The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

Structural investigation of human S. aureus-targeting antibodies that bind wall teichoic acid

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.     

Structural and Enzymatic Analysis of TarM Glycosyltransferase from Staphylococcus aureus Reveals an Oligomeric Protein Specific for the Glycosylation of Wall Teichoic Acid

Anionic glycopolymers known as wall teichoic acids (WTAs) functionalize the peptidoglycan layers of many Gram-positive bacteria. WTAs play central roles in many fundamental aspects of bacterial physiology, and they are important determinants of pathogenesis and antibiotic resistance. A number of enzymes that glycosylate WTA in Staphylococcus aureus have recently been identified. Among these is the glycosyltransferase TarM, a component of the WTA de novo biosynthesis pathway. TarM performs the synthesis of α-O-N-acetylglycosylated poly-5′-phosphoribitol in the WTA structure. We have solved the crystal structure of TarM at 2.4 Å resolution, and we have also determined a structure of the enzyme in complex with its substrate UDP-GlcNAc at 2.8 Å resolution. The protein assembles into a propeller-like homotrimer in which each blade contains a GT-B-type glycosyltransferase domain with a typical Rossmann fold. The enzymatic reaction retains the stereochemistry of the anomeric center of the transferred GlcNAc-moiety on the polyribitol backbone. TarM assembles into a trimer using a novel trimerization domain, here termed the HUB domain. Structure-guided mutagenesis experiments of TarM identify residues critical for enzyme activity, assign a putative role for the HUB in TarM function, and allow us to propose a likely reaction mechanism.     

Staphylococcus aureus Mutants Lacking the LytR-CpsA-Psr Family of Enzymes Release Cell Wall Teichoic Acids into the Extracellular Medium

The LytR-CpsA-Psr (LCP) proteins are thought to transfer bactoprenol-linked biosynthetic intermediates of wall teichoic acid (WTA) to the peptidoglycan of Gram-positive bacteria. In Bacillus subtilis, mutants lacking all three LCP enzymes do not deposit WTA in the envelope, while Staphylococcus aureus Δlcp mutants display impaired growth and reduced levels of envelope phosphate. We show here that the S. aureus Δlcp mutant synthesized WTA yet released ribitol phosphate polymers into the extracellular medium. Further, Δlcp mutant staphylococci no longer restricted the deposition of LysM-type murein hydrolases to cell division sites, which was associated with defects in cell shape and increased autolysis. Mutations in S. aureus WTA synthesis genes (tagB, tarF, or tarJ2) inhibit growth, which is attributed to the depletion of bactoprenol, an essential component of peptidoglycan synthesis (lipid II). The growth defect of S. aureus tagB and tarFJ mutants was alleviated by inhibition of WTA synthesis with tunicamycin, whereas the growth defect of the Δlcp mutant was not relieved by tunicamycin treatment or by mutation of tagO, whose product catalyzes the first committed step of WTA synthesis. Further, sortase A-mediated anchoring of proteins to peptidoglycan, which also involves bactoprenol and lipid II, was not impaired in the Δlcp mutant. We propose a model whereby the S. aureus Δlcp mutant, defective in tethering WTA to the cell wall, cleaves WTA synthesis intermediates, releasing ribitol phosphate into the medium and recycling bactoprenol for peptidoglycan synthesis.     

Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis

The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.     

Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. The Listeria monocytogenes Ply118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure of Listeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding using L. monocytogenes serovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundant tagO homologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of either tagO1 (EGDe lmo0959) or tagO2 (EGDe lmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficient Listeria cells. Substitution of tagO from an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from other Listeria phage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.     

Biosynthesis of lipophosphoglycan from Leishmania donovani: characterization of mannosylphosphate transfer in vitro.

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes and is composed of a capped polymer of repeating PO4-6Gal(beta 1,4)Man alpha 1 disaccharide units linked via a phosphosaccharide core to a lyso-1-O-alkylphosphatidylinositol anchor. An exogenous acceptor composed of the glycolipid anchor portion of LPG was shown to stimulate the enzymatic synthesis of the repeating phosphorylated disaccharide units of LPG in a cell-free system. Using the exogenous acceptor, GDP-[3H]Man, [beta-32P]GDP-Man, and unlabeled UDP-Gal as substrates, membrane preparations from an LPG-defective mutant of L. donovani that lacks endogenous acceptors catalyzed the incorporation of the doubly labeled mannosylphosphate unit into a product that exhibited the chemical and chromatographic characteristics of LPG. Analysis of fragments generated by mild acid hydrolysis of the radiolabeled product indicated that [3H]mannose-1-[32P]PO4 had been transferred from the dual-labeled sugar nucleotide. These results are consistent with the proposal that the repeating units of the L. donovani LPG are synthesized by the alternating transfer of mannose 1-phosphate and galactose from their respective nucleotide donors.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Glycans from streptococcal cell walls: The molecular structure and immunological properties of an antigenic glycan from Streptococcus bovis

The molecular structure and immunological properties of an antigenic glycan from the cell wall of Streptococcus bovis, strain C3, a member of the Group D Streptococci, have been determined by methylation analysis, periodate oxidation, and hapten inhibition methods. The glycan is shown to be a tetraheteroglycan composed of 6-deoxy-l-talose, l-rhamnose, d-galactose, and d-glucuronic acid. The sugar sequence and the types of glycosidic linkages of the glycan are: a main chain of l-rhamnosyl-(1,3)-d-galactosyl- (1,2)-l-rhamnosyl-(1,3)-6-deoxy-l-talosyl-(1,3)- units with d-glucuronosyl residues attached to position 4 of the first rhamnose of each repeating unit of the main chain. The d-glucuronic acid moiety is the primary immunodeterminant group of the glycan. On the basis of hapten inhibition data, it has been concluded that the binding of the antigen to the antibody occurs at the hydroxyl groups at positions 2 and 3 and the carboxyl group at position 6 of the d-glucuronic acid moieties. The antigen has been used to prepare antiserum with anti-glucuronic acid antibodies.     

Optimization of a small-molecule Lipid II binder

Lipid II is an essential precursor of bacterial cell wall biosynthesis and an attractive target for antibiotics. Lipid II is comprised of specialized lipid (bactoprenol) linked to a hydrophilic head group consisting of a peptidoglycan subunit (N-acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc) disaccharide coupled to a short pentapeptide moiety) via a pyrophosphate. We previously identified a (E)-2,4-bis(4-bromophenyl)-6-(4-(dimethylamino)styryl)pyrylium boron tetrafluoride salt, termed 6jc48-1, that interacts with the MurNAc moiety, the phosphate cage and the isoprenyl tail of Lipid II. Here, we report on the structure-activity relationship of 6jc48-1 derivatives obtained by de novo chemical synthesis. Our results indicate that bacterial killing is positively driven by bi-phenyl stacking with peptidoglycan units. Replacement of bromides by fluorides resulted in activity against S. aureus without affecting Lipid II binding and cytotoxicity. Antibacterial activity was affected negatively by extended interaction of the scaffold with Lipid II isoprenyl units.     

Synthesis of the repeating unit of the teichoic acid isolated from the cell wall of bacillus subtilis VAR. niger WM

Stereocontrolled synthesis of 1-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-2,3-O-isopropylidene-D-glycerol (6) was achieved in good yield by use of the modified, orthoester method. Compound 6 was then transformed into 1-deoxy-3-O-phosphono-D-glycerol-1-yl β-D-glucopyranoside (1), identical with the repeating unit of the teichoic acid isolated from the cell wall of Bacillus subtilis var. niger WM, in a regio-controlled way, unambiguous evidence for the assignment of the stereochemistry of the natural product being provided by the ¹³C-n.m.r. data for 1 and its L-glycerol-1-yl isomer.     

Characterization of a Lactobacillus gasseri JCM 1131T Lipoteichoic Acid with a Novel Glycolipid Anchor Structure

We determined the chemical structure of lipoteichoic acid (LTA) from Lactobacillus gasseri JCM 1131^T. The repeating unit was comprised of glycerolphosphate and 2-alanylglycerolphosphate. The glycolipid anchor was tetrahexosylglycerol with two or three acyl groups. To our knowledge, this is the first demonstration of a tetrahexose structure in an LTA glycolipid anchor.     

Cell-wall lipopolysaccharide of the 'Shigella-like' Escherichia coli 058. Structure of the polysaccharide chain.

Two lipopolysaccharide preparations were obtained from Escherichia coli 058 by extraction with 45% aqueous phenol and fractional precipitation with cetyltrimethyl ammonium bromide (Cetavlon). Chemical analysis and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. The O-specific polysaccharide was characterized with proton magnetic resonance and infrared spectroscopy, optical rotation and paper electrophoresis. Using gas-liquid chromatography and ion-exchange chromatography, it was shown to contain D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-O-(R-1'-carboxyethyl)-L-rhamnose (rhamnolactylic acid), and O-acetyl groups in the molar ratios of 2:1:1:1. The polysaccharide and oligosaccharides obtained from it were subjected to methylation and chromic acid oxidation. The results obtained indicated that the polysaccharide consists of tetrasaccharide repeating units in which the trisaccharide beta-GlcNAc1 - 4alphaMan-1 - 4(2/3-O-Ac)-Man is substituted at C-3 of the non-acetylated mannose with rhamnolactylic acid. The repeating units are joined through alpha-mannosyl-1 - 3-glucosamine bonds. This structure is identical with that of the cell wall polysaccharide of Shigella dysenteriae type 5.     

Fractionation of xyloglucan fragments and their interaction with cellulose.

Tamarind seed xyloglucan was partially degraded with a purified endoglucanase (endoV) from Trichoderma viride. Analysis by high-performance anion-exchange chromatography showed that this digest was composed of fragments consisting of 1 to 10 repeating oligosaccharide units ([xg]1-[xg]10). To study the adsorption of xyloglucan fragments to cellulose in detail, this digest was fractionated on BioGel P-6. Fragments were separated satisfactorily up to 5 repeating oligosaccharide units ([xg]5). The galactose substitution of the fragments increased with increasing molecular weight. The BioGel P-6 pools, as well as polymeric xyloglucan ([xg] infinity), were tested for their ability to interact with Avicel crystalline cellulose. Quantitative binding to cellulose occurred for sequences consisting of (at least) 4 repeating units. The adsorption of [xg]4 to Avicel was very high relative to that of [xg] infinity. The dimensions of these fragments were such that they could also penetrate the smaller pores of cellulose. Apparently, the effective surface area for the polymers is much smaller. Adsorption isotherms of [xg] infinity and [xg]4 showed a pattern that is typical for polydisperse systems. However, the mechanisms underlying these patterns were different. At high xyloglucan concentrations, this polydispersity resulted in preferential adsorption of the larger molecules in the case of [xg] infinity and a more extensive colonization of the smaller pores of cellulose in the case of [xg]4. The pH influenced the interaction between xyloglucan (fragments) and cellulose to only a small extent.     

Conformation of the branched O-specific polysaccharide of Shigella dysenteriae type 2: molecular mechanics calculations show a compact helical structure exposing an epitope which potentially mimics galabiose

Conformational analyses of the branched repeating unit of the O-antigenic polysaccharide of Shigella dysenteriae type 2 have been performed with molecular mechanics MM3. A filtered systematic search on the trisaccharide alpha-D-GalNAc-(1-->3)-[alpha-D-GlcNAc-(1-->4)]-alpha-D-GalNAc forming the branch, shows essentially a single favored conformation. Also, the downstream alpha-D-GalNAc-(1-->4)-alpha-D-Glc linkage is sterically constrained. The alpha-D-Glc-(1-->4)-beta-D-Gal moiety, however, forms a more flexible link region between the branch points, and shows a 90 degrees bend similar to what is known for the galabiose moiety occurring in globo-glycolipids. The calculations indicate that consecutive repeating units in their minimum energy conformation arrange in a helical structure with three repeating units per turn. This helix is very compact and appears to be stabilized by hydrophobic interactions involving the N-acetyl groups at the branch points. Random conformational search suggests the existence of another helical structure with four repeating units per turn. It appears possible that the alpha-D-Glc-(1-->4)-beta-D-Gal moiety, which is exposed on the surface of the helical structures, can evade recognition by the immune system of the host by the mimicry of globo structures.     

Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S.?aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S.?aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S.?aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S.?aureus cell envelope biogenesis.     

Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore β-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC_50s for tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations.     

Design and synthesis of glucose-templated proline-lysine chimera: polyfunctional amino acid chimera with high prolyl cis amide rotamer population

We describe the synthesis of two glucose-templated proline-lysine chimeras (GlcTProLysCs) that differ in the stereochemistry of the hydroxymethyl substituent at the C-5′ position of the pyrrolidine ring. The key synthetic steps involve C-glycosylation of an exocyclic glucose-based epoxide with allyltributylstannane, which affords functionalized C-ketosides containing an α-hydroxy ester moiety; introduction of an amino group at C-2 through stereoselective reductive amination; and regioselective installation of the azide group at C-6 on the glucose scaffold. Incorporation of these chimeras into the model peptides Ac-GlcTProLysC-NHMe and Ac-GlcTProLysC-OMe demonstrates that the stereochemistry of the hydroxymethyl substituent at the C-5′ position has a profound effect on the equilibrium constant of prolyl amide cis/trans isomerization. The equilibrium constant K_c/t for the peptide mimic Ac-GlcTProLysC-NHMe with C-5′(R) stereochemistry was determined to be 3.03 ± 0.04, while the K_t/c for the C-5′(S) diastereoisomer was 0.56 ± 0.04 in D₂O. Temperature coefficient experiments indicate that the origin of these effects is derived from two critical hydrogen bonds involving the C-5′ hydroxymethyl substituent: one to the N-terminal amide carbonyl group, and the other to the primary amino group in the glucose moiety.