Membrane Teichoic Acids Research Articles

The EssentialtacFGene Is Responsible for the Choline-Dependent Growth Phenotype ofStreptococcus pneumoniae

Streptococcus pneumoniae has an absolute nutritional requirement for choline, and the choline molecules are known to incorporate exclusively into the cell wall and membrane teichoic acids of the bacterium. We describe here the isolation of a mutant of strain R6 in which a single G-->T point mutation in the gene tacF (formerly designated spr1150) is responsible for generating a choline-independent phenotype. The choline-independent phenotype could be transferred to the laboratory strain R6 and to the encapsulated strain D39 by genetic transformation with a PCR product or with a plasmid carrying the mutated tacF gene. The tacF gene product belongs to the protein family of polysaccharide transmembrane transporters (flippases). A model is presented in which TacF is required for the transport of the teichoic acid subunits across the cytoplasmic membrane. According to this model, wild-type TacF has a strict specificity for choline-containing subunits, whereas the TacF present in the choline-independent mutant strain is able to transport both choline-containing and choline-free teichoic acid chains. The proposed transport specificity of parental-type TacF for choline-containing subunits would ensure the loading of the cell wall with teichoic acid chains decorated with choline residues, which appear to be essential for the virulence of this pathogen.

Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.

Introduction: Strategies for Prevention and Treatment of Sepsis in Post-Surgical Intensive Care

© E.I.F.T. srl Firenze ISSN 1120-009X Sepsis remains the main cause of morbidity and mortality in the critically ill patient. Death rates from severe sepsis range from 30 to 50% despite advances in critical care 1. In the US, approximately 750,000 cases occur each year, at least one-third are lethal 2. Sites of infection include most commonly the lung, the abdomen and the urinary tract. An important point is that in 20-30% of patients the infection site is never determined. Significant progress has been made in the development of consensus definitions for sepsis that had previously been utilized differently by different investigators. These definitions have facilitated clinical and epidemiological trials. They have, however, important limitations and continue to generate intense debate. The four criteria that define SIRS (the systemic inflammatory response syndrome) are non-specific measures of physiologic severity, rather than distinctive manifestations of a disease process. SIRS and MODS (multiple organ dysfunction syndrome) are not diseases or syndromes, but concepts. As such, they provide a useful intellectual framework for investigation, but the clinician has to treat diseases, not acronyms 3. The response to infection is triggered by bacterial toxins or by components of microbial cel ls, such as membrane fragments. Considerable progress has been made in recent years in understanding how bacterial products initiate sepsis. Gram-positive organisms can trigger sepsis by at least two mechanisms: producing exotoxins that act as “superantigens” or by releasing cell membrane fragments, including peptidoglycan, teichoic and lipoteichoic acids, that can activate the sequence of processes culminating in septic shock 7. In this supplement, Jonathan Cohen highlights the important differences between the cell walls of Gram-positive and Gram-negative organisms and how these differences translate into different host responses. For Gram-negative bacteria, endotoxin (LPS) triggers the sepsis syndrome. LPS binds to a specific protein (LPSbinding protein) in the plasma. The complex formed binds to a macrophage membrane receptor, CD14. This receptor presents LPS to a signal transducing receptor in the membrane, the Tol ll ike receptor (TLR) result ing in macrophage activation. Thierry Calandra explains the importance of Gram-negative sepIntroduction: Strategies for Prevention and Treatment of Sepsis in Post-Surgical Intensive Care

Autolysis and cell wall degradation in a choline-independent strain of Streptococcus pneumoniae.

Streptococcus pneumoniae has an auxotrophic requirement for choline, and choline residues that incorporate into the wall and membrane teichoic acids are intimately involved with the control of autolytic phenomena of this bacterium. We report here the re-examination of the role of choline in autolytic cell wall degradation using the choline-independent S. pneumoniae strain R6Cho- recovered from a heterologous cross with DNA from Streptococcus oralis. S pneumoniae Cho- cultured in choline-free medium grew with normal generation time but formed long chains, failed to undergo stationary-phase autolysis, and was also resistant to lysis induced by deoxycholate or penicillin. Cell walls produced under these conditions had reduced phosphorus content, contained no choline residues detectable by nuclear magnetic resonance, and had reduced binding capacity for the pneumococcal autolytic amidase, and complete hydrolysis of such walls by the amidase required prolonged incubation with high concentrations of the enzyme. Addition of choline to the growth medium reversed at these phenomena. High-performance liquid chromatography analysis of amidase digests of cell walls prepared from strain R6Cho- grown with or without choline produced identical stem peptide profiles, which were also similar to that of the parental S. pneumoniae strain R6. Peptidoglycans prepared by hydrofluoric extraction of cell walls from Cho- growth with or without choline or from the parental strain R6 were uniformly susceptible to the autolytic amidase and were fully degraded to the normal family of stem peptides, indicating that, in sharp contrast to the case of cell walls, the amidase degradation of teichoic acid-free peptidoglycan did not require the presence of choline residues in the substrate.

Bacteriophage receptors of Lactococcus lactis subsp. ?diacetylactis? F7/2 and Lactococcus lactis subsp. cremoris Wg2-1

Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L. lactis subsp. 'diacetylactis' F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L. lactis subsp. cremoris Wg2-1. Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions. However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments. This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors of strains F7/2 and Wg2-1.

Bacteriophage receptors ofLactococcus lactissubsp. ‘diacetylactis’ F7/2 andLactococcus lactissubsp.cremorisWg2–1

Bacteriophage P008 revealed irreversible and uniform adsorption to cell walls of L. lactis subsp. ‘diacetylactis’ F7/2, whereas phage P127 adsorbed reversibly to a limited number of receptor sites on cell walls of L. lactis subsp. cremoris Wg2–1. Neither extraction of lipids, cell wall- and membrane-teichoic acids nor enzymatic degradation of proteins altered the binding efficiencies of both cell wall fractions. However, phage binding was inhibited, when cell walls were subjected to lysozyme, metaperiodate, or acid treatments. This reflects that a carbohydrate component embedded in the peptidoglycan matrix is part of the phage receptors or strains F7/2 and Wg2–1.

Inter-chain transacylation of D-alanine ester residues of lipoteichoic acid: a unique mechanism of membrane communication.

Lipoteichoic acid is an important example of an amphiphile that appears to play a vital role in bacterial growth. A number of functions have been ascribed to this polymer. These include the regulation of autolytic enzymes, the binding of Mgz+ for enzyme function, and the assembly of wall polymers. One of the key components which has been proposed to modulate these functions is the substituent D-alanine. James Baddiley and his co-workers discovered that this amino acid is an important component of most teichoic acids (Armstrong et al., 1959). Thus, esterification of teichoic acids by D-alanine may be an important step in controlling the function of this polymer in wall assembly. In Lactobacillus casei the incorporation of D-alanine into lipoteichoic acid is accomplished in a two-step reaction sequence. The first step of this sequence was discovered in 1960 when Baddiley & Neuhaus showed that a variety of Gram-positive organisms have the ability to activate Dalanine. The second step of this sequence was not detected until Reusch & Neuhaus (1971) recognized that the incorporation of D-alanine into teichoic acids in vitro required the endogenous membrane teichoic acid to act as acceptor. All attempts to demonstrate the incorporation of D-alanine into isolated wall teichoic acid proved to be elusive and it was reasoned that the teichoic acid has to be intimately associated with the membrane in a particular conformation or environment (Neuhaus et aZ., 1974). On the basis of this hypothesis a system for the incorporation of D-alanine into membranes was found that required both a supernatant fraction and ATP, and was stimulated by metal ions. Linzer & Neuhaus (1973) showed that the supernatant fraction could be replaced by the D-alanine-activating enzyme and a protein factor designated D-alanine: membrane acceptor ligase. L. casei was chosen for these experiments because, firstly, it has a high concentration of the

Pneumococcal Forssman antigen: enrichment in mesosomal membranes and specific binding to the autolytic enzyme of Streptococcus pneumoniae.

The choline-containing pneumococcal membrane teichoic acid (Forssman antigen) can be isolated with the membrane fractions of the bacteria. The small vesicle (mesosomal) fraction generated during the formation of protoplasts seems to be highly enriched in this material. Forssman antigen was identified in cell fractions on the basis of (i) radioactive choline label, (ii) autolysin-inhibitory activity, and (iii) the sedimentation profile in sucrose density gradients with and without detergent. A membrane teichoic acid could also be isolated from pneumococci grown in medium in which choline was replaced by ethanolamine as the nutritionally required amino alcohol. This material contained radioactive ethanolamine label and behaved similarly to the choline-containing membrane teichoic acid during centrifugation in detergent-containing and detergent-free density gradients. On the other hand, the material had only low autolysin-inhibitory activity. Binding of pure pneumococcal autolysin to micelles of purified Forssman antigen could be demonstrated by mixing these components in vitro and analyzing them by sucrose density gradients and by agarose chromatography. No binding could be observed between the pneumococcal enzyme and the micellar forms of either cardiolipin or polyglycerophosphate-type lipoteichoic acid isolated from Streptococcus lactis.

On the multispecificity of carcinoscorpin, the sialic acid binding lectin from the horseshoe crab Carcinoscorpius rotundacauda: Recognition of glycerolphosphate in membrane teichoic acids

Our work [l-5] on the structure and function of carcinoscorpin, the sialic acid-specific lectin from the horseshoe crab Carcinoscorpius rotundacauda, has indicated that its binding to sialoglycoproteins is dependent not only on the sialic acid content, but also on the type of glycosidic linkage. This lectin recognizes 2-keto-3-deoxyoctonate residues in the lipopolysaccharide of certain Gram-negative bacteria [6]. Apart from this acidic sugar which is very similar to sialic acid, a structurally unrelated acidic sugar such as D-glucuronic acid was also found to be an effective inhibitor of carcinoscorpin-fetuin interaction [3,5]. Looking at the dimension of the multispecificity, we considered the possibility that certain acidic sugar derivatives such as phosphorylated sugars could also interact with this lectin. Of the various phosphorylated sugars and sugar alcohols tested, we came across an interesting specificity of this lectin towards glycerolphosphate which occurs in the membrane teichoic acids of Gram-positive bacteria.

ChemInform Abstract: A SIMPLE APPROACH TO THE SYNTHESIS OF A MEMBRANE TEICHOIC ACID FRAGMENT OF STAPHYLOCOCCUS AUREUS

Chemischer InformationsdienstVolume 13, Issue 26 Natural Products ChemInform Abstract: A SIMPLE APPROACH TO THE SYNTHESIS OF A MEMBRANE TEICHOIC ACID FRAGMENT OF STAPHYLOCOCCUS AUREUS J. J. OLTVOORT, J. J. OLTVOORTSearch for more papers by this authorC. A. A. VAN BOECKEL, C. A. A. VAN BOECKELSearch for more papers by this authorJ. H. DE KONING, J. H. DE KONINGSearch for more papers by this authorJ. H. VAN BOOM, J. H. VAN BOOMSearch for more papers by this author J. J. OLTVOORT, J. J. OLTVOORTSearch for more papers by this authorC. A. A. VAN BOECKEL, C. A. A. VAN BOECKELSearch for more papers by this authorJ. H. DE KONING, J. H. DE KONINGSearch for more papers by this authorJ. H. VAN BOOM, J. H. VAN BOOMSearch for more papers by this author First published: June 29, 1982 https://doi.org/10.1002/chin.198226302Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume13, Issue26June 29, 1982 RelatedInformation

Immunochemistry of the cell-surface carbohydrate antigens of Clostridium difficile.

Two carbohydrate cell-surface antigens were extracted from Clostridium difficile. One was extracted from pure cell walls by NaOH and contained glucose, mannose, galactosamine and phosphate in the approximate molar proportions of 2:0.65:1:0.63. The other antigen was extracted with phenol from the disrupted contents of whole cells and purified by chromatography on Sepharose 6B and an immunoabsorbent column; it contained glucose, glucosamine, phosphate and fatty acid in the approximate molar proportions of 2:1:1.6:0.04. Both antigens showed partial immunological identity and both cross-reacted with Clostridium sordellii antiserum. They are analogues of the wall and membrane teichoic acids of other Gram-positive bacteria.

Teichoic acid and lipid metabolism during sporulation of Bacillus megaterium KM.

The biochemistry of teichoic acid and lipid metabolism has been studied during sporulation of Bacillus megaterium KM. Measurements of cell-wall and membrane teichoic acid have shown that net synthesis of these polymers ceases at the onset of sporulation. Pulse-labelling studies show that the period of asymmetric septation and forespore engulfment is marked by an initiation of turnover of membrane teichoic acid but not of wall teichoic acid. This is reflected in the presence of inner-membrane teichoic acid and the virtual absence of wall teichoic acid in dormant spores. The total amount of lipid phosphorus in the sporulating cell increases by 70% as a result of asymmetric septation and subsequent engulfment of the forespore. The phosphorus requirement for this synthesis is derived from a pool formed during exponential growth, which is not exchangeable with extracellular Pi during sporulation. These results suggest that during sporulation a proportion of the glycerol 3-phosphate produced by preferential degradation of membrane teichoic acid formed during exponential growth is used for phospholipid synthesis during sporulation.

Lipoteichoic acids from d-glucosylation-defective, cell-wall mutants of Bacillus subtilis

Lipoteichoic acids from Bacillus subtilis 168 and its gta mutants were partially purified and characterized. The teichoic acids contain glycerol residues and phosphorus in equimolar ratios, and fatty acids. Some contaminating protein and nucleic acids were also present in the preparations. Gel electrophoresis of the preparations revealed the presence of common proteins, suggestive of functional protein-lipoteichoic acid complexes in bacterial membranes. All of the lipoteichoic acids were partially d-glucosylated, as evidenced by enzymic analysis. The d-gIucose residues were in the α anomeric configuration, as shown by precipitation with concanàvalin A. The data suggested that some, but not all, of the pathways leading to the d-glucosylation of the cell-wall teichoic acid are functional in the d-glucosylation of the membrane teichoic acids. The lipoteichoic acids were complexed with methylated bovine serum albumin, and injected into rabbits, in order to elicit a humoral immune-response. Antibodies were directed against poly(glycerol phosphate) and not against the d-glucosyl residues of the lipoteichoic acids. The antibody preparations cross-reacted with all of the membrane teichoic acids. Soluble, cell-wall teichoic acid was a potent inhibitor of passive hemmagglutination in a system containing lipoteichoic acid-sensitized, sheep red-blood cells and anti-lipoteichoic acid antiserum.

Structure of the cell wall of Bacillus species C.I.P. 76-111.

An unusual type of bacterial cell wall was encountered in a Bacillus strain referred to as Bacillus sp. C.I.P. 76-111. The major constituent of this cell wall is a high-molecular-weight anionic protein non-covalently associated to a peptidoglycan-polysaccharide complex. The cell wall appeared as a multilayered structure when sections of whole cells or of isolated cell walls fixed with glutaraldehyde and postfixed with osmium tetroxide were examined by electron microscopy. The correlation between the observed morphological features and the biochemical data suggested that a thin central electron-dense layer identifiable with the peptidoglycan-polysaccharide complex is located between two similar thick layers of protein. Furthermore, negative staining of isolated cell walls revealed that the outer protein layer has a regular surface array of subunits with hexagonal symmetry. Several structural properties of the cell wall peptidoglycan were investigated and were found to resemble those of other bacilli. Further characterization of the autolytic system showed that an N-acetylmuramyl-L-alanine amidase and a glycosidase, presumably a N-acetylmuramidase, were associated with the peptidoglycan-polysaccharide complex. It was also established that this strain is devoid of membrane teichoic acid.

Biosynthesis of the unit that links teichoic acid to the bacterial wall: Inhibition by tunicamycin

The walls of Gram-positive bacteria contain acidic polysaccharides and teichoic acids that are covalently linked through phosphodiesters to muramic acid residues in the peptidoglycan. It has been shown recently that in St@yZococcus agrees H [ 1 ] and in a mutant strain that lacks N-acetylglucosaminyl substituents on its wall teichoic acid [2] a ‘linkage unit’ that is a trimer of glycerol phosphate is interposed between the phosphate-terminal end of the teichoic acid chain and a muramic acid residue of the peptidoglycan to which it is attached. Evidence for similar linkage units has been found in Micrococcus sp. 2102 and in Bacillus subtilis W32 [J. Coley and E. Tarelli, unpublished work]. None of the bacteria examined contains a glycerol teichoic acid in its wall, and it has been demonstrated that the glycerol membrane teichoic acids are synthesized from phosphatidylglycerol [3,4]. Nevertheless, we have found [5] that membrane preparations from all of them catalyse the synthesis from CDP-glycerol of material containing a glycerol phosphate oligomer. Maximal synthesis of this product requires the addition of UDP-N-acetylglucosamine and the substrate for the synthesis of the backbone of the wall teichoic acid. The product is amphiphilic and appears to consist of the teichoic acid chain linked through the glycerol phosphate oligomer to a lipid acceptor. Bracha and Glaser [6] found that synthesis of the ribitol teichoic acid covalently linked to wall, in a wall-membrane preparation from S. aure’eus H, also required the addition of CDP-glycerol and UDP-N-acetylglucosamine. If therefore appears that teichoic acid attached to linkage unit is synthesized in

Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain.

Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids.

Soluble macromolecular complexes involving bacterial teichoic acids

Cell wall and membrane teichoic acids from several bacteria formed soluble complexes with polysaccharides and bovine plasma in alkyl alcohol solutions. Polysaccharides which contain different monomeric units and anomeric configurations complexed with the teichoic acids, suggesting that the interaction is relatively nonspecific. Teichoic acids complexed glycogen or bovine plasma albumin in 50 to 97% ethanol solutions. The macromolecular association between teichoic acids and polysaccharides or proteins was independent of teichoic acid size over a threefold molecular weight range. Glycerol phosphates or an acid hydrolysate of teichoic acid would not complex to either glycogen or bovine plasma albumin in ethanol. The optimal interaction between glycogen and the Bacillus subtilis lipoteichoic acid occurred between pH 4.5 and 8.2. The ability of teichoic acids to bind polysaccharides and proteins in moderate dielectric constant solvents suggests that these polymers may serve as complexing agents for hydrophilic molecules found in membranes.

Glycerol teichoic acid as an antigenic determinant in a Gram-negative bacterium Butyrivibrio fibrisolvens.

An antigenic determinant isolated from a strain of the Gram-negative bacterium Butyrivibrio fibrisolvens reacted with specific antisera to the polyglycerophosphate backbone of membrane teichoic acids of lactobacilli. It gave a reaction of identity with membrane glycerol lipoteichoic acid and glycerol teichoic acid preparations from lactobacilli, and with phenol extracts of other Gram-positive bacteria. The antigen-antibody reactions was strongly inhibited by glycerol-phosphoryl-glycerol-phosphoryl-glycerol and the chemical composition was consistent with glycerol teichoic acid. It was concluded that this Gram-negative bacterium contained a glycerol teichoic acid whose polyglycerophospate backbone was acting as antigenic determinant. Extracts of 33 out of 52 other strains of butyrivibrios examined gave similar reactions.

Membrane Lipoteichoic Acid Is Not a Precursor to Wall Teichoic Acid in Pneumococci

The distribution of a pulse of teichoic acid-specific radiolabel between wall and membrane teichoic acids in pneumococci was constant over a subsequent chase period, suggesting that wall and membrane teichoic acids are biosynthesized simultaneously and independently.

Incorporation of D-alanine into the membrane of Streptococcus pyogenes and its stabilized L-form.

A principal aim of this study was to explain our earlier finding of a lack of d-alanine in the glycerol teichoic acid from the membrane of a stabilized L-form of Streptococcus pyogenes (B. M. Slabyj and C. Panos, 1973. J. Bacteriol. 114:934-942). It was found that the incorporation of d-alanine into the membrane teichoic acid of S. pyogenes requires either supernatant fraction or two enzymes from supernatant fraction, stimulator (d-alanine activating enzyme) and d-alanine:membrane acceptor ligase, plus membrane fragments, ATP and Mg(2+). A similar system from the L-form is inoperative. Also, no incorporation is observed with L-form or coccal supernatant fractions when L-form membranes are used. However, d-alanine incorporation is observed when L-form enzymes are used with membrane fragments from the parental streptococcus. Thus, the L-form possesses the required soluble components for d-alanine incorporation but the L-form membrane cannot function as acceptor even though it contains d-alanine-deficient membrane teichoic acid. These results suggest that a defect has occurred in the membrane of this stabilized L-form for d-alanine incorporation into membrane teichoic acid.