Streptococcal Lipoteichoic Acid Research Articles

Antimicrobial activity of tea catechin against canine oral bacteria and the functional mechanisms.

Epigallocatechin gallate (EGCG) is the major polyphenolic compound of green tea. Polyphenolic compounds were extracted from the leaf of Camellia sinensis (Japanese green tea), and the minimum inhibitory concentration against canine oral bacteria was measured. Subsequently, we investigated the inhibitory effects of polyphenolic compounds and EGCG on the growth of canine oral bacteria. EGCG showed antimicrobial activity against a model bacterium, Streptococcus mutans. Our results indicate that EGCG can inhibit the growth and biofilm formation of S. mutans and that EGCG does not interact with streptococcal lipoteichoic acid (LTA). Furthermore, our findings suggest that EGCG interacts with other component(s) of the bacterial membrane aside from streptococcal LTA to inhibit biofilm formation and damage biofilms.

Decreased serum and pharyngeal antibody levels specific to streptococcal lipoteichoic acid in children with recurrent tonsillitis

Objective: Streptococcus ( S.) pyogenes is a common cause of primary as well as recurrent tonsillitis (RT). Lipoteichoic acid (LTA) has been proposed as a possible candidate for vaccine formulation against streptococcal infections, because LTA is a common constituent of streptococci and the antibody to LTA inhibits bacterial attachment to epithelial cells in vitro. Streptolysin-O and streptococcal whole cell body are highly immunogenic and the antibodies to these antigens are reported to be better parameters for streptococcal infections The objective of the present study is to investigate how systemic and local immune activities against S. pyogenes may be associated with RT. Methods: Sera from 178 children with or without RT aged 1–15 years with a median age of 5 years were investigated for the levels of total immunoglobulins and antibodies specific to streptococcal antigens such as whole cell body, LTA, and streptolysin-O. Pharyngeal secretions from 67 children with or without RT aged 2–14 years with a median age of 6 years were subjects to secretory IgA (SIgA) antibody levels to streptococcal LTA. The antibodies to whole cell body and LTA were measured by enzyme-linked immunosorbent assay. Total immunoglobins and the anti-streptolysin-O antibody were assayed by nephelometry. Results: An age-matched comparison revealed that either levels of serum IgG antibody or pharyngeal SIgA antibody to streptococcal LTA at 2–5 years of age were significantly lower in RT children than in non-RT children (1.39 vs. 5.14 μg/ml, P=0.001; 10.6 vs. 29.9 units/ng/ml total SIgA, P=0.015; respectively) and correlated inversely to episodes of tonsillitis ( r=−0.242, P=0.024; r=−0.3, P=0.024; respectively). Either serum total immunoglobulin levels of IgG or IgA correlated positively to episodes of tonsillitis in children aged 2–5 years ( r=0.293, P=0.011; r=0.361, P=0.002; respectively). No difference was found on either serum levels of IgG antibody to streptococcal whole cell body or antibody to streptolysin-O between RT and non-RT children in any age-matched comparisons. High serum antibody levels to whole cell body was associated with high antibody levels to streptococcal LTA in non-RT children ( r=0.198, P<0.05), but no association was found between these antibody levels in RT children. Conclusions: Selective immunologic failure in systemic and pharyngeal antibody response to streptococcal LTA may be a potential cause of RT in young children.

Cytokine production in an ex vivo whole blood model following induction by group B streptococcal polysaccharides and lipoteichoic acid.

An excessive release of pro-inflammatory cytokines, such as TNF-α and IL-1, during bacterial invasive infections can mediate several pathophysiologic phenomena2, 9, 11. Group B streptococci (GBS) are important causes of neonatal sepsis and invasive infections in adults, which are often rapidly fatal despite appropriate therapy. High TNF-α and IL-6 plasma levels were found in children with GBS sepsis2. Infection with GBS can induce the production of TNF-α, IL-1, IL-6 and interferon γ in neonatal rats10. Moreover, in these animals, by the use of heat-killed isogenic GBS mutant strains devoid of the capsular type-specific polysaccharide it was shown that this polysaccharide would not be the only factor which elicits the release of TNF-α6. Many of the host responses to gram-negative bacteria could be attributed to lipopolysaccharide (LPS), which triggers monocytes to release inflammatory cytokines5. Recent studies indicated that, aside from LPS, peptidoglycan and various polysaccharides, including uronic acid, polyuronic acid, rhamnose-glucose polymers, trigger monocytes to release cytokines1, 7, 8. This led us to investigate whether TNF-α, IL-1β, IL-6 and IL-8 could be induced in human whole blood by the addition of purified surface components of GBS, namely group (CHO-B), type Ia (CHO-Ia) and III (CHO-III) specific polysaccharides or lipoteichoic acid (LTA).

An oxygen-induced but protein F-independent fibronectin-binding pathway in Streptococcus pyogenes.

Protein F is an important fibronectin-binding adhesin of Streptococcus pyogenes (group A streptococcus). However, all previous analyses of protein F have been conducted in a mutant strain which expresses protein F under anaerobic conditions nonpermissive for expression in other strains. In this study, we have examined the fibronectin-binding properties of several protein F-deficient mutants cultured under aerobic conditions and have identified a second pathway for binding fibronectin. Unlike the case with protein F, exposure to an aerobic environment does not induce transcription of a new gene product. Rather, O2 is apparently required for the modification of a protease-resistant cell surface component into a binding-component form. Modification occurred preferentially at a pH of 6.0 or less, and the binding of the modified component to fibronectin required Zn2+. The oxidizing agent Fe(CN)6 could be substituted for O2 and stimulated expression of binding activity under O2-limiting conditions. Streptococcal fibronectin binding mediated by this pathway but not by protein F could be inhibited by laminin and by streptococcal lipoteichoic acid, a molecule previously implicated as the streptococcal adhesin for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism for fibronectin. The non-protein F-binding activity could also substantially enhance the binding of the organism to basement membrane. By using differential inhibition, analyses of binding to non-protein F mutant strains demonstrated that the total level of fibronectin bound under aerobic conditions reflects contributions from both pathways. Because of its dependence on Zn2+, an oxidant, and pH, this binding activity has been designated the ZOP binding pathway.

Competitive binding of calcium and magnesium to streptococcal lipoteichoic acid

Equilibrium dialysis was used to investigate the binding capacity and affinity of lipoteichoic acid (LTA) from the Gram-positive bacterium Streptococcus sanguis for calcium and magnesium by a competitive method. LTA was shown to bind approx. 1 mol of either calcium or magnesium per mole phosphate. Calcium and magnesium dissociation constants were found to be 8.39 ± 0.31 mmol/l and 15.01 ± 2.01 mmol/l respectively, indicating that S. sanguis LTA will preferentially bind calcium. LTA may act as a calcium buffer by reducing the free calcium concentration to which the cell is exposed. The capacity to produce large quantities of LTA could thus be as important as aciduricity in selection of species at caries-prone sites.

Macrophage Inflammatory Protein-1α Expression in Vivo and in Vitro: The Role of Lipoteichoic Acid

Lipoteichoic acid (LTA), a component of the cell wall of most gram-positive bacteria, has been shown to play a significant role in the initiation and progression of bacterial infection. However, little is known of its position in the cytokine network involved in the induction and perpetuation of inflammation. In this study, we assessed whether the macrophage activating and chemotactic cytokine macrophage inflammatory protein-1α (MIP-1α) was expressed in the setting of localized gram-positive infection. Furthermore, we determined whether LTA purified from either Staphylococcus aureus or Streptococcus pyogenes could induce the expression of MIP-1α mRNA and protein from human blood monocytes. Immunohistochemical staining of human endocardial samples obtained from patients with acute S. aureus endocarditis revealed cell-associated MIP-1α expression by neutrophils, macrophages, and fibroblasts. Treatment of human peripheral blood monocytes in vitro with LTA isolated from either S. aureus or S. pyogenes resulted in both the time- and dose-dependent expression of MIP-1α mRNA. Similarly, staphylococcal and streptococcal LTA induced the dose-dependent production of MIP-1α protein after 24 h in culture. These studies suggest that LTA may play an important role in triggering the recruitment and activation of leukocytes that characterizes the host response to gram-positive bacterial invasion.

Identification of a novel lectin inStreptococcus pyogenes and its possible role in bacterial adherence to pharyngeal cells

During investigation of the interaction of human lactoferrin (HLf) with variou bacteria, it was found that inStreptococcus pyogenes, HLf binding occurred to agar-rather than broth-grown cells irrespective of the nutrients used. Furthermore, binding of HLf to broth-grown, heat-killed bacteria was induced by overnight incubation on agar media or short-time exposure of the cells to water-soluble agar extract. The binding pattern was revealed in most of 92S. pyogenes strains representing various M-or T-types with no apparent type variation. The component thus bridging the attachment of HLf to the streptococcal cell surface was recovered in extracts of agar-grown cells and isolated by affinity chromatography on HLf-sepharose. By gel filtration in the presence of radiolabeled HLf, this component exhibited similar elution position as crude water-soluble agar extract. Chemical analysis identified the active HLf-binding agar component to be a galactose-rich polysaccharide (GRP). Further binding tests showed that the interaction between streptococci and GRP was stable in the presence of high molar NaCl, KSCN, or urea and was unaffected by various serum or matrix proteins or by streptococcal lipoteichoic acid; however, a moderate inhibition by heparin or bovine mucin was observed. Studies on isogenic mutants ofS. pyogenes did not support the involvement of M-protein or the hyaluronate capsule in the binding of GRP. SDS-PAGE and Western blot analyses revealed a GRP-binding protein of approximately 70 kDa in the cell-wall extracts of two strains ofS. pyogenes, types M19 and M55. Finally, the adherence of (broth-grown)3H-thymidine-labeledS. pyogenes, type M19, to the pharyngeal epithelial cell line DT-562 or to normal tonsillar epithelial cells was inhibited by GRP in a dose-related manner. We thus propose that the streptococcal GRP-binding component may represent a novel surface lectin acting as a mucosal adhesin forS. pyogenes, in accordance with previous data indicating that galactosecontaining sugar moieties may serve as ligands for the adherence of streptococci to pharyngeal cells. Our results also indicate that GRP-like components such as mucin or heparin might act to block epithelial adherence ofS. pyogenes at the mucosal level.

In vitro activation of the classical pathway of complement by a streptococcal lipoteichoic acid.

The purpose of this study was to find whether a glycerolphosphate-containing lipoteichoic acid prepared from Streptococcus sobrinus OMZ 176 cells would activate the classical pathway of complement while in solution. Reference activators were lipopolysaccharide from Escherichia coli 0111:B4 and heat-aggregated immunoglobulin G. Serum samples were taken from healthy students. Analysis through crossed immunoelectrophoresis showed that lipoteichoic acid caused an almost complete dissociation of the C1qrs macromolecule. All activators decreased the area of and slowed the electrophoretic mobility of the C4 protein peaks, with lipoteichoic acid causing the most pronounced alterations. Electroimmunoassays showed that lipoteichoic acid separately, yielded detectable amounts of free C1r2s2 subunits; it also generated significantly more trimer complexes between C1r, C1s and C1 inhibitor (C1INH) than did the other two activators. Lipoteichoic acid was, however, a comparatively weak inducer of tetramer C1INH-C1r-C1s-C1INH complexes. Analysis through Western blotting showed that all activators accelerated consumption of C1r, induced complex formations between C1INH and C1s and produced cleavage products of C2. Altogether, the immunochemical analysis gave clear evidence of classical pathway activation by lipoteichoic acid, but its activation profile differed from those seen with lipopolysaccharide and aggregated immunoglobulin G.

Identification of a Fibronectin-Binding Protein as Adhesin of Streptococcus pyogenes

The role of streptococcal fibronectin-binding protein (Sfb protein) in the adherence of Streptococcus pyogenes to epithelial cells was analyzed by using a recombinant Sfb fusion protein which was constructed by fusion of the binding domain of Sfb protein to MS2 polymerase. Sfb fusion protein was overexpressed in Escherichia coli, purified from E. coli lysates by FPLC and analysed in binding experiments with 125I-labelled fibronectin and adherence studies with HEp2 epithelial cells. Lysates from E. coli expressing the fusion protein as well as purified Sfb protein competitively inhibited fibronectin binding and epithelial cell adherence of S. pyogenes, whereas streptococcal lipoteichoic acid (LTA), previously suggested to be the adhesin of S. pyogenes, had no effects. Southern blot analysis of chromosomal DNA from various streptococci revealed that the gene coding for the binding domain of Sfb protein was also present in other S. pyogenes strains. These results indicate that fibronectin-binding protein is the major adhesin of S. pyogenes.

Effects of a streptococcal lipoteichoic acid on complement activation in vitro

This study describes activation of serum complement by lipoteichoic acid (LTA) from Streptococcus mutans OMZ 176, while in solution. Serum from 16 healthy students was taken. Test samples were incubated with increasing doses (1-5,000 micrograms/ml) of LTA or lipopolysaccharide (LPS) from Escherichia coli 0111:B4 for 1 h at 37 degrees C; then assayed for degradation of C3, C4 or factor B by crossed immunoelectrophoresis. Each preparation caused a significant (p < 0.05) dose-dependent conversion of C3. The response curves obtained were not statistically different. LPS was a stronger activator of the alternative pathway than LTA, as judged from analysis of C3 degradation in the presence of Mg2+/EGTA, and from their effects on factor B cleavage. LTA caused, however, pronounced alterations in the shape of C4 precipitation in the gels. Functional (hemolytic) assays showed that, when tested at 200 micrograms/ml, LTA and LPS triggered significant (p < 0.05) consumptions of both classical and alternative pathway proteins. LPS was a significantly (p < 0.05) stronger activator than LTA. Apparently, the C3 degradation found for this LTA involved the alternative pathway to a small extent; thus some other mechanism of fluid-phase C3 cleavage seemed also to be operative.

Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells.

The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to epithelial cells.

Deposition of circulating streptococcal lipoteichoic acid in mouse tissues

The tissue binding properties of streptococcal lipoteichoic acid (LTA) were studied using normal and passively immunized BALB/c mice. After intraperitoneal injection in non-immunized mice, 3H-LTA concentrations in blood, heart, kidney and liver were highest between 24 and 30 h post-injection. LTA deposits in heart remained high for the next 24 h, whereas other tissue levels decreased. Constant amounts of 3H-LTA were detected in urine throughout the 48 h period. In passively immunized mice, the amount of tissue deposition of 3H-LTA was inversely proportional to the ratio of antibodies to LTA. Autoradiography revealed focal deposits of 3H-LTA in heart, kidney and liver. These observations indicate that LTA, released by streptococci growing at remote body sites, can be carried by the blood to internal organs where it can accumulate and participate in pathogenesis.

Interaction of streptococcal lipoteichoic acid with artificial tooth pellicle

Artificial pellicles were prepared by coating hydroxyapatite beads with whole saliva. Radio-labelled lipoteichoic acid was isolated from Streptococcus sanguis NCTC 7863 by phenol extraction. Various concentrations of radiolabelled lipoteichoic acid were mixed with saliva-coated hydroxyapatite in the presence and absence of high ionic strength phosphate buffer, bovine serum albumin, gelatin, unlabelled lipoteichoic acid, Tween 20 and Triton X-100. The amount of lipoteichoic acid binding was measured by counting the residual radioactivity of the saliva-coated hydroxyapatite after thorough washing. In one experiment the binding was measured in the presence of a mucinous glycoprotein isolated from human saliva. The data were analysed by means of Scatchard and double reciprocal plots of the bound and unbound fraction of lipoteichoic acid. The lipoteichoic acid interacted hydrophobically with the saliva-coated hydroxyapatite; the interaction was complex with multiple binding sites exhibiting a range of affinities. The mean association constant was 1.1 × 10 9 M −1 and the minimum number of binding sites was 3.9 × 10 12/mm 2 of artificial pellicle. The salivary mucin competitively inhibited the interaction, which suggests that this may be one of the salivary components involved.

A sandwich adhesin on Streptococcus pneumoniae attaching to human oropharyngeal epithelial cells in vitro

Streptococcus pneumoniae attach to human pharyngeal epithelial cells through the specific interaction of bacterial surface adhesins with glycoconjugate receptors. The present study defines the adhesin as a molecule bridging between an anchoring site in the bacterial cell wall and the epithelial cell receptor. The nature of the adhesin was defined in three ways: First, the attachment of whole bacteria was reduced by trypsin, periodate and heat. Second, heat treatment of whole bacteria was shown to release material, which was able to reconstitute the adherence. The heat extract bound to epithelial cells, as shown by fluorescence labelling, and agglutinated latex beads covalently coupled with receptor oligosaccharide. Active material could be extracted by heat from both high and low adhering strains, but could reconstitute only attaching strains. Third, the bacterial component binding the adhesin was localized to protoplasts and cell wall fractions obtained by mechanical or deoxycholate induced lysis of pneumococci. Isolated pneumococcal surface components, which did not inhibit attachment, included peptidoglycan, C polysaccharide, Forssmann antigen, capsular polysaccharide and a phenol extract produced in analogy to streptococcal lipoteichoic acid. The procedure used to extract the adhesin was previously used to prepare the competence factor. The competence deficient mutant RA7 attached poorly compared to the competent R6 parent. The possible relatedness of attachment to competence is discussed.

Adherence of group B streptococci to adult and neonatal epithelial cells mediated by lipoteichoic acid.

We have investigated the role of lipoteichoic acid in mediating the adherence of different serotypes of group B streptococci to human adult and neonatal epithelial cells. Pretreatment of neonatal buccal and vaginal epithelial cells with lipoteichoic acid, but not with deacylated lipoteichoic acid, induced a marked inhibition in the adherence of all strains tested. Pretreatment of bacteria with substances known to bind lipoteichoic acid, such as monoclonal and polyclonal antipolyglycerophosphate antibodies and albumin, also resulted in adherence inhibition. Group B streptococci adhered in 6- to 10-fold-higher numbers to buccal epithelial cells from neonates older than 3 days than to those from neonates less than 1 day old. This increase in receptiveness for group B streptococci was paralleled by an increased ability of epithelial cells from older neonates to bind group B streptococcal lipoteichoic acid. These data suggest a role for the lipid portion of lipoteichoic acid in the adherence of different serotypes of group B streptococci to vaginal and neonatal epithelial cells.

A nontoxic tumour necrosis factor induced by streptococcal lipoteichoic acids.

A nontoxic tumour necrosis factor induced by streptococcal lipoteichoic acids.

Hybridoma Antibodies to the Lipid-Binding Site(s) in the Amino-Terminal Region of Fibronectin Inhibits Binding of Streptococcal Lipoteichoic Acid

In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain.

Lack of toxicity of oral and intrapulmonary group B streptococcal lipoteichoic acid.

Lipoteichoic acid (LTA) was prepared from type III group B streptococci and administered by topical oral application or intravenous or intratracheal injection in weanling and adult white New Zealand rabbits. Tritiated [3H]LTA in tissues and body fluids was measured by scintillation spectrometry. Five minutes to 120 h after intravenous injection of 10 mg (17 x 10(6) dpm) of [3H]LTA, none was present in blood. Combined urine and fecal excretion peaked at 24 h and decreased over 5 days. There was no effect on collagen-induced platelet aggregation. [3H]LTA concentrations were greatest in colon, bone, stomach, and skin 1 day after intravenous injection. After a 5-mg oral dose (8.5 x 10(6) dpm) in an adult animal, fecal excretion peaked at 24 h and decreased after 4 days. No systemic absorption was noted. No [3H]LTA was found in any of seven tissues examined at autopsy 3 days after 1 to 5 mg/kg oral doses in weanling animals with normal or traumatized buccal mucosa. No effect was noted on platelet aggregation or serum complement, there was no increase in the incidence of nephrocalcinosis and the buccal mucosa remained histologically normal. Intratracheal injection of 0.5 to 2.5 mg/kg of LTA resulted in no tachypnea or alteration in blood gases. All animals remained healthy after LTA administration. The absence of toxicity and absorption in animals suggests that studies could be performed in humans to evaluate the safety and efficacy of oral LTA.

Effect of streptococcal lipoteichoic acid on prolyl hydroxylase activity as related to collagen formation in mouse fibroblast monolayers.

Dried and wet mouse fibroblast monolayers with labeled collagenous substrate were used to study the effects of lipoteichoic acid (LTA) on cellular prolyl hydroxylase activity. LTA is a scavenger of cations, and Fe2+ is essential for prolyl hydroxylase activity. Surprisingly, addition of LTA to dried monolayers resulted in increased prolyl hydroxylase activity, whereas preincubation of Fe2+ with LTA only negated this increase. However, significant inhibition of enzyme activity by wet monolayers occurred whether LTA was added directly to the test system or whether it was used after preincubation with Fe2+. These data suggest that LTA causes membrane perturbations. Also, that the binding of LTA to the membrane of dried and wet monolayers appears to be decidedly different when based on the subsequent availability of Fe2+ for cellular prolyl hydroxylase activity. The ability of LTA to act as a cationic exchanger and the presence of intracellular Fe2+ inaccessible to LTA probably accounted for the lack of complete inhibition of prolyl hydroxylase activity by this amphiphile in the wet cell system. Considerably less iron was needed to negate the partial inhibition of prolyl hydroxylase activity by LTA in viable cells than was needed to restore the increased enzyme activity by this amphiphile in equivalent dried preparations. These and other results showed that, although LTA does not affect collagen polypeptide chain formation in wet monolayers, its involvement at the molecular level does result in a marked decrease in the hydroxylation of collagenous peptidyl prolyl residues through LTA interaction with Fe2+. This reduction in prolyl hydroxylase activity equaled the reduction in hydroxylation of collagenous protein in fibroblast monolayers caused by LTA reported earlier (O. Leon and C. Panos, Infect. Immun. 40:785-794, 1983). Therefore, these data suggest that partial inhibition of prolyl hydroxylase activity is directly related to the synthesis of defective collagen by wet fibroblast monolayers exposed to minute amounts of group A, type 12 streptococcal LTA. Use of LTA also showed that complete inhibition of hydroxyproline formation is not required for the continued formation and accumulation of defective collagenous protein by these monolayers.

Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid.

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.