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Soluble macromolecular complexes involving bacterial teichoic acids.

Cell wall and membrane teichoic acids from several bacteria formed soluble complexes with polysaccharides and bovine plasma in alkyl alcohol solutions. Polysaccharides which contain different monomeric units and anomeric configurations complexed with the teichoic acids, suggesting that the interaction is relatively nonspecific. Teichoic acids complexed glycogen or bovine plasma albumin in 50 to 97% ethanol solutions. The macromolecular association between teichoic acids and polysaccharides or proteins was independent of teichoic acid size over a threefold molecular weight range. Glycerol phosphates or an acid hydrolysate of teichoic acid would not complex to either glycogen or bovine plasma albumin in ethanol. The optimal interaction between glycogen and the Bacillus subtilis lipoteichoic acid occurred between pH 4.5 and 8.2. The ability of teichoic acids to bind polysaccharides and proteins in moderate dielectric constant solvents suggests that these polymers may serve as complexing agents for hydrophilic molecules found in membranes.

Bacillus subtilis

On the physiological functions of teichoic acids.

The choline-containing teichoic acids of pneumococci can be modified by biosynthetic replacement of the choline residues with certain structural analogues, such as ethanolamine (EA) or the N-monomethyl-(MEA) and N-dimethyl-(DEA) amino derivatives of ethanolamine. Cells containing such analogues in their teichoic acids develop pleiomorphic alterations in several physiological properties, which include resistance to detergent-induced lysis and inhibition of cell separation (chain formation). We report here the results of physiological studies on the mechanism of these two phenomena. Our results are summarized in the following: (a) Pneumococci grown on various amino alcohols produce cell walls of identical amino sugar and amino acid composition. (b) Both choline- and EA-containing teichoic acids seem to follow the same conservative pattern of segregation during growth and cell division.(c)Lysis sensitivity of pneumococci requires the juxtaposition oflysissensitive (choline-containing) cell walls and endogenous autolysin at the cell wall growth zone. (d) Upon readdition of choline to ethanolamine-containing cells, lysis sensitivity and catalytically active (C-type) autolysin reappear in the bacteria with the same kinetics. (e) The chains of EA-grown pneumococci contain fully compartmentalized cells and normal cross walls.

Amino Alcohols

Counterimmunoelectrophoretic detection of a high incidence of precipitin reactions in normal human sera against staphylococcal teichoic acids and protein A.

The use of counterimmunoelectrophoresis (CIE) for detection of serum antibodies to staphylococcal teichoic acids was evaluated against teichoic acids prepared by sonic treatment or lysostaphin extraction of Staphylococcus aureus (Lafferty strain). Of 54 patient sera from suspected cases of staphylococcal endocarditis, osteomyelitis, or septicemia, 33 (61.1%) were positive by CIE analysis; however, 128 of 291 sera (44.0%) from normal adult donors were also positive. Selected CIE-positive sera from patient and control groups were titered by Ouchterlony gel diffusion. In the control group of normal sera, 65% were also positive by gel diffusion, but only 15% had titers of >/=1:2. Of the patient sera, 44.4% had gel diffusion titers of >/=1:2. In addition to the specific teichoic acid band, a second precipitation band could be demonstrated with both patient or normal sera by CIE or gel diffusion. This second precipitin band was shown to involve interactions of test sera with staphylococcal protein A present in the teichoic acid extracts. The protein A precipitins were detected at high concentrations of the antigen extracts, whereas the anti-teichoic acid precipitins were optimally detected at lower antigen concentrations. The formation of protein A precipitin bands did not correlate with the presence of anti-teichoic acid antibodies, as most sera tested were positive for protein A regardless of anti-teichoic acid activity. This study suggests that a high incidence of normal people have levels of antibodies to teichoic acids which are detectable by the highly sensitive, but nonspecific, technique of CIE.

Adult

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.

Binding Sites

Immunochemical analysis of the teichoic acid from Staphylococcus hyicus.

The wall teichoic acid of Staphylococcus hyicus has been isolated and characterized. The teichoic acid is a glycerol phosphate polymer with glycosidically linked N-acetylglucosamine. Interaction with concanavalin A and susceptibility to alpha- but not to beta-N-acetylglucosaminidase showed that the sugar is in the alpha-configuration.

Acetylglucosaminidase

An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.

3,3'-Diaminobenzidine

Glycerol teichoic acid as an antigenic determinant in a Gram-negative bacterium Butyrivibrio fibrisolvens.

An antigenic determinant isolated from a strain of the Gram-negative bacterium Butyrivibrio fibrisolvens reacted with specific antisera to the polyglycerophosphate backbone of membrane teichoic acids of lactobacilli. It gave a reaction of identity with membrane glycerol lipoteichoic acid and glycerol teichoic acid preparations from lactobacilli, and with phenol extracts of other Gram-positive bacteria. The antigen-antibody reactions was strongly inhibited by glycerol-phosphoryl-glycerol-phosphoryl-glycerol and the chemical composition was consistent with glycerol teichoic acid. It was concluded that this Gram-negative bacterium contained a glycerol teichoic acid whose polyglycerophospate backbone was acting as antigenic determinant. Extracts of 33 out of 52 other strains of butyrivibrios examined gave similar reactions.

Antibodies, Bacterial

Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.

Acetylgalactosamine

Teichoic acid antibodies in chronic staphylococcal osteomyelitis.

Gel-diffusion and the enzyme-linked immunosorbent assay (ELISA) were used to quantify and to identify the immunoglobulin class of teichoic acid antibodies in patients with chronic staphylococcal osteomyelitis and a wide variety of other infections. Teichoic acid antibodies were identified by gel-diffusion in 14 of 23 patients with staphylococcal endocarditis, six of 30 with staphylococcal bacteremia without endocarditis, four of 35 with staphylococcal skeletal infections, and one of 45 with nonstaphylococcal infections. None of the 20 patients with chronic staphylococcal osteomyelitis had positive gel-diffusion assays, even though many had had their infections for several years. The ELISA method was more sensitive than gel-diffusion in measuring teichoic acid antibodies, but was also much less specific. Teichoic acid antibodies were detected predominantly in the IgG fraction of serum. Our findings suggest that the presence and degree of antigenemia are more important than the duration of the staphylococcal infection in stimulating production of teichoic acid antibodies.

Acute Disease

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

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

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

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