Pneumococcal Lipoteichoic Acid Research Articles

Lipoteichoic Acids Are Essential for Pneumococcal Colonization and Membrane Integrity.

The hydrophilic, polymeric chain of the lipoteichoic acid (LTA) of the Gram-positive pathobiont Streptococcus pneumoniae is covalently linked to the glycosylglycerolipid α-<sc>d</sc>-glucopyranosyl-(1,3)-diacylglycerol by the LTA ligase TacL, leading to its fixation in the cytoplasmic membrane. Pneumococcal LTA, sharing identical repeating units with the wall teichoic acids (WTA), is dispensable for normal growth but required for full virulence in invasive infections. Mutants deficient in TacL and complemented strains constructed were tested for their growth, resistance against oxidative stress, and susceptibility against antimicrobial peptides. Further, the membrane fluidity of pneumococci, their capability to adhere to lung epithelial cells, and virulence in a Galleria mellonella as well as intranasal mouse infection model were assessed. In the present study, we indicate that LTA is already indispensable for pneumococcal adherence to human nasopharyngeal cells and colonization in an intranasal mouse infection model. Mutants deficient for TacL did not show morphological defects. However, our analysis of pneumococcal membranes in different serotypes showed an altered membrane fluidity and surface protein abundance of lipoproteins in mutants deficient for LTA but not WTA. These mutants had a decreased membrane fluidity, exhibited higher amounts of lipoproteins, and showed an increased susceptibility to antimicrobial peptides. In complemented mutant strains, this defect was fully restored. Taken together, LTA is crucial for colonization and required to effectively protect pneumococci from innate immune defence mechanisms by maintaining the membrane integrity.

Commensal Streptococcus mitis produces two different lipoteichoic acids of type I and type IV.

The opportunistic pathogen Streptococcus mitis possesses, like other members of the Mitis group of viridans streptococci, phosphorylcholine (P-Cho)-containing teichoic acids (TAs) in its cell wall. Bioinformatic analyses predicted the presence of TAs that are almost identical with those identified in the pathogen Streptococcus pneumoniae, but a detailed analysis of S. mitis lipoteichoic acid (LTA) was not performed to date. Here, we determined the structures of LTA from two S. mitis strains, the high-level beta-lactam and multiple antibiotic resistant strain B6 and the penicillin-sensitive strain NCTC10712. In agreement with bioinformatic predictions, we found that the structure of one LTA (type IV) was like pneumococcal LTA, except the exchange of a glucose moiety with a galactose within the repeating units. Further genome comparisons suggested that the majority of S. mitis strains should contain the same type IV LTA as S. pneumoniae, providing a more complete understanding of the biosynthesis of these P-Cho-containing TAs in members of the Mitis group of streptococci. Remarkably, we observed besides type IV LTA, an additional polymer belonging to LTA type I in both investigated S. mitis strains. This LTA consists of β-galactofuranosyl-(1,3)-diacylglycerol as glycolipid anchor and a poly-glycerol-phosphate chain at the O-6 position of the furanosidic galactose. Hence, these bacteria are capable of synthesizing two different LTA polymers, most likely produced by distinct biosynthesis pathways. Our bioinformatics analysis revealed the prevalence of the LTA synthase LtaS, most probably responsible for the second LTA version (type I), among S. mitis and Streptococcus pseudopneumoniae strains.

2597. Dolichos biflorus Agglutinin Binds to Pneumococcal Teichoic Acid and Lipoteichoic Acid

Background Dolichos biflorus agglutinin (DBA) is a lectin with a binding specificity toward α-linked N-acetylgalactosamine (α-GalNAc). While DBA is known to bind some, but not all, pneumococci, its target molecule has not been identified. Pneumococcus teichoic acid (TA) and lipoteichoic acid (LTA) have repeating units with α-GalNAc-(1→3)β-GalNAc decorated with phosphorylcholine (PC) at the O-6 positions. Two PC transferases, LicD1 and LicD2, mediate the attachment of PC to GalNAc residues while phosphorylcholine esterase (Pce) removes PCs attached to the terminal GalNAcs. We examined if DBA binds to the terminal α-GalNAc-(1→3)β-GalNAc created by Pce.MethodsFifteen pneumococcus strains expressing 14 different serotypes, including one non-encapsulated strain (R36A), were studied with flow cytometry (FC) and confocal fluorescence microscopy (CFM) for DBA binding. Pce enzyme activity was detected with a colorimetric assay using p-nitrophenyl-phosphorylcholine as the substrate. Mutant strains with pce knocked-out were constructed in R36A and D39 by replacing pce with Janus cassette. Both licD genes were sequenced for some of the strains.ResultsTen of the 15 strains had Pce activity and all of them bound DBA (Table 1). When the pce gene was inactivated in two normally Pce-positive strains (R36A△pce and D39△pce), the strains did not show DBA binding by CFM (Figure 1). Thus, expression of Pce appears to be sufficient for expressing the DBA antigen. Of the five strains that had no Pce activity, two bound DBA. Sequencing of the licD genes in these two strains with positive DBA binding and negative Pce activity revealed one SNP in licD1 and four SNPs in licD2, resulting in a single amino acid difference each for LicD1 and LicD2, compared with R36A and D39.ConclusionDBA can bind to the terminal α-GalNAc-(1→3)β-GalNAc of pneumococcal TA and LTA, which is created by Pce. DBA binding is independent of capsule type. The unexpected binding of DBA to the two Pce-negative strains suggests that there is a Pce-independent mechanism for generating the target for DBA binding. Since LicD1 and LicD2 are involved in attaching PC to α-GalNAc-(1→3)β-GalNAc, we are now investigating their role in creating DBA targets independent of Pce. Disclosures All authors: No reported disclosures.

Identification of Pneumococcal Factors Affecting Pneumococcal Shedding Shows that the dlt Locus Promotes Inflammation and Transmission.

Host-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogen Streptococcus pneumoniae generated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism's transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was the dlt locus, which adds d-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence of d-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of the dlt locus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in a lysM-/- host, there was no longer an effect of the dlt locus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism's capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiased in vivo transposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by the dlt locus, previously shown to add d-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

Attachment of phosphorylcholine residues to pneumococcal teichoic acids and modification of substitution patterns by the phosphorylcholine esterase

The bacterial lung pathogen Streptococcus pneumoniae has a unique nutritional requirement for exogenous choline and attaches phosphorylcholine (P-Cho) residues to the GalpNAc moieties of its teichoic acids (TAs) in its cell wall. Two phosphorylcholine transferases, LicD1 and LicD2, mediate the attachment of P-Cho to the O-6 positions of the two GalpNAc residues present in each repeating unit of pneumococcal TAs (pnTAs), of which only LicD1 has been determined to be essential. At the molecular level, the specificity of the P-Cho attachment to pnTAs by LicD1 and LicD2 remains still elusive. Here, using detailed structural analyses of pnTAs from a LicD2-deficient strain, we confirmed the specificity in the attachment of P-Cho residues to pnTA. LicD1 solely transfers P-Cho to α-d-GalpNAc moieties, whereas LicD2 attaches P-Cho to β-d-GalpNAc. Further, we investigated the role of the pneumococcal phosphorylcholine esterase (Pce) in the modification of the P-Cho substitution pattern of pnTAs. To clarify the specificity of Pce-mediated P-Cho hydrolysis, we evaluated different concentrations and pH conditions for the treatment of pneumococcal lipoteichoic acid with purified Pce. We show that Pce can hydrolyze both P-Cho residues of the terminal repeat of the pnTA chain and almost all P-Cho residues bound to β-d-GalpNAc in vitro However, hydrolysis in vivo was restricted to the terminal repeat. In summary, our findings indicate that LicD1 and LicD2 specifically transfer P-Cho to α-d-GalpNAc and β-d-GalpNAc moieties, respectively, and that Pce removes distinct P-Cho substituents from pnTAs.

The Scavenger Receptor CD36 Downmodulates the Early Inflammatory Response while Enhancing Bacterial Phagocytosis during Pneumococcal Pneumonia

CD36 is a scavenger receptor that exhibits pleiotropic functions, including adhesion to thrombospondin, inhibition of angiogenesis, transport of long-chain fatty acids, and clearance of apoptotic cells. In addition, it has been implicated in the host immune response because it acts as a coreceptor for TLR2 and plays a role in Staphylococcus aureus infection. However, its role in other Gram-positive bacterial infections is unclear. In this study, using mice deficient in CD36, we sought to examine the role of CD36 in pneumococcal pneumonia, a major cause of morbidity and mortality worldwide. We show that CD36 is expressed on both alveolar macrophages and respiratory epithelial cells. Early in infection, CD36(-/-) mice have an exaggerated inflammatory response compared with wild-type littermate controls. In vitro studies using CD36(-/-) primary cells confirm the enhanced early inflammation in response to S. pneumoniae and its lipoteichoic acid, demonstrate that S. pneumoniae binds to cells via its phosphocholine residues, and suggest a role for CD36 in reducing inflammation induced by the phosphocholine residues of pneumococcal lipoteichoic acid. Later in infection, although CD36(-/-) mice exhibit impaired bacterial clearance, owing to a decreased capacity of CD36(-/-) macrophages to phagocytose S. pneumoniae, minor effects on mortality occur, in comparison with those in wild-type littermate control mice. These data show that CD36 contributes to the pulmonary host response during S. pneumoniae infection by virtue of its ability to act as a phagocytic receptor and as a modulator of the early innate immune response.

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids, but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H(2)O(2)) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam(3)CSK(4) and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam(3)CSK(4) and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam(3)CSK(4) or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam(3)CSK(4) and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H(2)O(2) for 6 h at 37 degrees C inactivates Pam(3)CSK(4), FSL-1, and LTA by more than 80%. Although HF, LPL, and H(2)O(2) treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.

A New Model of Pneumococcal Lipoteichoic Acid Structure Resolves Biochemical, Biosynthetic, and Serologic Inconsistencies of the Current Model

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.

Comparison of Lipoteichoic Acid from Different Serotypes of Streptococcus pneumoniae

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that of Staphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae:R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitol-bound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon gamma). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.

Platelet-Activating Factor-Acetylhydrolase Can Monodeacylate and Inactivate Lipoteichoic Acid

Bacterial lipoteichoic acid (LTA) shares a structural motif with platelet-activating factor (PAF). Both molecules are strong inflammatory agents and have a glycerol backbone with two lipid chains at the sn-1 and sn-2 positions. PAF is normally inactivated by PAF-acetylhydrolase (PAF-AH), a phospholipase A2 (PLA2), which removes a short acyl group at the sn-2 position. To investigate whether PAF-AH can similarly degrade LTA, we studied the effects of porcine PLA2, bee venom PLA2, and recombinant human PAF-AH on pneumococcal LTA (PnLTA) and staphylococcal LTA (StLTA). After incubation with a porcine or bee venom PLA2, a large fraction of PnLTA lost 264 Da, which corresponds to the mass of the oleic acid group at the sn-2 position. After incubation with recombinant human PAF-AH, PnLTA lost 264 Da; the reduction did not occur when PAF-AH was exposed to Pefabloc SC, an irreversible inhibitor of the PAF-AH active site. Following PAF-AH treatment, PnLTA and StLTA were not able to stimulate mouse RAW 264.7 cells to produce tumor necrosis factor alpha but could stimulate CHO cells expressing human TLR2. This stimulation pattern has been observed with monoacyl PnLTA prepared by mild alkali hydrolysis (22). Taking these data together, we conclude that PAF-AH can remove one acyl chain at the sn-2 position of LTA and produce a monoacyl-LTA that is inactive against mouse cells.

Monoacyl Lipoteichoic Acid from Pneumococci Stimulates Human Cells but Not Mouse Cells

We have developed a method for obtaining pneumococcal lipoteichoic acid (LTA) with none, one, or two acyl chains. Anion-exchange chromatography at pH 9.5 yields pneumococcal LTA (labeled LTA-9.5) that has a mass spectrum identical to that of pre-ion-exchange LTA and loses 500 mass units after deacylation by alkali hydrolysis. Anion exchange at pH 10.5 produces LTA (labeled LTA-10.5) with mass peaks that are 264 mass units lower than those of pre-ion-exchange LTA, and deacylation of LTA-10.5 by alkali hydrolysis reduces the mass by only 239 mass units. This result indicates that LTA-10.5 has lost one of the two acyl chains, whereas LTA-9.5 has both acyl chains. When the biological properties of LTA-9.5 and LTA-10.5 are examined with mouse cells, only LTA-9.5 (and not LTA-10.5) is able to stimulate mouse cells to produce tumor necrosis factor alpha, interleukin-1beta, and nitric oxide. In contrast, both LTA-9.5 and LTA-10.5 can stimulate human cells. LTA became inactive when both acyl chains were removed. Thus, acyl chains are critical for LTA function, and small variations in acyl chains can alter biological properties of LTA.

Pneumococcal Lipoteichoic Acid (LTA) Is Not as Potent as Staphylococcal LTA in Stimulating Toll-Like Receptor 2

Streptococcus pneumoniae is a leading cause of gram-positive sepsis, and lipoteichoic acid (LTA) may be important in causing gram-positive bacterial septic shock. Even though pneumococcal LTA is structurally distinct from the LTA of other gram-positive bacteria, the immunological properties of pneumococcal LTA have not been well characterized. We have investigated the ability of LTAs to stimulate human monocytes by using highly pure and structurally intact preparations of pneumococcal LTA and its two structural variants. The variants were pneumococcal LTA with only one acyl chain (LTA-1) and completely deacylated LTA (LTA-0). The target cells used in the study were peripheral blood mononuclear cells (PBMCs) and two model cell lines (CHO/CD14/TLR2 and CHO/CD14/TLR4) that express human CD25 protein in response to Toll-like receptor 2 (TLR2) and TLR4 stimulation, respectively. Intact pneumococcal LTA and LTA-1 stimulated PBMC and CHO/CD14/TLR2 cells in a dose-dependent manner but did not stimulate CHO/CD14/TLR4 cells. Pneumococcal LTA was about 100-fold less potent than Staphylococcus aureus LTA in stimulating the CHO/CD14/TLR2 cells and PBMCs. LTA-0 (or pneumococcal teichoic acid) stimulated neither CHO/CD14/TLR2 nor CHO/CD14/TLR4 cells even at high concentrations. Excess teichoic acid, LTA-0, antibodies to phosphocholine, or antibodies to TLR4 did not inhibit the LTA-induced TLR2 stimulation. However, antibodies to CD14, TLR1, or TLR2 suppressed tumor necrosis factor alpha (TNF-alpha) production by PBMCs in response to LTA or LTA-1. These results suggest that pneumococcal LTA with one or both acyl chains stimulates PBMCs primarily via TLR2 with the help of CD14 and TLR1.

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.

Teichoic acid and lipoteichoic acid of Streptococcus pneumoniae possess identical chain structures

Teichoic acid (C polysaccharide) was extracted and purified from Streptococcus pneumoniae R6 with standard procedures except that lipoteichoic acid was extracted first. The dephosphorylated repeating unit was isolated after hydrolysis with 48% (by mass) HF, the bis(phosphocholine)-containing repeating unit was isolated by alkali hydrolysis, anion-exchange chromatography and phosphomonoester cleavage. On the basis of compositional analysis, fast-atom-bombardment mass spectrometry and NMR spectroscopy the following structure is proposed: [formula: see text] where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. The repeating units are linked to each other by phosphodiester bonds between O5 of the ribitol and O6 of the glucopyranosyl residue of adjacent units. This chain structure is identical with that previously established for pneumococcal lipoteichoic acid [Behr, T., Fischer, W., Peter-Katalinić, J. & Egge, H. (1992) Eur. J. Biochem. 207, 1063-1075]. This represents a unique situation because in other Gram-positive bacteria teichoic and lipoteichoic acids are structurally unrelated.

The binding of Pneumococcal lipoteichoic acid to human erythrocytes

Previous studies have evaluated the binding characteristics of lipoteichoic acids (LTA) composed of polyglycerol phosphate to mammalian cell membranes. This present study was performed to evaluate the binding characteristics of Pneumococcal LTA, a polyribitol phosphate containing LTA, to human erythrocytes. Binding was found to be specific, reversible, and dependent on temperature and time. Maximum binding was achieved within 2 h at 37°C. Scatchard plot analysis of the binding revealed a biphasic plot which suggests two receptor sites with different affinities. Competitive inhibition studies with LTA composed of polyglycerol phosphate and isolated from Streptococcus pyogenes and Lactobacillus fermentum revealed significant inhibition of Pneumococcal LTA-binding to the erythrocyte membrane by these heterologous LTAs. These results demonstrate that Pneumococcal LTA binds specifically to human erythrocytes and that the receptor site is similar for other structurally different LTAs.

The structure of pneumococcal lipoteichoic acid. Improved preparation, chemical and mass spectrometric studies.

Pneumococcal lipoteichoic acid was extracted and purified by a novel, quick and effective procedure. Structural analysis included methylation, periodate oxidation, Smith degradation, oxidation with CrO3, and fast-atom-bombardment mass spectrometry. Hydrolysis with 48% (by mass) HF and subsequent phase partition yielded the lipid anchor (I), the dephosphorylated repeating unit of the chain (II) and a cleavage product of the latter (III). The proposed structures are: (I) Glc(beta 1----3)AATGal(beta 1----3)Glc(alpha 1----3)acyl2Gro, (II) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc(beta 1----1)ribitol and (III) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose, and all sugars are in the pyranose form and belong to the D-series. Alkaline phosphodiester cleavage of lipoteichoic acid, followed by treatment with phosphomonoesterase, resulted in the formation of II and IV, with IV as the prevailing species: [sequence: see text] The linkage between the repeating units was established as phosphodiester bond between ribitol 5-phosphate and position 6 of the glucosyl residue of adjacent units. The chain was shown to be linked to the lipid anchor by a phosphodiester between its ribitol 5-phosphate terminus and position 6 of the non-reducing glucosyl terminus of I. The lipoteichoic acid is polydisperse: the chain length may vary between 2 and 8 repeating units and variations were also observed for the fatty acid composition of the diacylglycerol moiety. Preliminary results suggest that repeating units II and IV are enriched in separate molecular species. All species were associated with Forssman antigenicity, albeit to a various extent when related to the non-phosphocholine phosphorus. Owing to its unique structure, the described macroamphiphile may be classified as atypical lipoteichoic acid.

Bacterial lipoteichoic acid sensitizes host cells for destruction by autologous complement.

Lipoteichoic acids (LTA) released by gram-positive bacteria can spontaneously bind to mammalian cell surfaces. In the present study, erythrocytes (E) sensitized with pneumococcal LTA (LTA-E) were used as a model system to determine if LTA could render host cells susceptible to damage by autologous complement. Complement (C)-mediated lysis of LTA-E from normal rats and normal humans occurred when these cells were incubated in their respective autologous sera in vitro. In addition, when LTA-E from a C2-deficient human and from C4-deficient guinea pigs were incubated in their autologous sera, there was significant lysis in vitro, demonstrating a role for the alternative pathway. The in vivo survival of 51Cr-labeled autologous LTA-E was also studied. Only 2.9% of autologous LTA-E remained in the circulation of normal rats after 90 min. In contrast, 31.2% of autologous LTA-E remained in the circulation of rats depleted of C3. Intravascular hemolysis accounted for the clearance of LTA-E in the normal rats, whereas liver sequestration was responsible for clearance in the C3-depleted rats. These results demonstrate that LTA can render the host's cells susceptible to damage by its own complement system, establishing this as a possible mechanism of tissue damage in natural bacterial infections.

988 INDUCTION OF COMPLEMENT-MEDIATED DESTRUCTION OF HOST CELLS BY BACTERIAL LIPOTEICHOIC ACID

Lipoteichoic acids (LTA) released by gram positive bacteria can spontaneously bind to mammalian cell surfaces. Cells bearing LTA can activate either the classical or alternative pathways of complement (C') in heterologous sera. These observations suggest that the tissue damage which occurs during the course of a bacterial infection might in part be mediated by the host's C' system directed against its own cells bearing surface LTA. A model system was established using erythrocytes (E) presensitized with pneumococcal LTA (LTA-E). When LTA-E from normal rats, normal humans, normal guinea pigs, a C2-deficient human and C4-deficient guinea pigs were each incubated in their autologous sera, there was significant C'-mediated lysis in vitro. The survival of 51Cr-labelled autologous LTA-E in normal rats or cobra venom factor-treated rats (CoVF-rats) (<3% normal C3 levels) was also studied. Only 2.9% of autologous LTA-E remained in the circulation of normal rats at 90 min. In contrast, 31.2% of autologous LTA-E remained in the circulation of CoVF-rats. Intravascular hemolysis accounted for the clearance of LTA-E in the normal rats, while liver sequestration was responsible for clearance in the CoVF-rats. The survival of untreated autologous E in normal or CoVF-rats was normal (93.6% and 96.4% respectively). The results demonstrate that LTA can render cells susceptible to damage by the host's own C' system, establishing this as a possible mechanism of tissue damage in natural infections.

Activation of the alternative complement pathway by pneumococcal lipoteichoic acid.

Cell wall teichoic acids of some gram-positive bacteria are potent activators of the alternative pathway of complement. It is unclear, however, whether the other form of teichoic acid, cell membrane lipoteichoic acid (LTA), can also activate the alternative pathway. In the present study, radiolabelled pneumococcal LTA was found to bind spontaneously to sheep erythrocytes in a temperature- and time-dependent fashion. In addition, the presence of pneumococcal LTA on the erythrocyte surface was verified by the fact that they could be agglutinated by a myeloma protein (TEPC-15) specific for choline, a constituent of pneumococcal LTA. Pneumococcal LTA when fixed to the surface of erythrocytes was able to activate the alternative pathway of complement in both guinea pig serum deficient in the fourth component of complement and human serum deficient in the second component of complement, resulting in lysis of the sensitized erythrocytes. The sensitizing principle of the LTA preparation was removed before erythrocyte sensitization by immunoabsorption, using the choline-specific TEPC-15 myeloma protein. These data demonstrate that purified pneumococcal LTA will bind to sheep erythrocytes and endow them with the ability to activate the alternative pathway.

Biological effects of lipoteichoic acids

Pneumococcal lipoteichoic acid (Forssman antigen) added to the medium of growing pneumococcal cultures caused chain formation, prevented culture lysis in the stationary phase of growth, and inhibited lysis by penicillin and by the pneumococcal bacteriophage Dp-1.