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Modulation of the immune response to sheep erythrocytes by lipid-free glycerol teichoic acid.

The 4-day response of C3H/HeJ mice to sheep erythrocytes was suppressed by a lipid-free teichoic acid with an average molecular weight of 2,900 when it was administered by the intraperitoneal route. Enhancement was not observed at that time, and neither suppression nor enhancement could be demonstrated by the intravenous route. Either suppression or enhancement of background plaques could be induced, depending upon the timing. Dosage influenced the degree of suppression from 8 to 100 micrograms, whereas suppression of background plaques required only 1 microgram of lipid-free teichoic acid. The kinetics of the sheep erythrocyte response was altered by treatment of the mice with lipid-free teichoic acid, delaying the peak until day 5 and producing enhancement at that time. Although lipid-free teichoic acid was shown to be toxic for mouse splenocytes (50% lethal dose, ca. 200 micrograms) in vitro, no effect at the levels employed was observed in vivo. The data presented indicate that modulatory activity is influenced by route, timing, dosage, and apparently the number of antibody-secreting cells.

Animals

Poly(glucosylglycerol phosphate) teichoic acid in the walls of Bacillus stearothermophilus B65.

1. Walls of Bacillus stearothermophilus B65 contain a glycerol teichoic acid in which repeating structures consisting of 1-O-alpha-D-glucopyranosylglycerol phosphate are held together by phosphodiester linkage between the glycerol and glucose moieties of adjacent units. 2. The walls are not agglutinated on incubation with concanavalin A, nor does the isolated teichoic acid form a precipitate with this lectin. 3. No evidence was obtained of the presence of the glucosylated (1 leads to 2)-poly(glycerol phosphate) teichoic acid which has previously been reported to occur in walls of this bacterium.

Borohydrides

Teichoic acids of group D streptococci with special reference to strains from pig meningitis (Streptococcus suis).

Immunoelectrophoresis revealed in phenol extracts from S. faecalis and S. faecium a mixture of free and lipid-bound teichoic acids, both reactive with Group D antisera. In phenol extracts from S. suis only lipid-bound teichoic acid, also reactive with Group D antiserum, was seen. This difference probably accounts for the low yield of Group D antigen from S. suis as compared with S. faecalis and S. faecium when heating at pH 2 is used for extraction. When phenol is used good yields are obtained from S. suis as well as from S. faecalis and S. faecium. Lipoteichoic acids from S. faecalis and S. faecium have a backbone structure the same as or similar to that of Group A streptococcal teichoic acid. Lipoteichoic acid from S. suis has a structure differing from that of S. faecalis and S. faecium, e.g., possibly in the attachment of its glucosyl substituents. Precipitation reactions between S. suis lipoteichoic acid and Group D antisera were specifically inhibited by glucose. Reactions between S. bovis phenol extracts and some Group D antisera were also specifically inhibited by glucose, but extracts from S. faecalis and S. faecium were not. This may indicate a monosaccharide glucosyl substituent in teichoic acid from S. suis and S. bovis instead of the di- or trisaccharide previously postulated as the glucosyl substituent in the teichoic acid of S. faecalis.

Animals

Teichoic acid antibody determination by agar-gel diffusion: effect of using dilute antigen preparations.

Because the reported frequency of teichoic acid precipitins in controls and various patient groups has varied considerably among laboratories, we studied the effect of using various concentrations of staphylococcal extracts in agar-gel diffusion tests for teichoic acid antibodies. Of 25 normal sera, only 1 was positive against an undiluted extract, but 4 were positive against a 16-fold-diluted extract. Of nine sera from patients with staphylococcal bacteremia, two were positive at a higher titer against the diluted extract. A false-positive serum against the undiluted extract had a twofold titer increase against the diluted extract. Because human immune serum globulin is generally used as a positive teichoic acid antibody control, the variability of five different lots was studied. Three lots ahd teichoic acid antibody titers of 1:4, whereas one each had titers of 1:2 and 1:8. Based on this study, we feel that staphylococcal extracts should not be diluted. If immune serum globulins are used to determine the adequacy of ultrasonic extracts, newly acquired globulin lots should be standarized against an ultrasonic extract of proven sensitivity and specificity.

Antibodies, Bacterial

Regulation of teichoic acid synthesis during phosphate limitation.

Bacillus subtilis W-23, when placed in phosphate-free medium, ceases to synthesize teichoic acid and synthesizes teichuronic acid. The enzymatic basis for the cessation of teichoic acid synthesis is the irreversible inhibition of the first membrane-bound enzyme involved in teichoic acid synthesis which catalyzes the reaction Undecapenol-P + UDP-GlcNAc leads to undecaprenol-P-P-GlcNAc + UMP.

Bacillus subtilis

Environmental origin of natural antibodies to teichoic acid.

In an effort to determine the origin of natural antibodies to teichoic acid, rats were fed a sterile liquid diet free of detectable teichoic acid and virtually free of gram-positive bacteria. Both germ-free and conventional Sprague-Dawley rats raised on this diet failed to produce antibodies to polyglycerophosphate, whereas 100% of their counterparts fed the usual teichoic acid-containing diet did produce these antibodies. The intestinal flora was similar in both groups of animals. When the test animals were immunized intraperitoneally or orally with gram-positive bacteria, 100% displayed immunocompetency by producing significant levels of antibody. These results demonstrate the environmental nature of the antigenic stimulus for these antibodies and suggest the importance of food as the major source of stimulation. The experimental model described here furnishes a valuable tool for studies of immunologic responses where a single known specificity and a controlled system would be advantageous.

Animals

Organization of teichoic acid in the cell wall of Bacillus subtilis.

The phytohemagglutinin, concanavalin A (Con A), interacts specifically and reversibly with the polyglucosyl glycerol phosphate teichoic acid of Bacillus subtilis 168 cell walls. Advantage has been taken of this interaction to examine the organization of the surface teichoic acid at the ultrastructural level. Con A-treated whole cells and cell walls contain an irregular, fluffy layer 25 to 60 nm thick which is absent in untreated or alpha-methyl glucoside-treated preparations. This discontinuous layer is present only on the outer profile of Con-A-treated cell walls. The surface teichoic acid is proposed to be oriented perpendicular to the long axis of the cell. Fixation and embedment for electron microscopy result in condensation of this layer which then contributes to the stainable portion of the wall. Con A treatment binds adjacent teichoic acid molecules in their native configuration producing the irregular, fluffy layer visualized.

Bacillus subtilis

Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis.

Although exponential growth of Bacillus subtilis 168 in a phosphate-limited medium halted with the exhaustion of inorganic phosphate, the bacteria continued to grow at a slower rate for a further 3 to 4 h at 37 degrees C. This postexponential growth in the absence of an exogenous phosphate supply was accompanied by a loss of teichoic acid from the cell walls of the bacteria. Quantitative analysis of walls and culture fluids showed that the phosphate loss from the walls could not be accounted for by an increase in phosphate-containing compounds in the medium, which implied that the cells were using their own wall teichoic acids to supply phosphate necessary for growth. Addition of exogenous teichoic acid to phosphate-starved cultures resulted in stimulation of growth and in the simultaneous disappearance of teichoic acid phosphate from the medium. It is proposed that teichoic acids, which can contain more than 30% of the total phosphorus of exponential-phase cells, can be used as a reserve phosphate source when the bacteria are starved for inorganic phosphate.

Bacillus subtilis

Defect in biosynthesis of the linkage unit between peptidoglycan and teichoic acid in a bacteriophage-resistant mutant of Staphylococcus aureus.

The biosynthesis of the linkage region between peptidoglycan and the ribitol teichoic acid was investigated in the bacteriophage-resistant, teichoic acid-less mutant Staphylococcus aureus 52A5 (Chatterjee et al., J. Bacteriol. 100:846--853, 1969). Membrane preparations of this strain were found to be incapable of forming the first intermediate of the biosynthetic pathway, namely, the transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to the acceptor molecule, which presumbably is undecaprenol phosphate (R. Bracha and L. Glaser, Biochem. Biophys. Res. Commun. 72:1091--1098, 1976). The addition of heat-inactivated membrane preparations of S. aureus 52A2 (which normally has ribitol teichoic acid) that had been preincubated with UDP-GlcNAc to membranes of strain 52A5 enabled the synthesis of teichoic acid. These data suggest that the mutational defect in the teichoic acid-less organism is in the synthesis of the first compound of the linkage unit, and this is apparently the reason for its absence in the cell walls.

Cell Wall

Synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species.

Examination of both supernatant culture medium and cell pellets after exponential- and stationary-phase growth of Streptococcus mutans strain FA-1 and Streptococcus faecalis ATCC 9790 (S. faecium) showed the presence of [-3H]glycerol-labeled material that possessed several of the properties of glycerol teichoic acid. In the supernatant medium of S. mutans FA-1, an apparently large-molecular-size material, which eluted from agarose columns with the Kd value expected of a lipoteichoic acid, was observed. Large amounts of this material were present in supernatants during the stationary phase. In contrast, with S. faecalis only an apparently lower-molecular-weight form, with a Kd consistent with deacylated glycerol teichoic acid, was found in the growth medium. Both organisms had high-molecular-weight lipoteichoic acid in the cells along with the deacylated glycerol teichoic acid. The presence of relatively large amounts of glycerol teichoic acids in the medium was considered to be a result of excretion of these compounds rather than a result of cellular lysis.

Acids

Teichoic acid serology in staphylococcal infections of infants and children.

Counterimmunoelectrophoresis and gel diffusion were utilized for the detection and titration of antibodies to staphylococcal teichoic acids in various disease states caused by coagulase-positive staphylococcus in infants and children. Serum samples were obtained on admission and serially for 2 to 12 weeks during illness. Teichoic acid antibodies were found by CIE in 12 of 21 patients (57%) with invasive CPS disease with bacteremia (Group A), in two of 17 patients (12%) with CPS infection without bacteremia (Group B), in none of 27 patients with bacteremia and/or invasive infections caused by organisms other than CPS (Group C), and in none of 24 noninfected, hospitalized patients or healthy children (Group D). Gel diffusion was useful for titrating antibodies in seropositive sera. Teichoic acid serology is a useful adjunct in the diagnosis of invasive CPS infections. The presence of these antibodies by CIE and gel diffusion may help to identify patients with endothelial or metastatic infections associated with staphylococcal bacteremia.

Antibodies, Bacterial

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.

Adsorption

The structure of teichoic acid from Bacillus subtilis var, niger WM as determined by C nuclear-magnetic-resonance spectroscopy.

The walls of Bacillus subtilis var. niger WM, grown in a Mg2+ -limited chemostat culture (carbon source glucose, dilution rate = 0.2 h(-1), 37 degrees C, pH 7) contained 45% (w/w) teichoic acid, a polymer composed of glycerol, phosphate[ and glucose in the molar ratio 1.00:1.00:0.88, respectively. Alkaline hydrolysis of this teichoic acid yeilded 1-O-beta-glucosylglycerol phosphate (together with small amounts of glycerol phosphate0 and 13C nuclear magnetic resonance spectra of this hydrolysis product, and its derivative after alkaline phosphate treatment, confirmed that the monomeric unit was 1-O-beta-glucosylglycerol-3-phosphate. Assignment of the resonances in the spectrum of undergraded teichoic acid revealed that the polymer was a poly [(2,3) glycerol phosphate 1, glucosidically substituted on C-1 of glycerol with beta-glucose.

Alkaline Phosphatase

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.

Calcium Chloride

Control of teichoic acid synthesis during phosphate limitation.

The synthesis of teichoic acids was examined in Bacillus subtilis Marburg grown under conditions of phosphate limitation. The results indicate that the inhibition of polyglycerolphosphate synthesis observed under these conditions is the result of two processes. The first process is reversible and is independent of new protein synthesis; the second process is irreversible and requires the synthesis of new protein. During growth, under conditions of phosphate limitation, there is a slow decrease in the level of CDP glycerol pyrophosphorylase activity which is by itself not sufficient to account for the decrease in the rate of polyglycerolphosphate synthesis.

Bacillus subtilis

Stimulation of reticuloendothelial system and toxicity to macrophages of Staphylococcus aureus cell wall, peptidoglycan, and teichoic acid.

Staphylococcus aureus cell wall possesses several biological activities. It is removed from the blood by reticuloendothelial system (RES) and persists there for a long time. The influence of cell wall components on RES cells in vivo and in vitro was investigated. RES activity was studied in mice by carbon clearance method. Intravenous injection of 10 microgram of cell walls or peptidoglycan caused early stimulation and subsequent suppression of RES activity, while teichoic acid was inactive. Four hundred microgram of peptidoglycan caused RES stimulation with maximum after three days, whereas 400 microgram of cell walls caused no such stimulation. Viability of mouse peritoneal macrophages was studied after four days of culture in the presence of cell walls, peptidoglycan, and teichoic acid. Fifty microgram/ml of cell walls or peptidoglycan caused death of all or 68% of macrophages respectively. Teichoic acid was inactive, exhibiting toxic effects at 400 microgram/ml level.

Animals

Control of teichoic acid synthesis in Bacillus licheniformis ATCC 9945.

Analysis of cell walls of Bacillus licheniformis ATCC 9945 grown under phosphate limitation showed that teichoic acid could be replaced by teichuronic acid under these conditions. Teichuronic acid, however, was always present in the walls to some extent irrespective of the growth conditions. The enzymes involved in teichoic acid synthesis were investigated and the synthesis of these was shown to be repressed when the intracellular Pi level fell. CDP-glycerol pyrophosphorylase was studied in some detail and evidence is presented to show that the enzyme is inactivated under phosphate-limited conditions. The mechanism of inactivation is unknown but it has been shown that it does not require protein synthesis de novo.

Bacillus