Enhancement of dengue virus type 2 replication in mouse macrophage cultures by bacterial cell walls, peptidoglycans, and a polymer of peptidoglycan subunits

The effects of bacterial cell walls, peptidoglycans, and a water-soluble polymer of peptidoglycan subunits on dengue virus type 2 replication in cultured mouse peritoneal macrophages were studied. Pretreatment of macrophage cultures with all of test cell walls isolated from seven bacterial species for 3 days significantly enhanced the virus production in the cultures. Peptidoglycans prepared from four of the above cell walls also exerted the virus production-enhancing effects in a similar manner as the walls. A water-soluble polymer of peptidoglycan subunits which was prepared by treatment of Staphylococcus epidermidis wall peptidoglycan with an interpeptide bridge-splitting enzyme (endopeptidase) also definitely enhanced the virus production in macrophage cultures, although its activity was weaker than that of the original wall and peptidoglycan. Macrophage cultures from athymic nude mice, when treated with cell walls and peptidoglycans of S. epidermidis and Lactobacillus plantarum for 3 days, also showed an increased ability to support dengue virus type 2 replication. The infectious center assay demonstrated that the virus replication enhancement by S. epidermidis cell wall and peptidoglycan was primarily due to an increase in the number of virus-infected cells. This finding did not seem to be in conflict with the observation that macrophages treated with the above cell wall or peptidoglycan phagocytized more latex particles than did untreated macrophages. The conclusions based on the above experiments are that the treatment of mouse peritoneal macrophage cultures with bacterial cell walls and their components increases the take of dengue virus type 2 by macrophages and thus raises the virus production in the macrophage cultures.     

Staphylococcus aureus peptidoglycan induces histamine release from basophil human leukocytes in vitro

Whole killed cells, cell walls, and peptidoglycans of Staphylococcus aureus were found to release histamine from human leukocytes and isolated rat mast cells in vitro. The histamine-releasing capability increased in the order of whole bacteria, cell walls, and peptidoglycans. Peptidoglycan was found to release histamine by a nonimmunological mechanism, as demonstrated by release in cells deprived of surface immunoglobulins, whereas whole bacteria and cell walls seemed to operate both by immunological and nonimmunological mechanisms. Histamine release was not a specific property of S. aureus; a wide range of whole bacterial species had this activity. We suggest that peptidoglycan may be a common factor responsible for histamine release by different bacteria.     

Mitogenic effects of bacterial cell walls, their fragments, and related synthetic compounds on thymocytes and splenocytes of guinea pigs.

Stimulation of [(3)H]thymidine incorporation of thymocytes and splenocytes from guinea pigs by various bacterial cell walls and their peptidoglycans, by enzymatic digests, and by synthetic muramyl dipeptides was studied as an indication of mitogenic activity. Cell wall and peptidoglycan preparations, isolated from 19 strains belonging to 18 different species, definitely increased [(3)H]thymidine incorporation of thymocytes as well as splenocytes, regardless of mycolic acid contents as a non-peptidoglycan component. Both the cell walls from Nocardia corynebacteriodes (containing mycolic acids) and those from Streptomyces gardneri (lacking mycolic acids) showed far stronger mitogenic activities on splenocytes than other cell walls (stimulation index, 25 to 30). Furthermore, water-soluble enzymatic digests, notably the endopeptidase digests, which generally were greater in degree of polymerization of peptidoglycan subunits than the glycosidase digests obtained from representative cell walls, were found to have as distinct a stimulating activity on splenocytes as the original cell walls. In contrast, solubilization of the cell walls by enzymes, irrespective of endopeptidases or glycosidases, was accompanied by disappearance of the mitogenic activity on thymocytes. On the other hand, studies with synthetic 6-O-acyl-MurNAc-l-Ala-d-isoGln preparations (6-O-acyl-MDPs) revealed that 6-O-stearoyl-MDP and 6-O-(2-tetradecylhexadecanoyl)-MDP, unlike MDP, had distinct mitogenic activity on thymocytes, whereas their activity on splenocytes was rather weaker than MDP itself. The findings presented here suggest that MDP is the minimal structure for the mitogenic activities of bacterial cell walls on guinea pig splenocytes, but that MDP, though distinctively active by itself, requires a polymerized form to exert effectively its inherent stimulating activities on splenocytes. On the other hand, on thymocytes, MDP, unless it takes a particular form or has appropriate additive groups, cannot exert its mitogenic activities.     

Stimulation of migration of human monocytes by bacterial cell walls and muramyl peptides.

Bacterial cell walls, water-soluble fragments of the wall peptidoglycan, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), and 6-O-acyl derivatives of MDP were examined for migration-stimulating activity on human peripheral blood monocytes by using a multiwell chemotaxis assembly. Cell walls isolated from 11 bacterial species caused a definite increase in monocyte migration, but the walls of Micrococcus lysodeikticus were scarely active. The migration-enhancing activity of Staphylococcus epidermidis cell walls was retained by a monomer as well as a polymer of disaccharide peptides which were prepared by digestion of the peptidoglycan with enzymes. It was finally revealed that the migration of monocytes was enhanced by MDP. 6-O-Octadecanoyl-MDP, 6-O-(2-tetradecylhexadecanoyl)-MDP, and 6-O-(3-hydroxy-2-docosylhexacosanoyl)-N-acetylmuramyl-L-seryl-D-isoglutamine were active, but to a lesser extent. A checkerboard assay demonstrated that the increased monocyte migration caused by S. epidermidis cell walls was directed toward a positive stimulus (chemotaxis).     

Inhibition of macrophage migration by muramyl peptides.

In the capillary tube migration system a synthetic muramyl dipeptide (MDP; N-acetylmuramyl-L-alanyl-D-isoglutamine), a part of bacterial cell wall peptidoglycans, inhibited the migration of peritoneal exudate macrophages from normal guinea pigs or rats. The migration inhibition was also caused by some MDP-containing peptidoglycan fragments from cell walls of Lactobacillus plantarum and Staphylococcus epidermidis. The migration inhibition could not be explained on the basis of macrophage migration inhibitory factor. A stereochemically highly specific structure of MDP required for its adjuvant activity was also required for the macrophage migration inhibition. These findings suggest that MDP and MDP-containing cell wall fragments may activate macrophages and that this activation may be important in the exertion of their adjuvant activity.     

Immunostimulating activity of cell walls from lactic acid bacteria and related species

In the present study the immunostimulating capacity of cell walls of lactic acid bacteria, present in a fermented milk (BIO MILK) with proven biotherapeutic activity, was analyzed. Cell walls and peptidoglycans were isolated, resuspended in water and thereafter administered orally (50 fig/day) to mice for different periods (2, 5 and 7 days). After these periods mice were sacrificed and phagocytic activity of the peritoneal macrophages and immunoglobulin A (IgA) levels in intestinal fluid were analysed. It was observed that Lactobacillus casei was able to stimulate phagocytosis both by the cell wall and the peptidoglycan, whereas it did not produce changes in IgA. L. acidophilus, on the other hand, produced an increase in the levels of IgA without modifying phagocytosis. Propionibacterium acidipropionici only showed immunostimulating activity with the cell wall, but not with the peptidoglycan. These results do not show any relationship with previously studied chemical composition of the cell walls. However, a relationship was found with the presence of molecules emerging from the surface (observed with electron microscopy).     

Chemical and biological properties of a peptidoglycan isolated from Treponema pallidum kazan.

A peptidoglycan layer of Treponema pallidum kazan was isolated by solubilization of whole cells with 1% warm sodium dodecyl sulfate and subsequent digestion of an insoluble residue with proteases. Electron microscopy revealed that the peptidoglycan was isolated as a single-layered sacculus of less than 5 nm in thickness, freed from axial filaments and an envelope sheath. An isolated peptidoglycan fraction was mainly composed of glucosamine, muramic acid, alanine, glutamic acid, ornithine, and glycine in molar ratios of 0.65:0.68:1.63:1.00:0.75:1.03. Amino (N)- and carboxyl (C)-terminal amino acid analyses suggested the involvement of at least a part of the glycine residue in cross-linking between the amino group of ornithine residue at one strand of the stem peptide subunit and the carboxyl group of alanine of the neighboring strand. The treponemal peptidoglycan lacked the immunoadjuvant activity both to stimulate antibody production and to induce delayed-type hypersensitivity against ovalbumin, as well as the properties necessary to stimulate guinea pig and mouse splenocytes and guinea pigs peritoneal macrophages, unlike the cell walls or peptidoglycans (group A type of Schleifer and Kandler's classification, Bacteriol. Rev. 36:407-477, 1972) isolated from many bacterial species parasitic to the mammal. However, the peptidoglycan activated the human complement system through the alternative pathway, as well as the classical one, and caused a liberation of 5-hydroxytryptamine in rabbit blood platelets in a similar manner to the cell wall peptidoglycans of both group A and B types.     

Bacteria-induced histamine release. Examination of the bacterial cell wall components peptidoglycan,teichoic acid and protein A

The histamine-releasing capability of whole bacteria was examined in leukocyte suspensions from normal individuals. Both gram-positive and gram-negative bacteria caused basophil histamine release. It is probably the bacterial cell wall which interacts with the basophil cell surface leading to release of histamine, since cell walls showed higher histamine releasing capability than the whole bacteria. The releasing effect of the bacterial cell wall components peptidoglycan, teichoic acid and protein A was examined. The peptidoglycan preparations were found to be more potent than the corresponding whole bacteria and cell walls. Since peptidoglycan is found in the cell wall of both gram-positive and gram-negative bacteria, it might be a common factor responsible for histamine release by different bacteria. No release was obtained by teichoic acid, whereas protein A caused histamine release in leukocytes from allergic patients, but only a poor release in normal individuals. The initial step in protein A-induced histamine release might be a binding of protein A to IgE on the cell surface, since removal of cell-bound IgE reduced the release and a high correlation was found between protein A- and anti-IgE-induced histamine release.     

Gram-positive cell walls stimulate synthesis of tumor necrosis factor alpha and interleukin-6 by human monocytes.

Purified cell walls representing a wide variety in teichoic acid and peptidoglycan structure prepared from eight different gram-positive bacterial species induced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 from human monocytes in the presence of 10% plasma or serum. Significant amounts of cytokines began to be produced at concentrations above 100 ng to 1 microgram of cell walls per ml, with maximal production requiring 10 to 100 micrograms of cell wall material per ml. In the absence of plasma, the cytokine-inducing capacity of cell wall preparations was lower by at least an order of magnitude. The serum-derived cofactor was inactivated by heating at 90 degrees C for 30 min, suggesting that the activity is associated with a protein. On the other hand, replacement of normal with hypogammaglobulinemic plasma, inactivation of complement (at 56 degrees C), and blockade by the monoclonal antibody MY4 of the CD14 receptors on monocytes did not inhibit the production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha in the presence of polymyxin B, and macrophages derived from the lipopolysaccharide-insensitive cell line of C3He/HeJ mice also produced this cytokine when stimulated by cell walls. Both peptidoglycan and the soluble glycan-teichoic acid component prepared by an enzymatic method from the same wall preparation exhibited a serum-dependent induction of TNF-alpha from monocytes, while stem peptides and disacharride peptides had only poor, if any, activity. Cell walls may contribute to the septic shock induced by gram-positive bacteria.     

Endogenous pyrogen production by human blood monocytes stimulated by staphylococcal cell wall components.

To determine the properties of Staphylococcus aureus contributing to its pyrogenicity, we compared, in human monocytes, endogenous pyrogen production stimulated by heat-killed S. aureus with that stimulated by purified S. aureus cell walls or by particulate peptidoglycan prepared from the same strain. Peptidoglycan, but not the purified cell wall preparation, was found comparable to S. aureus as an endogenous pyrogen stimulus. This finding was associated with a more effective monocyte phagocytosis of S. aureus and peptidoglycan as compared with that of purified cell walls. Lysostaphin digestion of peptidoglycan markedly reduced its pyrogenicity. To test whether the chemical composition of the ingested particles is important, latex particles were tested as possible stimuli for monocyte endogenous pyrogen release. Although 40 to 68% of monocytes ingested latex particles during the first hour, there was no evidence of endogenous pyrogen activity in the supernatant even when supernatants equivalent to 5.2 X 10(6) monocytes were tested. This study demonstrates that the pyrogenic moiety of the S. aureus cell wall resides in the peptidoglycan component. Phagocytosis is not in itself a pyrogenic stimulus, but rather serves as an effective mechanism to bring about contact between the chemical stimulus and the monocyte.     

Preparation of arthritogenic hydrosoluble peptidoglycans from both arthritogenic and non-arthritogenic bacterial cell walls.

Cell wall lytic enzyme (Kyowa lytic no. 2 enzyme) liberated arthritogenic hydrosoluble peptidoglycans from both arthritogenic and non-arthritogenic bacterial cell walls. From these cell walls, mutanolysin (peptidoglycan-degrading enzyme) also liberated hydrosoluble peptidoglycans which, however, lacked arthritogenicity. Based on the chemical composition of these peptidoglycans, it was suggested that their arthritis-inducing ability depends on a relatively long chain of glycan units that consists of repeated units of N-acetylglucosaminyl-N-acetylmuramic acid. However, the glycan chain lengths on these peptidoglycans appeared to be related to their adjuvancy rather than to an antigen(s) responsible for development of arthritis in rats.     

Induction of release of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans.

The role of cytokines in gram-positive infections is still relatively poorly defined. The purpose of this study was to establish whether or not intact staphylococci and purified peptidoglycans and peptidoglycan components derived from staphylococci are capable of stimulating the release of tumor necrosis factor (TNF) by human monocytes. We show here that intact staphylococci and purified peptidoglycans, isolated from three Staphylococcus epidermidis and three S. aureus strains, were indeed able to induce secretion of TNF by human monocytes in a concentration-dependent fashion. TNF release was detected by both enzyme immunoassay and the L929 fibroblast bioassay. In the enzyme immunoassay, a minimal concentration of peptidoglycan of 1 micrograms/ml was required to detect TNF release by monocytes, whereas in the bioassay a peptidoglycan concentration of 10 micrograms/ml was needed to detect a similar amount of TNF release. Peptidoglycan components such as the stem peptide, tetra- and pentaglycine, and muramyl dipeptide were unable to induce TNF release from human monocytes.     

Quantitative analysis of cell walls of nutritionally variant streptococci grown under various growth conditions.

Strains of nutritionally variant streptococci are usually isolated from patients with subacute bacterial endocarditis. Only recently have these strains been subdivided into three serotypes; however, no group-specific antigen has been described. To understand the immunochemical basis for the serology of these microorganisms as well as set the groundwork for adherence studies, quantitative analysis of the cell walls of nutritionally variant streptococci was undertaken. The bacteria were grown in semisynthetic medium or pyridoxal-supplemented Todd-Hewitt broth and harvested during the exponential or stationary phase. Cell walls were isolated and analyzed for amino sugars, sugars, polyalcohols, amino acids, and phosphorus by gas chromatography, high-pressure liquid chromatography, or colorimetric assays. The peptidoglycans of the cell walls of the prototype strains from the three serotypes were representative of other streptococcal cell walls, including the presence of alanine as the possible cross-bridge. The composition of the peptidoglycan was similar for all three strains and included a decreased concentration of peptidoglycan in their cell walls during the stationary phase. Glucosamine, glucose, galactose, ribitol, and a small amount of rhamnose were found in each of the cell wall polysaccharides. Galactosamine was only found in serotype II and III cell walls and might be responsible for the previously described cross-reaction between these strains. The concentration of the other sugars and amino sugars varied in each of the cell wall preparations, depending on the growth conditions. Finally, all three strains expressed both ribitol and phosphorus in their cell walls, characteristic of the presence of a ribitol teichoic acid. Therefore the cell wall composition of the nutritionally variant streptococci varies depending on the growth conditions, and their composition appears similar to that of strains of Streptococcus mitis.     

Cell walls, peptidoglycans, and teichoic acids of gram-positive bacteria as polyclonal inducers and immunomodulators of proliferative and lymphokine responses of human B and T lymphocytes.

In this study, the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated. Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 46 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferative and to produce leukocyte migration inhibitory factor. Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations acted as mitogens. Cell walls and peptidoglycans had a modulating effect on purified protein derivative-induced and protein A-induced proliferation. In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response. However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation. Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells. Solubilization had no effect on immunomodulating capacity.     

Preparation of Cell Wall Antigens of Staphylococcus aureus

Cell walls were prepared from Staphylococcus aureus strains Copenhagen and 263 by high-speed mixing in the presence of glass beads followed by differential centrifugation. Insoluble peptidoglycan complexes were derived from cell walls by extraction of teichoic acid with 10% trichloroacetic acid. Intact teichoic acid was prepared from each strain by digestion of cell walls with lysostaphin and isolated by column chromatography. Soluble glycopeptide (peptidoglycan in which only the glycan has been fragmented) and the stable complex of teichoic acid with glycopeptide were prepared by digestion of cell walls with Chalaropsis B endo-N-acetylmuramidase and were separated by column chromatography. Amino acid and amino sugar contents of walls and subunits of walls were comparable to those reported by others.     

Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains.

Immunoglobulin A (IgA) antibodies in human sera with binding specificity for the C-terminal R-D-Ala-D-Ala sequence of the precursor peptide from bacterial cell wall peptidoglycan were detected by an enzyme-linked immunosorbent assay (ELISA). Specificity of the test system was proved by comparing the high binding of specific IgA to albumin-(D-Ala3) as an antigen with the failure to bind to albumin-(L-Ala3), by binding inhibition studies with L-Ala3, D-Ala3, or peptides with structural analogy to peptidoglycan peptide subunit peptides as inhibitors, and by excluding binding of peroxidase-labeled anti-human IgA to immunoglobulin classes others than IgA. Interference of rheumatoid factors of IgA class was excluded by an ELISA for assaying IgA-rheumatoid factor and by the fact that an IgA fraction essentially free of IgG and IgM was isolated from a serum reacting strongly positive in the ELISA for measuring specific IgA to the peptide subunit of peptidoglycan. This isolated IgA again exhibited binding specificity in the ELISA, thus corroborating the existence of specific IgA in human serum to the C-terminal R-D-Ala-D-Ala sequence of peptidoglycan precursor peptide. The existence of IgA antibodies with specificity for bacterial peptidoglycan was further proved by preadsorption of serum to peptidoglycans and subsequent measurement of specific IgA in the ELISA. Screening of human sera for IgA antibodies with specificity for R-D-Ala-D-Ala peptides revealed that specific antibodies directed against this sequence of bacterial cell wall peptidoglycan may be detected in several human sera.     

Effect of peptidoglycans on erythrocyte survival

Peptidoglycans (PGNs) from bacterial cell walls belong to 'pathogen-associated molecular patterns' (PAMP), which modify the course of an infection with bacterial pathogens. Bacterial infections may lead to anaemia, which at least partially could result from accelerated erythrocyte death. The present study explored the effect of PGNs on eryptosis, a stress-induced suicidal death of erythrocytes, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Eryptosis is triggered by an increase in the cytosolic Ca(2+) concentration and by formation of ceramide. Erythrocyte Ca(2+) activity was estimated from Fluo3 fluorescence, ceramide formation by fluorescent antibodies, phosphatidylserine exposure from annexin V-binding, and erythrocyte volume from forward scatter in fluorescence activated cell sorting (FACS) analysis. Exposure of erythrocytes to PGNs increased cytosolic Ca(2+) concentration, increased ceramide formation, enhanced the percentage of annexin V-binding erythrocytes, decreased erythrocyte forward scatter, and lowered the intracellular ATP concentration. The effect of peptidoglycans was significantly blunted in the absence of extracellular Ca(2+). The clearance of erythrocytes exposed to PGNs was significantly enhanced in vivo. In conclusion, peptidoglycans induce eryptosis at least partially through an increase in the cytosolic Ca(2+) concentration, an effect presumably contributing to the development of anaemia during bacterial infections.     

Contractile effects of bacterial cell walls, their enzymatic digests, and muramyl dipeptides on ileal strips from guinea pigs.

Cell walls isolated from four bacterial species (Streptococcus pyogenes, Lactobacillus plantarum, Streptomyces gardneri, and Nocardia corynebacteriodes), which exhibited the adjuvant effect of stimulating cellular and humoral immune responses against ovalbumin in guinea pigs, caused the slow-starting and long-lasting contraction of guinea pig ileal strips suspended in Tyrode solution. In contrast to these cell walls active in immunoadjuvancy, those isolated from five bacterial species (Micrococcus lysodeikticus, Staphylococcus epidermidis, Arthrobacter atrocyaneus, Corynebacterium insidiosum, and Ampullariella regularis), which lacked immunoadjuvancy at least in intact walls, caused no or very weak contraction of the ileal strips. Further study demonstrated that both a monomer and a polymer of disaccharide-stem peptides, which were obtained by enzymatic degradation of S. epidermis cell wall peptidoglycans, displayed similar contractile effects. It was finally revealed that guinea pig ileum strips showed a definite contractile response to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and 6-O-stearoyl- and 6-O-(2-tetradecylhexadecanoyl)-MDPs, but not to their analogs, whose C-terminal amino acid was L-isoglutamine or D-isoasparagine in place of D-isoglutamine and which lacked adjuvancy. 6-O-(3-Hydroxy-2-docosylhexacosanoyl)-MDP, on the other hand, caused a slow and lasting relaxation of the ileum strips, but its L-isoglutamine and D-isoasparagine analogs did not.     

Surface fibrils (fimbriae) of Actinomyces viscosus T14V.

Surface antigens of Actinomyces viscosus T14V were released from cell walls by digestion with lysozyme. These were separated by ion-exchange and gel filtration chromatography into fractions rich in carbohydrate or protein. The former contained a polysaccharide high in 6-deoxytalose, along with a peptide fragment from the cell wall. In the protein-rich fractions, material of high molecular weight was present, which contained some carbohydrate and up to 14.3% nitrogen. Aspartic acid, threonine, glutamic acid, lysine, alanine, and glycine were detected in these fractions, along with smaller amounts of 10 other amino acids. Most of the alanine was present as the L isomer and thus was not from peptidoglycan. Electron microscopy of the high-molecular-weight material revealed long fibrils, 3.5 to 4.5 nm in diameter, which resembled those seen on bacterial cells. V-specific antiserum, prepared by absorbing anti-A. viscosus T14V serum with cell walls of the avirulent strain (A. viscosus T14AV), did not react with the 6-deoxytalose polysaccharide but reacted well with isolated fibrils, and this was not inhibited by 6-deoxytalose.     

Evidence for the secretion of soluble peptidoglycans by clinical isolates of Staphylococcus aureus.

Four isolates of Staphylococcus aureus from patients with endocarditis and bacteremia were capable of secreting high-molecular-weight soluble peptidoglycans when grown in a minimal cell wall medium containing penicillin G. Vancomycin was not able to substitute for penicillin G in triggering this secretion. Secretion reflected de novo synthesis of soluble peptidoglycan and was strongly dependent on time of incubation (30 to 60 min), and number of bacteria (2 X 10(8) to 5 X 10(8) colony-forming units per ml), but not on penicillin G concentration (10 to 250 micrograms/ml). The incorporation of alanine into the peptidoglycans secreted in vitro by these isolates incubated in the presence of penicillin G under optimal conditions was variable. The least incorporation of alanine into peptidoglycan occurred with an isolate from a patient treated with nafcillin who had no detectable antipeptidoglycan titer.