Streptococcal histone induces murine macrophages To produce interleukin-1 and tumor necrosis factor alpha.

The histone-like protein (HlpA) is highly conserved among streptococci. After lysis of streptococci in infected tissues, HlpA can enter the bloodstream and bind to proteoglycans in the glomerular capillaries of kidneys, where it can react with antibodies or stimulate host cell receptors. Deposits of streptococcal antigens in tissues have been associated with localized acute inflammation. In this study, we measured the ability of purified HlpA (5 to 100 microg/ml), from Streptococcus mitis, to induce the production of proinflammatory cytokines by cultured, murine peritoneal macrophages. The release of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) was time and concentration dependent and was not diminished by the presence of polymyxin B. Exposure of macrophages to a mixture of HlpA and lipoteichoic acid resulted in a synergistic response in the production of both TNF-alpha and IL-1. Stimulation with a mixture of HlpA and heparin resulted in reduced cytokine production (50% less IL-1 and 76% less TNF-alpha) compared to that by cells incubated with HlpA alone. The inclusion of antibodies specific to HlpA in macrophage cultures during stimulation with HlpA did not affect the quantity of TNF-alpha or IL-1 produced. These observations suggest that streptococcal histone may contribute to tissue injury at infection sites by promoting monocytes/macrophages to synthesize and release cytokines that initiate and exacerbate inflammation. Streptococcus pyogenes, which can infect tissues in enormous numbers, may release sufficient amounts of HlpA to reach the kidneys and cause acute poststreptococcal glomerulonephritis.

Influence of environmental conditions on hydrogen peroxide formation by Streptococcus gordonii.

Hydrogen peroxide generated by viridans group streptococci has an antagonistic effect on many bacterial species, including a number of pathogens, in the oral environment. This study examines the influence of a variety of environmental conditions on rates of hydrogen peroxide synthesis by Streptococcus gordonii. Hydrogen peroxide was synthesized at every concentration of glucose and sucrose tested from 10 microM to 1 M, with the highest rates occurring at 0.1 mM sucrose and 1 mM glucose. S. gordonii appeared to have an intracellular store of polysaccharide which supported hydrogen peroxide formation even when the assay buffer contained no carbohydrate. Most heavy metal ions inhibited peroxidogenesis, and anaerobic conditions induced adaptive down-regulation of hydrogen peroxide synthesis; however, peroxidogenesis was generally insensitive to moderate increases in salt concentration, alteration of the mineral content of the assay solution, and changes in pH between 5.0 and 7.5. In contrast, stimulation of peroxidogenesis occurred in 1 mM Mg(2+) and 10 to 50 mM potassium L-lactate. Maximum peroxidogenesis occurred during the mid-logarithmic and late-logarithmic phases of bacterial growth. These bacterial responses may have significant implications for oral ecology and oral health.

An extracellular protease of Streptococcus gordonii hydrolyzes type IV collagen and collagen analogues.

Streptococcus gordonii is a frequent cause of infective bacterial endocarditis, but its mechanisms of virulence are not well defined. In this study, streptococcal proteases were recovered from spent chemically defined medium (CDM) and fractionated by ammonium sulfate precipitation and by ion-exchange and gel filtration column chromatography. Three proteases were distinguished by their different solubilities in ammonium sulfate and their specificities for synthetic peptides. One of the enzymes cleaved collagen analogs Gly-Pro 4-methoxy-beta-naphthylamide, 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), and p-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (pZ-peptide) and was released from the streptococci while complexed to peptidoglycan fragments. Treatment of this protease with mutanolysin reduced its 180- to 200-kDa mass to 98 kDa without loss of enzymatic activity. The purified protease cleaved bovine gelatin, human placental type IV collagen, and the Aalpha chain of fibrinogen but not albumin, fibronectin, laminin, or myosin. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. Maximum production of the 98-kDa protease occurred during growth of S. gordonii CH1 in CDM containing 0.075% total amino acids at pH 7.0 with minimal aeration. Higher initial concentrations of amino acids prevented the release of the protease without reducing cell-associated enzyme levels, and the addition of an amino acid mixture to an actively secreting culture stopped further enzyme release. The purified protease was stored frozen at -20 degreesC for several months or heated at 50 degreesC for 10 min without loss of activity. These data indicate that S. gordonii produces an extracellular gelatinase/type IV collagenase during growth in medium containing minimal concentrations of free amino acids. Thus, the extracellular enzyme is a potential virulence factor in the amino acid-stringent, thrombotic, valvular lesions of bacterial endocarditis.

Streptococcal histone-like protein: primary structure of hlpA and protein binding to lipoteichoic acid and epithelial cells.

In addition to its role in the nucleoid, the histone-like protein (HlpA) of Streptococcus pyogenes is believed to act as a fortuitous virulence factor in delayed sequelae by binding to heparan sulfate-proteoglycans in the extracellular matrix of target organs and acting as a nidus for in situ immune complex formation. To further characterize this protein, the hlpA genes were cloned from S. pyogenes, S. gordonii, S. mutans, and S. sobrinus, using PCR amplification, and sequenced. The encoded HlpA protein of S. pyogenes has 91 amino acids, a predicted molecular mass of 9,647 Da, an isoelectric point of 9.81, and 90% to 95% sequence identity with HlpA of several oral streptococci. The consensus sequence of streptococcal HlpA has 69% identity with the consensus sequence of the histone-like HB protein of Bacillus species. Oral viridans group streptococci, growing in chemically defined medium at pH 6.8, released HlpA into the milieu during stationary phase as a result of limited cell lysis. HlpA was not released by these bacteria when grown at pH 6.0 or below. S. pyogenes did not release HlpA during growth in vitro; however, analyses of sera from 155 pharyngitis patients revealed a strong correlation (P < 0.0017) between the production of antibodies to HlpA and antibodies to streptolysin O, indicating that the histone-like protein is released by group A streptococci growing in vivo. Extracellular HlpA formed soluble complexes with lipoteichoic acid in vitro and bound readily to heparan sulfate on HEp-2 cell surfaces. These results support a potential role for HlpA in the pathogenesis of streptococcus-induced tissue inflammation.

Lectin inhibition of bacterial adhesion to animal cells.

Adhesion of bacteria to epithelial cells of the respiratory, gastric, and genitourinary mucosa is generally considered to be the initial step in the pathogenesis of many bacterial infections (1). Adhesion enables the bacteria to localize near a food source and to resist being washed away by the fluids that constantly bathe mucosal surfaces Bacteria that persist at the site of attachment can proliferate and thus establish a stable colonization. If the bacteria produce the necessary exoenzymes and/or exotoxins to overcome other host defenses, they may invade deeper into the tissues and cause chmcal symptoms.

The alpha-hemolysin of Streptococcus gordonii is hydrogen peroxide.

The alpha-hemolysin of viridans group streptococci, which causes greening of intact erythrocytes, is a potential virulence factor as well as an important criterion for the laboratory identification of these bacteria; however, it has never been purified and characterized. The alpha-hemolysin of Streptococcus gordonii CH1 caused characteristic shifts in the A403, A430, A578, and A630 of sheep hemoglobin. A spectrophotometric assay was developed and used to monitor purification of alpha-hemolysin during extraction in organic solvents and separation by reverse-phase high-performance liquid chromatography (HPLC). The alpha-hemolysin was identical to hydrogen peroxide with respect to its effects on erythrocyte hemoglobin, oxygen-dependent synthesis by streptococci, insensitivity to proteases, inactivation by catalase, differential solubility, failure to adsorb to ion-exchange chromatography resins, and retention time on a reverse-phase HPLC column. The amount of hydrogen peroxide present in HPLC-fractionated spent culture medium was sufficient to account for all alpha-hemolytic activity observed.

Beta-adrenergic receptor regulation of macrophage-derived tumor necrosis factor-alpha production from rats with experimental arthritis.

Prostaglandin E2 (PGE2) and beta-adrenergic agonists can suppress lipopolysaccharide-induced tumor necrosis factor-alpha (TNF) production from elicited macrophages. We assessed the responsiveness of rat peritoneal macrophages to PGE2 and the beta-adrenergic agonist isoproterenol during immunologically-mediated arthritis. We assessed macrophage sensitivity to these mediators from resident macrophages and macrophages elicited with either streptococcal cell wall or complete Freund's adjuvant. Peritoneal macrophages were obtained from female Lewis rats that were (1) injected with complete Freund's adjuvant and non-arthritic (CFA); (2) injected with streptococcal cell wall and arthritic (ART); (3) injected with streptococcal cell wall and non-reactive (NON) and (4) non-elicited resident macrophages (RES). When challenged with graded concentrations of lipopolysaccharide (0.1 to 10,000 ng/ml), macrophages obtained from each group of rats released TNF in a concentration-dependent manner, with macrophages from arthritic rats (ART) producing the greatest amount of TNF (p < 0.001). While PGE2 suppressed lipopolysaccharide (100 ng/ml) stimulated TNF production in a concentration-dependent manner in all groups, the greatest sensitivity to PGE2 was observed with macrophages obtained from rats which received streptococcal cell wall when compared to both complete Freund's adjuvant-elicited and resident macrophages (p < 0.05). The beta-adrenergic agonist isoproterenol also inhibited lipopolysaccharide-stimulated TNF production from macrophages in all groups. In addition, the specific beta 2-adrenergic antagonist, ICI 118.551, shifted isoproterenol concentration-effect curves to the right (p < 0.01). Minimal responsiveness to isoproterenol was observed with resident peritoneal macrophages. Maximum isoproterenol-induced inhibition of TNF production was observed with complete Freund's adjuvant-elicited macrophages, and significantly less in macrophages of streptococcal cell wall-injected rats. Of particular interest, macrophages obtained from streptococcal cell wall-injected rats, which became arthritic, were significantly less sensitive to isoproterenol than those which did not develop arthritis (p < 0.02). In addition, these changes in sensitivity were not reflected by changes in the sensitivity of both CFA and ART groups to dibutyryl cAMP. The present study demonstrates a shift in the balance between inhibitory mediator responses in rats inoculated with one of two different adjuvants. These investigations support the role of PGE2 and a neurotransmitter as immunomodulating compounds which may effectively maintain an inflammatory lesion such as arthritis.

Dynamics of streptococcus histone retention by mouse kidneys.

To study the interaction of streptococcal histone-like protein with renal tissue, 2 groups of 12 mice were injected intravenously with either radioiodinated histone or bovine serum albumin. At intervals over 48 hr, 2 mice from each group were anesthetized and perfused with tissue culture medium and the amounts of radioactivity were measured in blood, urine, and kidneys. The streptococcal protein rapidly disappeared from the blood and accumulated in renal tissue. Kidney radioactivity was maximal at 2 hr and then declined steadily over the ensuing 46 hr. Retention of streptococcal protein in renal tissue was 2 orders higher than that of BSA throughout the experiment. Immunofluorescence staining of kidney sections showed that the histone protein was adsorbed to the basement membranes of the glomeruli and collecting tubules. There were similar rates of excretion of radioactivity in urine by the two groups of mice. Injection of preformed complexes of streptococcal histone and rabbit antibodies into a third group of mice resulted in deposits of immune complexes in glomeruli but did not change the overall rate at which the radiolabeled streptococcal protein was distributed. The accumulation of streptococcal histone in renal tissue, independently of antibodies or while contained in circulating immune complexes, makes it a potential virulence factor in the pathogenesis of poststreptococcal glomerulonephritis. Its pathogenic properties remain to be studied in an appropriate animal model.

Glucosyltransferase mediates adhesion of Streptococcus gordonii to human endothelial cells in vitro.

Human umbilical vein endothelial cells (HUVEC) were used as an experimental host model to investigate the mechanism(s) of streptococcal adhesion in infective endocarditis. Adhesion activity of Streptococcus gordonii was maximal during the logarithmic phase of growth and was greatly reduced or eliminated by pretreatment of bacteria with heat, formaldehyde, or trypsin. At saturating numbers of streptococci, an average of 81 bacteria were bound per HUVEC. Streptococcal adhesion was inhibited by low-molecular-weight dextran and heparin but not by sucrose, fibronectin, or laminin. Adhesion was also prevented by pretreatment of HUVEC with proteins dissociated from the surface of S. gordonii with 10 mM EDTA or isolated from spent culture medium. Western blot (immunoblot) assays detected a single adhesion protein of 153 kDa (AP153) on HUVEC after incubation with unfractionated extracts of streptococci. The adhesin exhibited glucosyltransferase (GTF) activity when incubated with sucrose and Triton X-100 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AP153 was purified by affinity chromatography on dextran beads and show to have binding activity for HUVEC, GTF activity, an amino acid composition similar to that reported for GTF of S. gordonii, and the ability to inhibit S. gordonii adhesion. Incubation of the streptococci with antibodies to the adhesin inhibited bacterial attachment to HUVEC monolayers. These results indicate that surface-localized GTF mediates adhesion of S. gordonii to HUVEC in vitro and may serve as a mechanism for colonization of the endocardium in infective endocarditis.

Streptococcal M6 protein binds to fucose-containing glycoproteins on cultured human epithelial cells.

M6 protein of Streptococcus pyogenes binds directly to HEp-2 cell surfaces and helps to mediate bacterial adhesion. Two epithelial cell receptors for M protein were identified as 97- and 205-kDa glycoproteins. Purified recombinant M6 protein (rM6) showed a dose-dependent and saturable binding to isolated HEp-2 membranes in an enzyme immunoassay. The HEp-2 cell receptors were selectively denatured by pretreatment of isolated membranes at 80 degrees C or with chymotrypsin; binding activity for rM6 was reduced 83 and 80%, respectively. Pretreatment of the HEp-2 membranes with neuraminidase-N-glycosidase, neuraminidase-O-glycosidase, alpha-L-fucosidase, or Ulex lectin caused 33, 42, 73, and 80% reduction of rM6 binding, respectively. Quantitative analysis of HEp-2 cells pretreated with alpha-L-fucosidase showed that the 97- and 205-kDa glycoproteins lost 70 and 62% of their abilities to bind M6 protein and that 33% of the HEp-2 cell's ability to bind whole streptococci was also lost. These results indicated that binding of M6 protein to HEp-2 cell surfaces is highly selective for certain fucose-containing oligosaccharides on these glycoproteins.

M protein mediates streptococcal adhesion to HEp-2 cells.

Streptococcus pyogenes adheres to human epithelial cells in vitro and in vivo. To identify adhesins, cell wall components were extracted from S. pyogenes M6 with alkali or by treatment with mutanolysin and lysozyme. HEp-2 cells were incubated with extracts of S. pyogenes M6 and then analyzed by Western blot (immunoblot) assays, using antibodies to S. pyogenes. Only one streptococcal component (62 kDa) was bound to HEp-2 cells and was identified serologically as M6 protein. Experiments with pepsin-cleaved fragments of M protein indicated that the binding site was located at the N-terminal half of the molecule. M protein was bound selectively to two trypsin-sensitive surface components, 97 and 205 kDa, of HEp-2 cells on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. Tritium-labeled lipoteichoic acid bound to different HEp-2 cell components, 34 and 35 kDa, in a parallel experiment, indicating that lipoteichoic acid was not complexed with M protein and does not mediate M-protein binding. The four HEp-2 components were unrelated to fibronectin since they did not react with specific antibodies. An M-protein-deficient (M-) strain of streptococcus (JRS75), grown in chemically defined medium, showed 73% less adhesion activity to HEp-2 monolayers than an M+ strain (JRS4). Streptococcal adhesion was insensitive to competitive inhibition by selected monosaccharides. These results indicate that M protein binds directly to certain HEp-2 cell membrane components and mediates streptococcal adhesion.

Isolation and characterization of a Streptococcus pyogenes protein that binds to basal laminae of human cardiac muscle.

A 9-kDa glycosaminoglycan-binding protein (GAG-BP) was isolated from Streptococcus pyogenes and purified to homogeneity by affinity chromatography on heparin-agarose. The protein selectively bound to the basal laminae of human cardiac muscle and had an apparent dissociation constant of 2.5 x 10(-7) M. Chemical analyses indicated that the GAG-BP was rich in alanine, lysine, and arginine (pI 9.5) and devoid of tyrosine, methionine, histidine, and half-cystine. There were no detectable carbohydrate or phosphate substituents. The amino acid sequence of the N terminus of GAG-BP showed homology with those of histone-like DNA-binding proteins of several other bacteria. Circular dichroism spectroscopy indicated that the protein was made up of 50% beta-sheet and 50% beta-turn and random coil in aqueous solution; however, when the protein complexed with heparin, it adopted a more ordered structure containing 25% alpha-helix, 50% beta-sheet, and 25% beta-turn and random coil. The GAG-BP cross-reacted serologically with a component of similar size in extracts of other group A streptococci and was present in the culture medium during late logarithmic growth.

Modulation of intergeneric adhesion of oral bacteria by human saliva.

Porphyromonas gingivalis adheres in vitro to biofilms containing Streptococcus and Actinomyces species. On initial entry to the mouth, this interbacterial adhesion may enable P. gingivalis to colonize dental plaque and to avoid clearance by saliva flow. Saliva may also interfere directly with P. gingivalis colonization of dental plaque; a 43-kDa glycoprotein in human submandibular-sublingual saliva binds to P. gingivalis surfaces and diminishes interbacterial adhesion activity. To avoid fouling of its surface by host components, P. gingivalis produces surface-localized proteases that can degrade adsorbed proteins and may serve to unmask bacterial adhesins. Successful management of P. gingivalis colonization might be achieved in the future by devising artificial methods to block its surface adhesins or to prevent bacterial proteolysis of native salivary molecules that have protective functions.

Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by human submandibular-sublingual saliva.

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibular-sublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 micrograms of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60 degrees C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity.

Direct demonstration of engraftment of human peripheral blood leukocytes in SCID mice.

One of the major problems using man-mouse chimeras is still the difficulty to demonstrate unequivocally engraftment of human cells in murine tissues. Using supravital labelling of human leukocytes with the Hoechst dye H33342, it was possible to demonstrate directly their engraftment and to assess their distribution in the tissues of the severe combined immunodeficiency (SCID) mice. The human cells can be traced for a period of 4-5 weeks. In contrast to earlier reports, combined marker and labelled-cell studies suggest that T-cell surface marker CD3 with reported specificity for human lymphocytes are indeed found, also in man-mouse chimeras, only on human cells. The ratio of B and T cells of human origin changes significantly after transfer into SCID mice and differs among various SCID tissues. The simple staining procedure using the supravital nuclear dye H33342 opens new possibilities for the study of cellular interactions and host responses of the human immunoreactive cells in an increasingly well-characterized animal model.

Binding of a Streptococcus mutans cationic protein to kidney in vitro.

An 8-kDa protein, with binding activity for heparin and heparan sulfate of basal laminae of animal tissues, was isolated from Streptococcus mutans MT703 and purified to homogeneity. Binding of radioiodinated 8-kDa protein to rabbit kidney tissue in vitro showed a high degree of specificity, as indicated by saturation kinetics, time dependence, and competitive inhibition by unlabeled protein. Binding activity for kidney tissue was competitively inhibited by selected glycosaminoglycans and polyanions in the following order: heparin greater than dextran sulfate greater than heparan sulfate greater than chondroitin sulfate greater than lipoteichoic acid greater than keratan sulfate greater than hyaluronic acid. Binding of the streptococcal protein to rabbit kidney tissue was also strongly inhibited by protamine sulfate, polylysine, and a random copolymer of lysine and alanine. Among the monosaccharides tested at 50 mM, glucosamine 2,3- or 2,6-disulfate, glucuronic acid, glucose 6-phosphate, and glucose 6-sulfate inhibited 50% or more of the binding activity, whereas N-acetylglucosamine 3-sulfate, glucosamine 6-sulfate, N-acetyl-glucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and a selection of neutral sugars were not inhibitory. The heparin-binding protein was detected on the cell wall of S. mutans and in the culture medium following growth. Several other species of streptococci produce an immunologically related protein of similar size.

Adherence of Porphyromonas (Bacteroides) gingivalis to Streptococcus sanguis in vitro.

Intergeneric bacterial adherence is responsible for the complexity of the microbiota in human dental plaque and is believed to enable some extraneous bacteria to initially colonize the human oral cavity. Some current evidence indicates that Streptococcus sanguis, an early colonizer of teeth, enhances subsequent colonization by Porphyromonas (Bacteroides) gingivalis, a bacterium associated with advanced adult periodontitis. In this study, selected strains of P. gingivalis and S. sanguis were tested for their adherence activities in vitro. A differential filtration assay was devised in which one member of the test pair was radiolabeled. Heterogeneous aggregates that formed in mixed suspensions were collected on polycarbonate filters (8-microns pore size) and were washed free of individual bacteria and small homologous clumps. P. gingivalis 381, W50, JKG7, and 33277 adhered to S. sanguis G9B, M5, Challis 6, and 38. P. gingivalis A7A1-28 did not adhere well to S. sanguis under these conditions. More precise measurements of intergeneric adherence were obtained with an alternative assay with radiolabeled P. gingivalis and an artificial dental plaque composed of S. sanguis coupled to cyanogen bromide-activated agarose beads. CNBr-agarose was selected as the supporting matrix for the plaque because it was uniformly and permanently coated with S. sanguis and because P. gingivalis had negligible adherence activity for streptococcus-free beads. P. gingivalis W50 grown to the early stationary phase adhered to S. sanguis-coated beads in higher numbers than either midlogarithmic- or late-stationary-phase cells. Intergeneric adherence was not inhibited or reversed by the presence of lactose or other monosaccharides or disaccharides. Pretreatment of either bacterium with trypsin or proteinase K reduced subsequent adherence by 86 to 100%. Neuraminidase treatment of P. gingivalis caused 98% reduction of adherence, whereas similar treatment of S. sanguis caused only a 2% loss. Preincubation of P. gingivalis at 60 degrees C for 30 min decreased subsequent adherence to S. sanguis-coated beads by 94%. Adherence was reduced by 96% when bacteria were assayed while suspended in human whole saliva or when pretreated with saliva and subsequently assayed in buffer. The concentration of whole human saliva required to inhibit 50% adherence in this assay was 23 micrograms per ml (1:200 dilution). Suspension of the bacteria in normal rabbit serum resulted in 94% inhibition of adherence. These data indicate that saliva and serum may be important host defense factors for controlling Porphyromonas-Streptococcus adherence.

Purification of a Streptococcus mutans protein that binds to heart tissue and glycosaminoglycans.

Proteins of Streptococcus mutans MT703 were isolated by differential filtration from chemically defined culture medium following growth of the bacteria. Incubation of this preparation with cryostat-cut sections of fresh rabbit cardiac muscle resulted in deposition of streptococcal components on basement membranes of sarcolemmal sheaths and capillary walls, as indicated by indirect immunofluorescence assay. Binding of radioiodinated streptococcal proteins to heart in vitro was time dependent and saturable. Unlabeled S. mutans proteins competitively inhibited 72% of heart binding by the radiolabeled proteins, indicating a high level of binding specificity. A selection of components common to tissue basement membranes was tested for their abilities to inhibit the binding of streptococcal proteins to heart tissue. Of the glycosaminoglycans, heparin was the most effective inhibitor, followed by heparan sulfate and chondroitin sulfate. Hyaluronic acid was not inhibitory. Of the glycoproteins tested, laminin and collagen type IV were weakly inhibitory, whereas fibronectin was ineffective. A single polypeptide was purified to homogeneity by affinity chromatography on a column of heparin-agarose. Gel filtration chromatography of the purified protein under nondissociating conditions showed a single component at 31 kilodaltons (kDa), whereas in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one band appeared at 8 kDa. This indicates that the tissue-binding protein may either be a linear polypeptide or be released into the environment by the bacterium as a tetramer of the 8-kDa polypeptide. The purified protein had an isoelectric point of 9.5 and showed binding activity for basement membranes in thin sections of heart. Chemical analyses of the purified binding protein showed it to have high contents of lysine and alanine and to be devoid of half-cystine, methionine, tyrosine, histidine, and both neutral and amino sugars.

Heparin-inhibitable basement membrane-binding protein of Streptococcus pyogenes.

Solubilized surface proteins of Streptococcus pyogenes serotype M6 were found by indirect immunofluorescence assays to bind selectively to proteoglycan-containing regions of basement membranes of kidney and cardiac muscle in vitro. Epithelial, endothelial, and interstitial cells were unstained. Binding of streptococcal protein to basement membranes was competitively inhibited by heparin and, to a lesser extent, by heparan sulfate. Weak inhibition was also observed with other glycosaminoglycans, including dermatan sulfate, chondroitin sulfate, and hyaluronic acid. Type IV collagen, gelatin, serum fibronectin, glucuronic acid, and a selection of monosaccharides had no significant effects on binding. The heparin-inhibitable basement membrane-binding protein was purified by affinity chromatography on heparin-Sepharose 6-B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea dissociated the affinity-purified protein into two polypeptides of 9,000 and 15,000 mrs. Chemical analyses revealed that the purified protein was devoid of cysteine, amino and neutral sugars, and phosphate. Thus, the polypeptides are not glycosylated or complexed with trace amounts of lipoteichoic acid or polysaccharide. Binding of purified protein to tissue was determined by direct radioassay and indirect immunofluorescence and was inhibitable by heparin. Although the in vivo effects of this streptococcal component remain to be determined, its deposition on basement membranes in vitro supports the hypothesis that it contributes to the pathogenesis of poststreptococcal glomerulonephritis or acute rheumatic fever.

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans.

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.