Streamlined Synthesis and Evaluation of Teichoic Acid Fragments.
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ABSTRACT:
SUBMITTER: van der Es D
PROVIDER: S-EPMC5887911 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; β-GlcNAc at the RboP C4; β-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.
| S-EPMC8361686 | biostudies-literature
Project description:1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.
| S-EPMC1166238 | biostudies-other
Project description:The regulation of bile acid pathways has become a particularly promising therapeutic strategy for a variety of metabolic disorders, cancers, and diseases. However, the hydrophobicity of bile acids has been an obstacle to clinical efficacy due to off-target effects from rapid drug absorption. In this report, we explored a novel strategy to design new structure fragments based on lithocholic acid (LCA) with improved hydrophilicity by introducing a polar "oxygen atom" into the side chain of LCA, then (i) either retaining the carboxylic acid group or replacing the carboxylic acid group with (ii) a diol group or (iii) a vinyl group. These novel fragments were evaluated using luciferase-based reporter assays and the MTS assay. Compared to LCA, the result revealed that the two lead compounds 1a-1b were well tolerated in vitro, maintaining similar potency and efficacy to LCA. The MTS assay results indicated that cell viability was not affected by dose dependence (under 25 µM). Additionally, computational model analysis demonstrated that compounds 1a-1b formed more extensive hydrogen bond networks with Takeda G protein-coupled receptor 5 (TGR5) than LCA. This strategy displayed a potential approach to explore the development of novel endogenous bile acids fragments. Further evaluation on the biological activities of the two lead compounds is ongoing.
| S-EPMC10383687 | biostudies-literature
Project description:Streptococcus pneumoniae has unusually complex cell wall teichoic acid and lipoteichoic acid, both of which contain a ribitol phosphate moiety. The lic region of the pneumococcal genome contains genes for the uptake and activation of choline, the attachment of phosphorylcholine to teichoic acid precursors, and the transport of these precursors across the cytoplasmic membrane. The role of two other, so far uncharacterized, genes, spr1148 and spr1149, in the lic region was determined. TarJ (spr1148) encodes an NADPH-dependent alcohol dehydrogenase for the synthesis of ribitol 5-phosphate from ribulose 5-phosphate. TarI (spr1149) encodes a cytidylyl transferase for the synthesis of cytidine 5'-diphosphate (CDP)-ribitol from ribitol 5-phosphate and cytidine 5'-triphosphate. We also present the crystal structure of TarI with and without bound CDP, and the structures present a rationale for the substrate specificity of this key enzyme. No transformants were obtained with insertion plasmids designed to interrupt the tarIJ genes, indicating that their function could be essential for cell growth. CDP-activated ribitol is a precursor for the synthesis of pneumococcal teichoic acids and some of the capsular polysaccharides. Thus, all eight genes in the lic region have a role in teichoic acid synthesis.
| S-EPMC2631998 | biostudies-literature
Project description:BackgroundStaphylococcus aureus or MRSA (Methicillin Resistant S. aureus), is an acquired pathogen and the primary cause of nosocomial infections worldwide. In S. aureus, teichoic acid is an essential component of the cell wall, and its biosynthesis is not yet well characterized. Studies in Bacillus subtilis have discovered two different pathways of teichoic acid biosynthesis, in two strains W23 and 168 respectively, namely teichoic acid ribitol (tar) and teichoic acid glycerol (tag). The genes involved in these two pathways are also characterized, tarA, tarB, tarD, tarI, tarJ, tarK, tarL for the tar pathway, and tagA, tagB, tagD, tagE, tagF for the tag pathway. With the genome sequences of several MRSA strains: Mu50, MW2, N315, MRSA252, COL as well as methicillin susceptible strain MSSA476 available, a comparative genomic analysis was performed to characterize teichoic acid biosynthesis in these S. aureus strains.ResultsWe identified all S. aureus tar and tag gene orthologs in the selected S. aureus strains which would contribute to teichoic acids sythesis. Based on our identification of genes orthologous to tarI, tarJ, tarL, which are specific to tar pathway in B. subtilis W23, we also concluded that tar is the major teichoic acid biogenesis pathway in S. aureus. Further analyses indicated that the S. aureus tar genes, different from the divergon organization in B. subtilis, are organized into several clusters in cis. Most interesting, compared with genes in B. subtilis tar pathway, the S. aureus tar specific genes (tarI,J,L) are duplicated in all six S. aureus genomes.ConclusionIn the S. aureus strains we analyzed, tar (teichoic acid ribitol) is the main teichoic acid biogenesis pathway. The tar genes are organized into several genomic groups in cis and the genes specific to tar (relative to tag): tarI, tarJ, tarL are duplicated. The genomic organization of the S. aureus tar pathway suggests their regulations are different when compared to B. subtilis tar or tag pathway, which are grouped in two operons in a divergon structure.
| S-EPMC1458327 | biostudies-literature
Project description:UnlabelledNasal colonization by the human pathogen Staphylococcus aureus is a major risk factor for hospital- and community-acquired infections. A key factor required for nasal colonization is a cell surface-exposed zwitterionic glycopolymer, termed wall teichoic acid (WTA). However, the precise mechanisms that govern WTA-mediated nasal colonization have remained elusive. Here, we report that WTA GlcNAcylation is a pivotal requirement for WTA-dependent attachment of community-acquired methicillin-resistant S. aureus (MRSA) and emerging livestock-associated MRSA to human nasal epithelial cells, even under conditions simulating the nutrient composition and dynamic flow of nasal secretions. Depending on the S. aureus strain, WTA O-GlcNAcylation occurs in either α or β configuration, which have similar capacities to mediate attachment to human nasal epithelial cells, suggesting that many S. aureus strains maintain redundant pathways to ensure appropriate WTA glycosylation. Strikingly, a lack of WTA glycosylation significantly abrogated the ability of MRSA to colonize cotton rat nares in vivo. These results indicate that WTA glycosylation modulates S. aureus nasal colonization and may help to develop new strategies for eradicating S. aureus nasal colonization in the future.ImportanceNasal colonization by the major human pathogen Staphylococcus aureus is a risk factor for severe endogenous infections and contributes to the spread of this microbe in hospitals and the community. Here, we show that wall teichoic acid (WTA) O-GlcNAcylation is a key factor required for S. aureus nasal colonization. These data provide a mechanistic explanation for the capacity of WTA to modulate S. aureus nasal colonization and may stimulate research activities to establish valuable strategies to eradicate S. aureus nasal colonization in high-risk hospitalized patients and in the general community.
| S-EPMC4488942 | biostudies-literature
Project description:Peptidoglycan and teichoic acids are the major cell wall components of Gram-positive bacteria that obtain and sequester metal ions required for biochemical processes. The delivery of metals to the cytoplasmic membrane is aided by anionic binding sites within the peptidoglycan and along the phosphodiester polymer of teichoic acid. The interaction with metals is a delicate balance between the need for attraction and ion diffusion to the membrane. Likewise, metal chelation from the extracellular fluid must initially have strong binding energetics that weaken within the cell wall to enable ion release. We employed atomic absorption and equilibrium dialysis to measure the metal binding capacity and metal binding affinity of wall teichoic acid and Mg2+. Data show that Mg2+ binds to WTA with a 1:2Mg2+ to phosphate ratio with a binding capacity of 1.27 μmol/mg. The affinity of Mg2+ to WTA was also found to be 41×10(3) M(-1) at low metal concentrations and 1.3×10(3) M(-1) at higher Mg2+ concentrations due to weakening electrostatic effects. These values are lower than the values describing Mg2+ interactions with peptidoglycan. However, the binding capacity of WTA is 4 times larger than peptidoglycan. External WTA initially binds metals with positive cooperativity, but metal binding switches to negative cooperativity, whereas interior WTA binds metals with only negative cooperativity. The relevance of this work is to describe changes in metal binding behavior depending on environment. When metals are sparse, chelation is strong to ensure survival yet the binding weakens when essential minerals are abundant.
| S-EPMC4554814 | biostudies-literature
Project description:An efficient route to various vancoresmycin-type tetramic acids has been developed. The modular route is based on an effective Fries-type rearrangement to introduce various appending acetyl residues. The minimum inhibitory concentration (MIC) values of the new tetramic acids against Staphylococcus aureus and Escherichia coli were determined, revealing that three of the new compounds exhibit antimicrobial activity against S. aureus. These bioactive compounds were structurally most closely related to the authentic vancoresmycin building block. Additionally, the compounds induced a lial-lux bioreporter, which responds to cell wall stress induced by antibiotics that interfere with the lipid II biosynthesis cycle. These data suggest the tetramic acid moiety to be a part of the vancoresmycin pharmacophore.
| S-EPMC7496136 | biostudies-literature
Project description:Lipoteichoic acids (LTA) are amphiphilic polymers that are important constituents of the cell wall of many Gram-positive bacteria. The chemical structures of LTA vary among organisms, albeit in the majority of Gram-positive bacteria the LTAs feature a common poly-1,3-(glycerolphosphate) backbone. Previously, the specificity of opsonic antibodies for this backbone present in some Gram-positive bacteria has been demonstrated, suggesting that this minimal structure may be sufficient for vaccine development. In the present work, we studied a well-defined synthetic LTA-fragment, which is able to inhibit opsonic killing of polyclonal rabbit sera raised against native LTA from Enterococcus faecalis 12030. This promising compound was conjugated with BSA and used to raise rabbit polyclonal antibodies. Subsequently, the opsonic activity of this serum was tested in an opsonophagocytic assay and specificity was confirmed by an opsonophagocytic inhibition assay. The conjugated LTA-fragment was able to induce specific opsonic antibodies that mediate killing of the clinical strains E. faecalis 12030, Enterococcus faecium E1162, and community-acquired Staphylococcus aureus strain MW2 (USA400). Prophylactic immunization with the teichoic acid conjugate and with the rabbit serum raised against this compound was evaluated in active and passive immunization studies in mice, and in an enterococcal endocarditis rat model. In all animal models, a statistically significant reduction of colony counts was observed indicating that the novel synthetic LTA-fragment conjugate is a promising vaccine candidate for active or passive immunotherapy against E. faecalis and other Gram-positive bacteria.
| S-EPMC4205086 | biostudies-literature
Project description:Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with D-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only alpha-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA alpha-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria.
| S-EPMC2859500 | biostudies-literature