Lipoteichoic Acid: a Specific Inhibitor of Autolysin Activity in Pneumococcus (Physiological Role of Lipoteichoic Acid/Regulation of Autolytic Enzymes)
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Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp. 1690-1694, May 1975 Lipoteichoic Acid: A Specific Inhibitor of Autolysin Activity in Pneumococcus (physiological role of lipoteichoic acid/regulation of autolytic enzymes) JOACHIM-V. HOLTJE AND ALEXANDER TOMASZ The Rockefeller University, New York, N.Y. 10021 Communicated by Maclyn McCarty, February 18, 1976 ABSTRACT The choline-containing pneumococcal li- Institute, National Institutes of Health, Bethesda, Md.). poteichoic acid (Forssman antigen) is a powerful inhibitor of the homologous autolytic enzyme, an N-acetylmura- The activity of pneumococcal autolysin (N-acetylmuramyl- myl-L-alanine amidase (EC 3.5.1.28, mucopeptide amido- L-alanine amidase; EC 3.5.1.28, mucopeptide amidohydrolase) hydrolase). Low concentrations of deoxycholate can re- was determined in the following manner: 2.36 ,ug of isotope- verse the inhibition. Wall teichoic acid preparations are labeled cell walls ([methyl-3H]choline, 1.8 ,uCi/mg) and 10 ,ul inactive at several hundred-fold higher concentrations. of amidase (containing about 1.5 X 108 cell equivalent units Activation of an inactive form of autolysin by in vitro incubation with choline-containing cell walls is also in- of crude amidase extract, 15 Mug of protein per 10 Mul) were hibited by lipoteichoic acid. Addition of lipoteichoic acid mixed in a final volume of 250 Mul of 0.05 M Tris-maleate buffer to the growth medium of pneumococcal cultures causes (pH 6.7). After incubation at 370, 20 ,ul of 38% formaldehyde chain formation, resistance to stationary phase lysis, and and 20 Mul of 0.5% bovine serum albumin (BSA; Armour frac- penicillin tolerance. It is suggested that a physiological role of lipoteichoic acids may be in the in vivo control of tion IV) solution were added and the unreacted cell wall ma- autolysin activity. terial was removed by centrifugation (12,000 X g, 10 min; The participation of bacterial autolytic enzymes in a variety of TABLE 1. Inhibition of conversion of the E-form to the C-form important physiological phenomena has been postulated by amidase by lipoteichoic acid several authors. It has been suggested that replication and Choline-cell walls + E-form amidase + lipoteichoic acid enlargement of the cell walls involves the balanced action of hydrolytic and synthetic enzymes (1). Autolytic enzymes may play a role in cell division and cell separation (2-4), com- Preincubated at 00, 5 min petence for genetic transformation (5-7), and-possibly-in the infection by a pneumococcal bacteriophage (8). In addi- tion, an essential role of the autolytic system in the bacteri- Deoxycholate added cidal and bacteriolytic action of antibiotics has been demon- strated in pneumococci (9) and in Bacillus subtilis (10). Incubation at 370 Any physiological functioning of autolysins must involve careful endogenous control of their activity. In search for po- Concentration of tential cellular inhibitors of these enzymes we observed a lipoteichoic acid Amidase activity powerful inhibitory effect of the pneumococcal lipoteichoic (Jsg/ml) (% of control) acid. Various aspects of this finding are described in this com- 0.63 102 munication. 1.26 99 3.15 68 MATERIALS AND METHODS 6.30 40 The common laboratory strain of Diplococcus pneumoniae 15.75 31 R36A was used in all studies. Several of the experimental 31.50 18 procedures used have been described in previous publications. These include: the in Inactive form (E-form) of amidase was obtained from pneumo- growth of bacteria chemically defined cocci grown on ethanolamine-containing medium (14). A prepa- medium (11) with choline or ethanolamine as the amino ration of E enzyme (10 ul; containing about 1.5 X 108 of cell alcohol components; preparation and biosynthetic labeling equivalents of bacterial lysate) was mixed with choline-containing (with radioactive isotopes) of cell walls (12); preparation of cell walls (236 jug in 1 ml of 0.15 M saline containing 0.01 M lipoteichoic acid (13) and the C- and E-forms of pneumococcal potassium phosphate buffer, pH 8.0) and various amounts of autolytic enzyme (14). Formamide extraction of wall teichoic lipoteichoic acid (LTA); these components were added in rapid acid (15); the preparation of C-polysaccharide (16); periodate succession in the order indicated. "Conversion" of the E-form oxidation and nitrous acid degradation (12); and disaggrega- to the C-form amidase was allowed to proceed for 5 min at 00 tion of the lipoteichoic acid by sodium dodecyl sulfate (17) (14). Deoxycholate (25 Mul of 2% solution) was added to each reac- were all done by published procedures. The myeloma protein tion mixture (in order to annul the effect of lipoteichoic acid on (TEPC-15) was a gift of Dr. the assay of amidase activity) and the samples were incubated at M. Potter (National Cancer 370 in order to determine the activity of amidase, as described in Materials and Methods. No enzyme activity could be detected Abbreviation: LTA, lipoteichoic acid. without "conversion." 1690 Downloaded by guest on September 27, 2021 Proc. Nat. Acad.'Sci. USA 72 (1975) Lipoteichoic Acid: Inhibitor of Autolysin 1691 -j 0rz 0 h. 0 z -I J hi 0hi 0 -i hi IC o 3.5 7.0 10.5 14.0 17.5 21.0 35.0 LIPOTEICHOIC ACID (1&g/ml) FIG. 1. The inhibition of amidase action by lipoteichoic acid. Lipoteichoic acid (LTA) was added to the reaction mixtures in the amounts (dry weight) indicated on the abscissa. Enzyme was added last. Amidase assays were performed as described in Materials and Methods. Eppendorf microcentrifuge). Radioactivity in the supernatant active "C-form" enzyme by in vitro incubation with choline- solution was determined by pipetting 200 Ml portions into 5 containing cell walls (14). Table 1 shows that the pneumo- ml of "Ready-Solv" scintillator (Beckman) and counting the coccal LTA preparations can also inhibit this enzyme activa- samples in a Mark II (Nuclear Chicago) scintillation tion process. spectrometer. RESULTS Effect of Lipoteichoic Acid on Amidase Activity and on the In Vitro Activation ofAmidase. Fig. 1 illustrates the inhibitory ef- fect of lipoteichoic acid (LTA) preparations on the activity of amidase. Concentrations of LTA as low as about 1.0 sg/ml 100 o caused 50% inhibition; 2-3 pg/ml was sufficient to inhibit loo 80% of the enzyme activity. The somewhat less than com- 50 8 plete inhibition of enzyme activity (see Fig. 1) may be more 0 apparent than real, since 10-20% of the choline residues may zk - be removed from the cell walls by the choline esterase (18) that is known to be present in the crude amidase preparations. x 2. Fig. 2 shows that the addition of low concentration (0.2%) of deoxycholate can completely reverse the inhibitory effect of 0 LTA. I I Pneumococci grown on ethanolamine-containing medium F contain an abnormal form of autolytic amidase ("E-form," I.) low-molecular-weight and low-specific-activity) that can be "converted" to the high-molecular-weight and catalytically 0 100 - 02%DEOXYCHOLATE 1 5 10 15 0 FRACTION NUMBER FIG. 3. Sucrose density gradient centrifugation of a lipotei- W 50 \ 0.1% DEOXYCHOLATE choic acid preparation in the presence of absence of sodium do- co50- decyl sulfate. Lipoteichoic acid labeled with [methyl-3H]choline (0.9 mg; total radioactivity: 104 cpm) was applied to a linear 5-20% sucrose gradient containing no detergent (A) or contain- ing 0.4% sodium dodecyl sulfate (B). Centrifugation was per- cC 10_ formed in polyallomer tubes in an SW 50.1 rotor of a Spinco model L3-50 ultracentrifuge at 35,000 rpm at 140 for 18 hr. 085 1.75 425 8.50 Fractions (250 ,d) were collected through a pinhole pierced LPOTECHOIC ACMD (mg /l) through the bottom of the tubes. Fraction 1 represents the bottom FIG. 2. Deoxycholate reverses the inhibition of amidase reac- of the gradient. Detergent was removed by precipitation at 0° tion by lipoteichoic acid. Enzyme was added to the reaction mix- (17). Solid line: lipoteichoic acid (radioactivity) in 100 Ad frac- tures last. tions. Dashed line: inhibitory effect; 10 ,A fractions were tested. Downloaded by guest on September 27, 2021 1692 Biochemistry: Holtje and Tomasz Proc. Nat. Acad. Sci. USA 72 (1975) -i z 0 I. 0 8It U)f oor x 4c UA. i 0 a. C.) I>- I- C7, U Ii %'.. 7 8 9 10 11 12 13 14 6 7 8 9 10 11 12 13 14 FRACTION NUMBER FIG. 4. Fragmentation of lipoteichoic acid by nitrous acid and periodate treatments. Lipoteichoie acid (LTA) labeled with [methyl-3H1- choline was used in both experiments. Nitrous acid treatment: 80 ,g (3 X 104 cpm) of LTA in 1 ml 0.4 M sodium acetate buffer (pH 3.5) was mixed with 1 ml of 2% (w/v) NaNO2 solution and was allowed to react at room temperature for 12 hr. The reaction mixture was passed through a Sephadex G-10 column (0.9 X 28.5 cm) in 0.15 M saline (frame a in figure). Frame A shows an untreated LTA preparation. Periodate treatment: 200 ,ug (7.5 X 104 cpm) of LTA in 50 ,l of water was mixed with 1 ml of paraperiodic acid (0.025 M) in 0.5 M sodium acetate buffer, pH 4.5, at 40 and incubated in the dark for 60 hr. Excess periodate was consumed by the addition of 0.2 ml of 10% glycerol and 1.5 hr continued incubation. The reaction mixture was passed through a Sephadex G-10 column to reisolate the de- graded LTA (frame b of figure). Frame B shows control LTA after reisolation from a Sephadex G-50 column. Fractions of 25 drops were collected in each case.