global deprotection afforded the target hexamer kojibiosyl-glycerol containing TA (1).
Asunto(s)
Disacáridos/química , Ácidos Teicoicos/síntesis química , Dimerización , Disacáridos/síntesis química , Enterococcus faecalis/química , Glicerol , Compuestos Organofosforados , Ácidos Teicoicos/químicaRESUMEN
Lipoteichoic acid (LTA) from gram-positive bacteria is the counterpart to lipopolysaccharide from gram-negative bacteria. LTA, which activates Toll-like receptor 2 (TLR2), induces a unique cytokine and chemokine pattern. The chemical synthesis of LTA proved its immunostimulatory properties. To determine the minimal active structure of LTA, we reduced synthetic LTA in a number of steps down to the synthetic anchor and employed these molecules to stimulate interleukin-8 (IL-8) release in human whole blood. Ten times more of the synthetic structures with four to six d-alanine-substituted polyglycerophosphate units (50 nM) than of the native LTA preparation was required to induce IL-8 release. A further reduction to three backbone units with two or no d-alanine residues resulted in cytokine induction only from 500 nM. The synthetic anchor was not able to induce IL-8 release even at 5 muM. When the LTA derivatives were used at 500 nM, they induced increasing levels of IL-8 and tumor necrosis factor alpha with increasing elongation of the backbone. Peritoneal macrophages were less responsive than human blood to the synthetic structures. Therefore, TLR2 dependency could be shown only with cells from TLR2-deficient mice for the two largest synthetic structures. This was confirmed by using TLR2-transfected HEK 293 cells. Taken together, these data indicate that although the synthetic anchor (which, unlike the native anchor, contains only myristic acid) cannot induce cytokine release, the addition of three backbone units, even without d-alanine substituents, confers this ability. Lengthening of the chain with d-alanine-substituted backbone units results in increased cytokine-inducing potency and a more sensitive response.
Asunto(s)
Citocinas/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Ácidos Teicoicos/química , Ácidos Teicoicos/farmacología , Animales , Línea Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Humanos , Riñón/citología , Lipopolisacáridos/síntesis química , Ratones , Ratones Noqueados , Modelos Químicos , Staphylococcus aureus/química , Estadística como Asunto , Ácidos Teicoicos/síntesis química , Receptor Toll-Like 2/metabolismo , TransfecciónRESUMEN
For the investigation of the minimal structural requirements for cytokine induction, Staphylococcus aureus lipoteichoic acid derivatives with two, three, four, and five glycerophosphate backbone moieties, carrying each a d-alanyl residue, were needed. Based on two different glycerophosphate building blocks and 6b-O-phosphitylated gentiobiosyl diacylglycerol the desired target molecules (compounds 1-4) could be readily obtained and provided for biological studies.
Asunto(s)
Citocinas/biosíntesis , Lipopolisacáridos/síntesis química , Ácidos Teicoicos/síntesis química , Disacáridos/química , Glicerofosfatos/química , Staphylococcus aureus/química , Relación Estructura-ActividadRESUMEN
Based on 1,2-O-isopropylidene-sn-glycerol five chiral building blocks containing differently modified glycerol residues were required for the synthesis of the target molecule 2. One of these building blocks is diacylglyceryl beta-gentiobioside carrying a phosphite residue at 6b-O position. Ligation of these five building blocks led to the desired glycerol phosphate backbone to which d-alanyl residues were attached, thus generating after O-deprotection the target molecule 2, a bisamphiphilic structural variant of Staphylococcus aureus LTA. This compound displayed higher potency in terms of cytokine release by human blood leukocytes than the monoamphiphilic variant LTA.
Asunto(s)
Diglicéridos/química , Lipopolisacáridos/química , Staphylococcus aureus/química , Ácidos Teicoicos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Citocinas/biosíntesis , Citocinas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Leucocitos/efectos de los fármacos , Lipopolisacáridos/síntesis química , Lipopolisacáridos/farmacología , Conformación Molecular , Datos de Secuencia Molecular , Estereoisomerismo , Relación Estructura-Actividad , Ácidos Teicoicos/síntesis química , Ácidos Teicoicos/farmacologíaRESUMEN
The controversy about the immune stimulatory properties of lipoteichoic acid (LTA) from Staphylococcus aureus was solved recently by showing decomposition and inactivation of LTA obtained by conventional purification strategies, as well as pronounced LPS contamination of commercial preparations. By introducing a novel preparation method, the structure of bioactive LTA was elucidated. This structure was confirmed by chemical synthesis. In this work, synthetic LTA derivatives were employed to study the structure-function relationship of cytokine induction in human monocytes. Synthetic LTA induced the same cytokine pattern as highly purified natural LTA. The gentiobiose core could be omitted without affecting bioactivity. The polyglycerophosphate backbone amplified the response to the lipid anchor ( approximately 100-fold) only when substituted with D-alanine, whereas alpha-D-N-acetylglucosamine substituents could be omitted. Replacing D-alanine substituents with L-alanine reduced the activity of the molecule at least 10-fold, indicating stereoselectivity. These results define for the first time the crucial patterns required for the immune recognition of LTA.