Multi-spectrometric analyses of lipoteichoic acids isolated from Lactobacillus plantarum

Lipoteichoic acid is a major cell wall virulence factor of gram-positive bacteria. LTAs from various bacteria have differential immunostimulatory potentials due to heterogeneity in their structures. Although recent studies have demonstrated that LTA isolated from Lactobacillus plantarum (pLTA) has anti-inflammatory properties and is less inflammatory than LTAs from pathogenic bacteria, little is known about the structure of pLTA. In this study, high-field NMR spectra of the pLTA were compared with those of LTA from pathogenic bacterium, Staphylococcus aureus (aLTA). The 2D NMR results demonstrated that pLTA possesses α-linked hexose sugar substituents on the poly-glycerophosphate backbone instead of N-acetylglucosamine substituents, and unsaturated fatty acids in its glycolipids. The sugar substituents were revealed as an approximately 29:1 molar ratio of the glucose to galactose by HPAEC-PAD analysis. MALDI-TOF/TOF MS analyses identified the presence of unsaturated fatty acids in the glycolipid moieties of pLTA. In addition, the glycolipid structure was found to be composed of trihexosyl-diacyl- and/or trihexosyl-triacyl-glycerol ceramide units by means of unique fragment ions of the glycolipids. These results enabled us to elucidate the pLTA structure, which is distinctively different from canonical LTA structure, and suggest that the unique immunological property of pLTA might be caused by the pLTA structure.     

Lipoteichoic acid isolated from Lactobacillus plantarum inhibits lipopolysaccharide-induced TNF-alpha production in THP-1 cells and endotoxin shock in mice

In this study, the effect of Lactobacillus plantarum lipoteichoic acid (pLTA) on LPS-induced MAPK activation, NF-kappaB activation, and the expression of TNF-alpha and IL-1R-associated kinase M (IRAK-M) was examined. The expression of the pattern recognition receptor and the survival rate of mice were also examined. pLTA pretreatment inhibited the phosphorylation of ERK, JNK, and p38 kinase. It also inhibited the degradation of IkappaBalpha and IkappaBbeta, as well as the activation of the LPS-induced TNF-alpha factor in response to subsequent LPS stimulation. These changes were accompanied by the suppression of the LPS-induced expression of TLR4, NOD1, and NOD2, and the induction of IRAK-M, with a concurrent reduction of TNF-alpha secretion. Furthermore, the overexpression of pattern recognition receptors such as TLR4, NOD1, and NOD2 and the degradation of IRAK-M by transient transfection were found to reinstate the production of TNF-alpha after LPS restimulation. In addition, the i.p. injection of pLTA suppressed fatality, and decreased the level of TNF-alpha in the blood, in LPS-induced endotoxin shock mice. In conclusion, these data extend our understanding of the pLTA tolerance mechanism, which is related to the inhibition of LPS-induced endotoxin shock, and suggest that pLTA may have promise as a new therapeutic agent for LPS-induced septic shock.     

Protein expression changes in human monocytic THP-1 cells treated with lipoteichoic acid from Lactobacillus plantarum and Staphylococcus aureus

Lipoteichoic acid (LTA) from Staphylococcus aureus (aLTA) and from Lactobacillus plantarum LTA (pLTA) are both recognized by Toll-like receptor 2 (TLR2), but cause different stimulatory effects on the innate immune and inflammatory responses, and their underlying cellular mechanisms are unknown. In this study, comparative proteome analysis was performed using two-dimensional gel electrophoresis and mass spectrometry on protein extracts from human monocyte THP-1 cells stimulated with either aLTA or pLTA. Differentially expressed proteins might be involved in innate immunity and inflammation. Cells treated with aLTA and with pLTA showed different protein expression profiles. Of 60 identified proteins, 10 were present only in treated cells (8 in aLTA-treated only, and 2 in pLTA-treated only), 1 protein (IMPDH2) was suppressed by pLTA, and 49 were up- or down-regulated more than three-fold by aLTA- or pLTA- stimulation. Several proteins involved in immunity or inflammation, antioxidation, or RNA processing were significantly changed in expression by aLTA- or pLTA-stimulation, including cyclophilin A, HLA-B27, D-dopachrome tautomerase, Mn- SOD, hnRNP-C, PSF and KSRP. These data demonstrated that aLTA and pLTA had different effects on the protein profile of THP-1 cells. Comparison of the proteome alterations will provide candidate biomarkers for further investigation of the immunomodulatory effects of aLTA and pLTA, and the involvement of aLTA in the pathogenesis of Staphylococcus aureus sepsis.     

Lactobacillus plantarum lipoteichoic acid alleviates TNF-α-induced inflammation in the HT-29 intestinal epithelial cell line

We recently observed that lipoteichoic acid (LTA) isolated from Lactobacillus plantarum inhibited endotoxin-mediated inflammation of the immune cells and septic shock in a mouse model. Here, we examined the inhibitory role of L. plantarum LTA (pLTA) on the inflammatory responses of intestinal epithelial cells (IEC). The human colon cell line, HT-29, increased interleukin (IL)-8 expression in response to recombinant human tumor necrosis factor (TNF)-alpha, but not in response to bacterial ligands and interferon (IFN)-gamma. TNF-α also increased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and intercellular adhesion molecule 1 (ICAM-1) through activation of p38 mitogen-activated protein kinase (MAPK) from HT-29 cells. However, the inflammatory response of HT-29 on TNF-α stimulation was significantly inhibited by pLTA treatment. This pLTA-mediated inhibition accompanied the inhibition of nuclear factor (NF)-kappa B and MAPKs. Our data suggest that pLTA regulates cytokine-mediated immune responses and may be a good candidate for maintaining intestinal homeostasis against excessive inflammation.     

CD14 plays no major role in shock induced by Staphylococcus aureus but down-regulates TNF-alpha production

Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed similar symptoms of shock. In addition, inflammatory cytokines such as TNF-alpha and IL-6 were readily detectable in the serum of CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-alpha in CD14-deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus, suggesting that CD14 down-regulates TNF-alpha. A similar increase in serum TNF-alpha occurred when CD14-deficient animals were injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-alpha.     

Intrinsic nitric oxide-stimulatory activity of lipoteichoic acids from different Gram-positive bacteria

Lipoteichoic acid (LTA) is a structural component of the cell walls of Gram-positive bacteria. Similar to lipopolysaccharide (LPS) which is expressed in Gram-negative bacteria, LTA exhibits immunostimulatory properties. Frequently observed positive response of LTA in the Limulus amebocyte lysate (LAL) assay has been interpreted as a sign of LPS contamination, raising doubts about the intrinsic immune activities of LTA. Regarding many similarities in immunobiological and physicochemical properties of LTA and LPS, we hypothesized that similar to LPS, the LAL reactivity of LTA might be due to its ability to bind to LAL. Our data confirm the positivity of Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis and Streptococcus pyogenes LTAs in the LAL test. The estimates of suspected LPS content were 605, 10.3, 6.2 and 127 pg/μg LTA, respectively. The effectiveness of LTAs to induce the NO production in rat peritoneal cells was remarkably higher than that of equivalent concentrations of reference LPS (Escherichia coli). The LPS-induced NO was inhibited by polymyxin B (PMX), the IC₅₀ of PMX:LPS concentration ratio (pg:pg) being 1050:1. Many fold higher concentrations of PMX were needed to partially suppress the NO-augmenting effects of LTAs, applied at concentrations representing the equivalents of LPS. Transposed to the concentrations of LTAs per se, the IC₅₀s of the PMX:LTA ratios (μg:μg) ranged from 0.3:1 (S. aureus) to 7.5:1 (B. subtilis). It is concluded that LTA is not necessarily contaminated with LPS. The results prove the intrinsic immunostimulatory properties of LTAs of Gram-positive bacteria. The positive response of LTA in the LAL assay results from its capacity to bind to LAL. In addition, LTA binds with high affinity to PMX.     

Beta 2 integrins are involved in cytokine responses to whole Gram-positive bacteria

Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-alpha was measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly increased and decreased, respectively, TNF-alpha responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-alpha release induced by heat-killed group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or S. aureus. Using either of the latter bacteria, significant inhibition of TNF-alpha release was produced by Abs to CD11c, one of the subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3 but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14 transfectants. Collectively our data indicate that beta 2 integrins, such as CR3 and CR4, may be involved in cytokine responses to Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative to Gram-positive ones.     

Lipoteichoic acid isolated from Lactobacillus plantarum suppresses LPS-mediated atherosclerotic plaque inflammation

Chronic inflammation plays an important role in atherogenesis. Experimental studies have demonstrated the accumulation of monocytes/macrophages in atherosclerotic plaques caused by inflammation. Here, we report the inhibitory effects of lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) on atherosclerotic inflammation. pLTA inhibited the production of proinflammatory cytokines and nitric oxide in lipopolysaccharide (LPS)-stimulated cells and alleviated THP-1 cell adhesion to HUVEC by down-regulation of adhesion molecules such as intracellular adhesion molecule-1 (ICAM-I), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The inhibitory effect of pLTA was mediated by inhibition of NF-κB and activation of MAP kinases. Inhibition of monocyte/macrophage infiltration to the arterial lumen was shown in pLTA-injected ApoE^−/− mice, which was concurrent with inhibition of MMP-9 and preservation of CD31 production. The anti-inflammatory effect mediated by pLTA decreased expression of atherosclerotic markers such as COX-2, Bax, and HSP27 and also cell surface receptors such as TLR4 and CCR7. Together, these results underscore the role of pLTA in suppressing atherosclerotic plaque inflammation and will help in identifying targets with therapeutic potential against pathogen-mediated atherogenesis.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.     

Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds

Lipoteichoic acid (LTA) from several Gram-positive microorganisms, Forssman antigen (Fag) from $\text{Diplococcus pneumoniae}$ R36A, and an acidic lipopolysaccharide (ALP) from $\text{Micrococcus luteus}$ were examined for effects on four wall lysis systems. The LTAs inhibited the N-acetylmuramidases of $\text{Streptococcus faecalis}$ and $\text{Lactobacillus acidophilus}$, and the amidase of $\text{Bacillus subtilis}$ 168. Deacylated LTA failed to inhibit. LTAs failed to inhibit the amidase of pneumococcus. Fag inhibited the pneumococcal amidase, but had no effect on the other three systems.     

Lipoteichoic acid inhibits lipopolysaccharide-induced adhesion molecule expression and IL-8 release in human lung microvascular endothelial cells

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-alpha, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3-30 microg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 microg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-alpha on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 microg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to approximately 2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.     

Myricetin blocks lipoteichoic acid-induced COX-2 expression in human gingival fibroblasts

Periodontitis is an infectious disease caused by microorganisms present in dental bacterial plaque. Lipoteichoic acid (LTA) is a component of the external membrane of Gram-positive bacteria. It causes septic shock. Ingested flavonoids have been reported to directly affect the regulation of cyclooxygenase-2 (COX-2) expression induced by bacterial toxins. In this study, we examined the effects of four flavonoids (luteolin, fisetin, morin and myricetin) on the activation of ERK1/2, p38 and AKT, and on the synthesis of COX-2 in human gingival fibroblasts treated with LTA from Streptococcus sanguinis. We found that luteolin and myricetin blocked AKT and p38 activation and that myricetin blocked LTA-induced COX-2 expression. The results of our study are important for elucidating the mechanism of action of flavonoid regulation of inflammatory responses.     

Lipoteichoic acid-induced lung inflammation depends on TLR2 and the concerted action of TLR4 and the platelet-activating factor receptor

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.     

Characterization of lipoteichoic acid structures from three probiotic Bacillus strains: involvement of d-alanine in their biological activity

Probiotics represent a potential strategy to influence the host’s immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with d-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure–activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove d-alanine. The molecular structure of native and modified LTAs was determined by ¹H NMR and GC–MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their d-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on d-alanine substitutions. d-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.     

The role of toll-like receptors (TLRs) in bacteria-induced maturation of murine dendritic cells (DCS). Peptidoglycan and lipoteichoic acid are inducers of DC maturation and require TLR2

Toll-like receptors (TLRs) have been found to be key elements in pathogen recognition by the host immune system. Dendritic cells (DCs) are crucial for both innate immune responses and initiation of acquired immunity. Here we focus on the potential involvement of TLR ligand interaction in DC maturation. TLR2 knockout mice and mice carrying a TLR4 mutation (C3H/HeJ) were investigated for DC maturation induced by peptidoglycan (PGN), lipopolysaccharide (LPS), or lipoteichoic acids (LTAs). All stimuli induced maturation of murine bone marrow-derived DCs in control mice. TLR2(-)/- mice lacked maturation upon stimulation with PGN, as assessed by expression of major histocompatibility complex class II, CD86, cytokine, and chemokine production, fluorescein isothiocyanate-dextran uptake, and mixed lymphocyte reactions, while being completely responsive to LPS. A similar lack of maturation was observed in C3H/HeJ mice upon stimulation with LPS. DC maturation induced by LTAs from two different types of bacteria was severely impaired in TLR2(-)/-, whereas C3H/HeJ mice responded to LTAs in a manner similar to wild-type mice. We demonstrate that DC maturation is induced by stimuli from Gram-positive microorganisms, such as PGN and LTA, with similar efficiency as by LPS. Finally, we provide evidence that TLR2 and TLR4 interaction with the appropriate ligand is essential for bacteria-induced maturation of DCs.     

Lactobacillus rhamnosus GG decreases TNF-alpha production in lipopolysaccharide-activated murine macrophages by a contact-independent mechanism

Animal studies and human clinical trials have shown that Lactobacillus can prevent or ameliorate inflammation in chronic colitis. However, molecular mechanisms for this effect have not been clearly elucidated. We hypothesize that lactobacilli are capable of downregulating pro-inflammatory cytokine responses induced by the enteric microbiota. We investigated whether lactobacilli diminish production of tumour necrosis factor alpha (TNF-alpha) by the murine macrophage line, RAW 264.7 gamma (NO-), and alter the TNF-alpha/interleukin-10 (IL-10) balance, in vitro. When media conditioned by Lactobacillus rhamnosus GG (LGG) are co-incubated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA), TNF-alpha production is significantly inhibited compared to controls, whereas IL-10 synthesis is unaffected. Interestingly, LGG-conditioned media also decreases TNF-alpha production of Helicobacter-conditioned media-activated peritoneal macrophages. Lactobacillus species may be capable of producing soluble molecules that inhibit TNF-alpha production in activated macrophages. As overproduction of pro-inflammatory cytokines, especially TNF-alpha, is implicated in pathogenesis of chronic intestinal inflammation, enteric Lactobacillus-mediated inhibition of pro-inflammatory cytokine production and alteration of cytokine profiles may highlight an important immunomodulatory role for commensal bacteria in the gastrointestinal tract.     

Monocyte and macrophage activation by lipoteichoic Acid is independent of alanine and is potentiated by hemoglobin

Lipoteichoic acids (LTAs) are Gram-positive bacterial cell wall components that elicit mononuclear cell cytokine secretion. Cytokine-stimulating activity is thought to be dependent on retaining a high level of ester-linked D-alanine residues along the polyglycerol phosphate backbone. However, Streptococcus pyogenes LTA essentially devoid of D-alanine caused human and mouse cells to secrete as much IL-6 as LTA with a much higher D-alanine content. Furthermore, hemoglobin (Hb) markedly potentiates the stimulatory effect of various LTAs on mouse macrophages or human blood cells, regardless of their d-alanine content. LTA and Hb appear to form a molecular complex, based on the ability of each to affect the other's migration on native acrylamide gels, their comigration on these gels, and the ability of LTA to alter the absorption spectra of Hb. Because S. pyogenes is known to release LTA and secrete at least two potent hemolytic toxins, LTA-Hb interactions could occur during streptococcal infections and might result in a profound alteration of the local inflammatory response.     

Effect of alanine ester substitution and other structural features of lipoteichoic acids on their inhibitory activity against autolysins of Staphylococcus aureus.

Native substitution with the D-alanine ester of lipoteichoic acids (LTAs) affects their immunological properties, the capacity to bind divalent cations, and LTA carrier activity. In this study we tested the influence of the D-alanine ester on anti-autolytic activity, using extracellular autolysin from Staphylococcus aureus and nine LTAs with alanine/phosphorus molar ratios of between 0.23 and 0.71. The inhibitory activity, highest with alanine-free LTA, exponentially decreased with increasing alanine content, approaching zero at substitutions of greater than 0.6. Correspondingly, dipolar ionic phospholipids were not inhibitory, in contrast to negatively charged ones. Glycosylation of LTA up to an extent of 0.5 did not depress inhibitory activity, and even at a degree of 0.8 the effect was comparatively small. On comparison of LTAs from various sources, differences in lipid structures and chain lengths were without effect. The inhibitory activity drastically decreased when the glycolipid carried a single glycerophosphate residue or the hydrophilic chain had the unusual structure [6 leads to Gal(alpha 1--6)Gal(alpha 1--3)Gro-(2 comes from 1 alpha Gal)-P]n, in which digalactosyl moieties connect the alpha-galactosylated glycerophosphate units. Principally, the same results were obtained with the more complex system of autolysis of S. aureus cells. We hypothesize that the anti-autolytic activity of LTA resides in a sequence of glycerophosphate units and that the negative charges of appropriately spaced phosphodiester groups play a crucial role. The alanine ester effect is discussed with respect to the putative in vivo regulation of autolysins by LTA.