D-alanine Ester Research Articles

Dissecting the Bacterial Cell Wall with Solid-State NMR

Gram-positive bacteria surround themselves with a macromolecular cell wall that is essential to cell survival and growth and serves as a major target for antibiotics. While the cell wall of S. aureus is comprised of only two major structural components, peptidoglycan and teichoic acids, it is a thick and heterogeneous matrix that is difficult to characterize with traditional biophysical techniques like x-ray crystallography and mass spectrometry. We use solid-state NMR spectroscopy to study the composition of isolated, but intact cell walls and also whole cells to measure and quantitatively compare chemical and atomic-level structural parameters across different strains and conditions, particularly to examine the modes of action of old and new antibiotics. We have introduced a new approach to measure the relative balance of teichoic acids and peptidoglycan within the cell wall structure. We extend this analysis to whole cells, where using spectral signatures of the cell wall, we identify distinct resonances to measure teichoic acid levels in intact bacterial cells. The distinguishing chemical shifts of the sugars and peptides in peptidoglycan, along with the phosphoribitol backbone and D-alanine ester decorations of teichoic acids provide insight into peptidoglycan and teichoic acid levels, respectively, in whole cell NMR samples. This analysis did not require isotopic labeling and can be performed with any bacterial strain. We do often couple such measurements with isotopic labeling and selective NMR detection schemes to fully dissect the atomic-level structure of the cell wall. We will also report on a focused set of results regarding the influence of growth state and the influence of inhibitors of teichoic acid synthesis on overall cell wall composition.

DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects.

The dlt operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes dltXABCD but the involvement of the first ORF (dltX) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in Bacillus thuringiensis. We, therefore constructed an in frame deletion mutant of dltX, without affecting the expression of the other genes of the operon. The growth characteristics of the dltX mutant and those of the wild type strain were similar under standard in vitro conditions. However, disruption of dltX drastically impaired the resistance of B. thuringiensis to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the dltX mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of B. thuringiensis in insects. To our knowledge, this work is the first report examining the involvement of dltX in the D-alanylation of TAs.

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus, Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

Chiral layered molecular spaces with amino acids

Novel chiral layered molecular space L-(+)-leucine amidepropylamidephenylsilica (L-(+)-Leu-APAPhS) and D-(−)-alanine amidepropylamidephenylsilica (D-(−)-Ala-APAPhS) were synthesized through grafting L-leucine tert-butyl ester (L-Leu-OtBu) and D-alanine tert-butyl ester (D-Ala-OtBu), in layered carboxylpropylamidephenylsilica (CPAPhS), which was developed in our previous research, with regular carboxyl groups, and the tert-butyl ester as protective groups of carboxyls in amino acids was cleaved with dilute trifluoroacetic acid (TFA). The improved XRD response indicated that the two dimensional layered structure had been retained in L-Leu-APAPhS and D-Ala-APAPhS. The chiral behaviors of layered molecular spaces CPAPhS, L-(+)-Leu-APAPhS and D-(−)-Ala-APAPhS were investigated. DL-Alanine tert-butyl ester (DL-Ala-OtBu) was grafted in the achiral layered molecular space CPAPhS as enantiomer pair, even if 70% L-Ala-OtBu and 30% D-Ala-OtBu existed in the reaction solution. L-(+)-Leu-APAPhS and D-(−)-Ala-APAPhS represented better chiral selectivity for amino acids both in chiral selective reactions and chiral column chromatography experiments, and exhibited the potential of chiral layered molecular space with an amino acid structure for chiral molecular recognition and selection processes. The formation of L-Leu-APAPhS-L-Ala-OMe also exhibited potential for that multi-amino acid (peptide) can be grafted in CPAPhS to form layered molecular spaces with peptides.

Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci

It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide 107R-A-W-V-A-W-R-N-R115 (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators (rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA/dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity (oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA/dltA or oatA/graRS double mutants are much more susceptible to lysozyme than each of the single mutants.

D‐Alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100‐23 results in impaired colonization of the mouse gastrointestinal tract

The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TA). D-alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of D-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of D-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that D-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.

Structure/function relationships of lipoteichoic acids

The role of lipoteichoic acids (LTAs) from Gram-positive bacteria as immunostimulatory molecules was controversial for many years, as inadequate preparation methods as well as heterogeneous and endotoxin-contaminated commercial preparations led to conflicting results. An improved purification methodology for LTA now yields potent bioactive and chemically defined material, which is currently being characterized in various models. A synthetic analogue of Staphylococcus aureus LTA has proven the structure/function relationship. The key role of D-alanine esters for the immune response of LTA was confirmed by synthetic derivatives. The glycolipid anchor of LTA plays a central role analogous to the lipid A of LPS. Methodological aspects and criteria for quality assessment of LTA preparations are discussed.

Structure/function relationships of lipoteichoic acids

The role of lipoteichoic acids (LTAs) from Gram-positive bacteria as immunostimulatory molecules was controversial for many years, as inadequate preparation methods as well as heterogeneous and endotoxin-contaminated commercial preparations led to conflicting results. An improved purification methodology for LTA now yields potent bioactive and chemically defined material, which is currently being characterized in various models. A synthetic analogue of Staphylococcus aureus LTA has proven the structure/function relationship. The key role of D-alanine esters for the immune response of LTA was confirmed by synthetic derivatives. The glycolipid anchor of LTA plays a central role analogous to the lipid A of LPS. Methodological aspects and criteria for quality assessment of LTA preparations are discussed.

Dynamic Kinetic Resolution of α‐Bromo Carboxylic Acid Derivatives in Asymmetric Nucleophilic Substitution with Chiral α‐Amino Esters.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Dynamic Kinetic Resolution of α-Bromo Carboxylic Acid Derivatives in Asymmetric Nucleophilic Substitution with Chiral α-Amino Esters

Dynamic kinetic resolution of -bromo carboxylic acid derivatives in nucleophilic substitution with chiral -amino ester nucleophiles in the presence of TBAI and DIEA has been investigated for stereoselective syntheses of 1,1'-iminodicarboxylic acid derivatives. Nucleophilic substitutions with various chiral -amino esters gave iminodiacetates 2-8 with stereoselectivities up to 87 : 13 dr. Also, the reactions of N-(-bromo--phenylacetyl)-L-alanine methyl ester with L-alanine, D-alanine and glycine methyl ester nucleophiles afforded N-carboxyalkyl dipeptide analogues 10-12 up to 90 : 10 dr.�⨀ԀĀ衺�⨀ԀĀऐ돀졽�⨀堘�⨀ꢐ�⨀돐좐�⨀��⨀킐�⨀뤏덐�⨀렏頏࢑�⨀਀࢑�⨀ᢑ�⨀؀ကᢑ�⨀?麆Ѐက䂑�⨀䂑�⨀

Key role of teichoic acid net charge in Staphylococcus aureus colonization of artificial surfaces.

Staphylococcus aureus is responsible for a large percentage of infections associated with implanted biomedical devices. The molecular basis of primary adhesion to artificial surfaces is not yet understood. Here, we demonstrate that teichoic acids, highly charged cell wall polymers, play a key role in the first step of biofilm formation. An S. aureus mutant bearing a stronger negative surface charge due to the lack of D-alanine esters in its teichoic acids can no longer colonize polystyrene or glass. The mutation abrogates primary adhesion to plastic while production of the glucosamine-based polymer involved in later steps of biofilm formation is not affected. Our data suggest that repulsive electrostatic forces can lead to reduced staphylococcal biofilm formation, which could have considerable impact on the design of novel implanted materials.

The D-alanine residues of Staphylococcus aureus teichoic acids alter the susceptibility to vancomycin and the activity of autolytic enzymes.

Recently, Staphylococcus aureus strains with intermediate resistance to vancomycin, the antibiotic of last resort, have been described. Multiple changes in peptidoglycan turnover and structure contribute to the resistance phenotype. Here, we describe that structural changes of teichoic acids in the cell envelope have a considerable influence on the susceptibility to vancomycin and other glycopeptides. S. aureus cells lacking D-alanine esters in teichoic acids exhibited an at least threefold-increased sensitivity to glycopeptide antibiotics. Furthermore, the autolytic activity of the D-alanine mutant was reduced compared to the wild-type, and the mutant was more susceptible to the staphylolytic enzyme lysostaphin. Vancomycin inhibited autolysis at very high concentrations but neither in the wild-type nor in the mutant was the autolytic activity influenced in the range of the MIC. Mutant cells had a considerably higher capacity to bind vancomycin.

Unique poly(glycerophosphate) lipoteichoic acid and the glycolipids of a Streptococcus sp. closely related to Streptococcus pneumoniae.

The Streptococcus sp. studied here is closely related to Streptococcus pneumoniae with 98.6% 16S rRNA similarity and 65% DNA/DNA homology. We isolated the lipoteichoic acid and the membrane glycolipids whose structures were established using conventional procedures and NMR spectroscopy. The lipoteichoic acid contains a linear 1,3-linked poly(glycerophosphate) chain which is partly substituted with D-alanine ester and is phosphodiester-linked to O6 of beta-D-Galf(1-->3)acyl2Gro. This lipoteichoic acid is the first example in which a monohexosylglycerol serves as the glycolipid anchor; and with an average chain length of 10 glycerophosphate residues it is the shortest known to date. MS analysis, applied for the first time to a native acylated lipoteichoic acid, revealed a continuous increase in chain length from seven to 17 glycerophosphate residues with a maximum at 10, and allowed identification of the fatty acid combinations. Membrane glycolipids consisted of beta-D-Galf(1-->3)acyl2Gro (9%), alpha-D-Glcp(1-->3)acyl2Gro (22%), alpha-D-Galp(1-->2)-alpha-D-Glcp(1-->3)acyl2Gro (64%) and alpha-D-Galp(1-->2)-(6-O-acyl)-alpha-D-Glcp(1-->3)acyl2Gro (5%). It is noteworthy that in lipoteichoic acid biosynthesis, Galfacyl2Gro, a less abundant membrane glycolipid, is selected as the lipid anchor. Despite the genetic relatedness to Streptococcus pneumoniae, the lipoteichoic acid structure is quite different to the complex structure of pneumococcal lipoteichoic acid [T. Behr et al. (1992) Eur. J. Biochem. 207, 1063-1075], thus providing an example that minor differences in DNA sequence exert major changes in macromolecular structure.

Inactivation of the dlt Operon inStaphylococcus aureus Confers Sensitivity to Defensins, Protegrins, and Other Antimicrobial Peptides

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.

The absence of D-alanine from lipoteichoic acid and wall teichoic acid alters surface charge, enhances autolysis and increases susceptibility to methicillin in Bacillus subtilis.

In Bacillus subtilis the physiological consequences of depriving lipoteichoic acid and wall teichoic acid of D-alanine ester were analysed using insertional inactivation of the genes of the dlt operon. Mutant strains which lacked positively charged D-alanine ester in teichoic acids bound more positively charged cytochrome C than other strains. These mutant strains also showed enhanced autolysis and a higher susceptibility to methicillin, which was expressed as accelerated wall lysis, a faster loss of viability and a slower recovery in the postantibiotic phase. The effects of methicillin could be suppressed by simultaneous addition of magnesium ions at low concentrations. The degradation of whole bacteria by bone-marrow-derived macrophages was not influenced by the surface charge and alanylation of the teichoic acids had no protective effect.

D-alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity.

Using insertional inactivation of the different genes of the dlt operon in Bacillus subtilis, we searched for metabolic and morphological changes caused by D-alanine ester deprivation of lipoteichoic acid and wall teichoic acid. There were no alterations of cell growth, basic metabolism, cellular content of phosphorus-containing compounds, ultrastructure, cell separation, and surface charge. The only alteration observed was an enhancement of endogenous and beta-lactam-induced cell lysis. Since this enhancement is doubtless correlated with the D-alanine ester deprivation of the teichoic acids, the present view based on in vitro experiments, that negatively charged LTA is inhibitory to autolysins, may be questioned. We propose that negatively charged lipoteichoic acid and/or wall teichoic acid serve in vivo to fix the cationic autolysins within the cell wall-membrane complex by electrostatic interaction. Positively charged D-alanine ester substituents decrease the binding capacity of the teichoic acids for autolysins by charge compensation.

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Basic structure.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic-interaction chromatography. The preparations from both strains have an identical structure: a 1,3-linked poly(glycerophosphate) chain phosphodiester-linked to Glc-(alpha 1-2)Glc(alpha 1-3)acyl2Gro as the lipid anchor. The chain is substituted with D-alanine ester and glycosyl residues which comprise mono-, di-, tri- and tetra-alpha-D-glucopyranosyl residues with (1-6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H-NMR and 13C-NMR spectroscopy. The alpha-isomalto-oligosaccharides add a novel motif to lipoteichoic-acid chain substituents. 1H-NMR and 13C-NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3-glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor. Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Microheterogeneity and distribution of chain substituents.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 contains a poly(glycerophosphate) chain, with 49% of the glycerophosphate residues being substituted with D-alanine ester, 35% with alpha-D-glucopyranosyl and alpha-isomalto-oligosaccharide residues. Analysis of molecular species by affinity chromatography on concanavalin A showed all chains to be substituted and alanine ester and glycosyl residues to be present on the same rather than on separate chains. Molecular species varied in the length of the poly(glycerophosphate) chain, the extent of glycosylation, and had a constant alanine-ester content. An alkali-hydrolysis procedure revealed a distribution pattern between random and regular for the glycosyl substituents and suggested a similar distribution for the alanyl residues which occupy the free positions between the glycosyl substituents.

Heterogeneity of lipoteichoic acid detected by anion exchange chromatography.

A complex polydispersity became apparent when the poly(glycerophosphate) lipoteichoic acid of Enterococcus faecalis was chromatographed on DEAE-Sephadex. The chain length varied between 13 and 33 glycerophosphate residues per lipid anchor. In parallel, the extent of chain glycosylation increased from 0.2 to 0.4 diglucosyl residues per glycerophosphate unit. Substitution with D-alanine ester showed a reverse distribution dropping with increasing chain length from 0.53 to 0.23 mol D-alanine per mol phosphorus. Variations in the fatty acid composition were also observed. The results extent and modify the current picture of lipoteichoic acid biosynthesis. They further suggest that during infection the mammalian organism may be confronted particularly with long-chain less hydrophobic molecular species.

Separation of the poly(glycerophosphate) lipoteichoic acids of Enterococcus faecalis Kiel 27738, Enterococcus hirae ATCC 9790 and Leuconostoc mesenteroides DSM 20343 into molecular species by affinity chromatography on concanavalin A

This study shows for the first time microheterogeneity of 1,3-linked poly(glycerophosphate) lipoteichoic acids. The lipoteichoic acids investigated were those of Enterococcus faecalis Kiel 27738 (I), Enterococcus hirae (Streptococcus faecium) ATCC 9790 (II), and Leuconostoc mesenteroides DMS 20343 (III). Lipoteichoic acids II and III are partially substituted by mono-, di-, tri-, and tetra-alpha-D-glucopyranosyl residues with (1----2) interglycosidic linkages. Lipoteichoic acid I is substituted with alpha-kojibiosyl residues only. Lipoteichoic acids I and III additionally carry D-alanine ester. Lipoteichoic acids were separated on columns of concanavalin-A-Sepharose according to their increasing number of glycosyl substituents per chain. It was evident that all molecular species are usually glycosylated and that alanine ester and glycosyl residues occur on the same chains. The chain lengths of lipoteichoic acid I and II vary between 9-40 glycerophosphate residues, whereas those of lipoteichoic acid III appear to be uniform (33 +/- 2 residues). Molecular species differ in the extent of glycosylation but their content of alanyl residues is fairly constant. All lipoteichoic acids contain a small fraction (5-15%) different in composition from the bulk and most likely reflecting an early stage of biosynthesis. Two procedures for chain length determination of poly(glycerophosphate) lipoteichoic acids are described.