Lipoteichoic Acid: a Specific Inhibitor of Autolysin Activity in Pneumococcus (Physiological Role of Lipoteichoic Acid/Regulation of Autolytic Enzymes)

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 addition, 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

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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 effect 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 containing 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)

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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. Solid lines: radioactivity; dashed line: inhibitory effect on the activity of amidase, assayed in 100 aliquots of the fractions.

The Chemical Nature of Amidase Inhibitor Present in the Periodate oxidation and incubation with nitrous acid cause LTA Preparations. The results of a series of experiments- degradation of LTA and elimination of the inhibitory ac- summarized in Figs. 3 and 4 and Tables 2 and 3-indicate that tivities of the preparations (Fig. 4). Table 2 shows that a the factor responsible for the inhibitory effects is the pneumo- phosphocholine-specific myeloma protein (19) known to pre- coccal LTA itself. Fig. 3 demonstrates that the inhibitory ac- cipitate the pneumococcal LTA (17) also precipitates the fac- tivity co-migrates with the LTA during sedimentation in de- tor responsible for amidase inhibition. tergent-containing and detergent-free sucrose gradients (17). Specificity. The inhibitory activity seems to be specific for the since the related wall teichoic acid TABLE 2. Precipitation of lipoteichoic acid by a LTA, structurally (12, phosphocholine-specific myeloma protein (TEPC-15) 20) showed no effect on the amidase activity even at several Amidase TABLE 3. Effect of choline-containing wall teichoic acid Lipoteichoic activity preparations on the amidase activity acid in in presence of Dilution of supernatant supernatant Amidase antiserum (Cpm/10 IA) (epm/150,A) Concentration activity 1:0 131 2935 Compound tested (Mg/mi) (% of control) 1:2 622 2876 Wall-teichoic acid 1:5 3191 1760 (Formamide) 1:10 6126 1072 Prep I 89 100 No TEPC-15 (maxi- 179 108 mal lipoteichoic Prep II 45 104 acid effect) 9692 579 89 110 C-carbohydrate 84 103 A 50 Ml portion of a lipoteichoic acid preparation labeled with 336 102 [methyl-3H] choline (1.1 mg and 5 X 106 cpm/ml) was incubated with 50 MAl portions of diluted TEPC-15 sera, at 40 for 8 hr. After Lipoteichoic acid 0.5 50 precipitation with 100 ,l of saturated ammonium sulfate solution 4.0 10 (40, 12 hr), the precipitates were removed by centrifugation (12,000 X g, 10 min). The supernatant solutions were assayed for Formamide-extracted pneumococcal wall teichoic acid and C- lipoteichoic acid (radioactivity in 10 jM1 aliquots) and for inhibi- carbohydrate were prepared by procedures described in Materials tory activity in the amidase reaction. and Methods. Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Lipoteichoic Acid: Inhibitor of Autolysin 1693

CYTOPLASM MEMBRANE WALL

(2-CHOLINE ESTERASE

Lipo- toichoic Acid

300 |(I)-HOI RI ®D-CHOLINE ESTERASE

/ K ; 200 PENICILLIN + LIPOTEICHOIC ACID

100 _ FIG. 6. A hypothetical scheme for the regulation of the activity of pneumococcal amidase.

50 PENICILLIN The existence of endogenous autolysin inhibitors in bacteria has been postulated on theoretical grounds and the presence of some low-molecular-weight as yet unidentified autolysin in- __ hibitor(s) has been reported in Streptococcusfaecalis (24). 1 2 3 4 5 6 7 At least two facts make the pneumococcal lipoteichoic acid a HOURS good candidate for the role of endogenous autolysin inhibitor. FIG. 5. Prevention of penicillin-induced lysis of pneumococci One is the cellular localization at the outer surface of the by lipoteichoic acid. Pneumococcal cultures at a concentration of plasma membrane (17), i.e., in an area where autolysin mole- 3.75 X 107 viable units/ml were given penicillin (0.1 unit/ml) cules engaged in their postulated physiological activity are dot or (0.1 unit/ml) plus lipoteichoic (dash and line) penicillin to reside. The other advantageous feature is the struc- acid (4 mg/ml) (dashed line). A control culture was also grown likely for comparison. Bacterial lysis and growth were measured by tural similarity to the wall teichoic acid (17, 23). The ability monitoring the light scattering of the culture with a Coleman of the pneumococcal autolysin to specifically recognize the nephelometer (N: nephelos units). homologous choline-containing wall teichoic acid has already been well documented (12). hundred-fold higher concentrations (Table 3). High concentra- The observations with autolysin-defective pneumococci tions (140 ,g/ml) of pneumococcal LTA had no inhibitory ef- illustrate clearly the potentially suicidal consequences of un- fect on the hydrolytic activity of egg white lysozyme or on the controlled autolysin action (9). The existence of a variety of autolytic activity(s) of crude B. subtilis 168 extracts. regulatory mechanisms is alsQ suggested by the postulated role of autolysins in the complex processes of cell wall enlarge- Mechanism. The mechanism of inhibitory action of LTA ment and cell division. Studies on the pneumococcal system does not seem to involve association with the cell wall ma- suggest several possible levels of regulation and these are de- terial, since there was no demonstrable attachment of radio- picted in the scheme in Fig. 6. Small amounts of the low- active LTA to pneumococcal cell walls (Table 4). molecular-weight and inactive E-form autolysin (typical of In Vivo Effects of LTA. Fig. 5 shows the protective effect of TABLE 4. Binding studies of [3H]choline-labeled lipoteichoic LTA (added to the growth medium of a growing pneumo- acid to pneumococcal cell walls coccal culture) against the bacteriolytic activity of penicillin G. Under the same LTA also gave conditions, protection Lipoteichoic acid against vancomycin (30 Mg/ml). In addition, bacteria growing concentration in the presence of LTA showed chain formation and resistance totg/165 w)C Radioactivity in 100 to spontaneous culture lysis in the stationary phase of growth. ,l .a Cel wal of supernatant (cpm) DISCUSSION radioactivity: concentration 2 X 104 cpm (MAg/i65 Il) Trial I Trial II In spite of their widespread occurrence, the physiological 5.6 0 14,450 12,190 function(s) of lipoteichoic acids are not well understood. A 5.6 30 14,130 12,640 possible role in the binding of Mg++ ions at the cell surface 5.6 75 13,590 12,200 has been proposed (20). More recently, Glaser and his as- sociates have presented evidence for the participation of Various amounts of cell walls were incubated with lipoteichoic lipoteichoic acids in the biosynthesis of cell wall teichoic acid acid (in 0.15 M NaCl solutions containing 0.01 M potassium phos- of Staphylococcus aureus (22). The observations described in phate buffer, pH 8) at 00 for 2 hr. After centrifugation (12,000 X this paper suggest an additional and novel physiological func- g, 10 min) the amount of lipoteichoic acid remaining in the super- tion for a lipoteichoic acid-as a natural inhibitor of autolysin natant solutions was determined by measuring radioactivity in activity. 100 Ml aliquots. Downloaded by guest on September 27, 2021 1694 Biochemistry: H6ltje and Tomasz Proc. Nat. Acad. Sci. USA 72 (1975) ethanolamine-grown pneumococci) (14) have been detected in oxycholate and wild-type autolysin (26). It is conceivable choline-grown cells also (unpublished observation). Thus, the that the mechanism of induction of bacterial lysis by deoxy- E-form autolysin may represent the precursor (proenzyme) cholate and other detergents may involve a specific dissocia- for this enzyme, synthesized on the ribosomes. By analogy to tion of inhibited autolysin-lipoteichoic acid complexes, rather the in vitro conversion of E-form to C-form autolysin, the ac- than a nonspecific removal of some membrane barrier that tivation of this proenzyme may occur after transport through may separate autolysin molecules from their endogenous the plasma membrane by interaction with choline residues in substrate in vivo. the wall teichoic acid. The existence of inactive autolysin It is not clear at the present time to what extent these con- precursor and its conversion to a catalytically active enzyme clusions might also apply to other species of bacteria. How- by proteolysis has been described by Shockman and his as- ever, the ubiquitous occurrence of lipoteichQic acids among sociates in the case of the Strep. faecalis muramidase (24). most Gram-positive species (27) and the potential general The pneumococcal enzyme activation as well as the cat- importance of our observations for antibiotic sensitivity alytic activity of the "converted" autolysin are both inhibited would seem to warrant the testing of other bacterial species by lipoteichoic acid-as the data presented in this paper in- too for the type of lipoteichoic acid effects described in this dicate. The recently described teichoic acid phosphocholine paper for pneumococci. esterase (18) offers an additional potential regulatory mech- This investigation has been supported by a grant from the anism through the removal of a critical set of phosphocholine U.S. National Institutes of Health. residues from either the cell wall or from the lipoteichoic acid (which is also a substrate for this enzyme). Since the catalytic 1. Weidel, W. & Pelzer, H. (1964) Advan. Enzymol. 26, 193- activity of autolysin seems to have an absolute requirement for 228. 2. Rogers, H. J. (1970) Bacteriol. Rev. 34, 194-214. choline moieties in the substrate (28), the removal of a fraction 3. Higgins, M. L. & Shockman, G. D. (1971) CRC Crit. Rev. of choline residues by the esterase is likely to create autolysin- Microbiol. 1, 29-72. resistant zones in the cell wall. Similarly, the inhibitory power 4. Shockman, G. D., Daneo-Moore, L. & Higgins, M. L. (1974) of lipoteichoic acid may be affected by the removal of phos- Ann. N.Y. Acad. Sci. 235, 161-197. 5. Young, F. E., Tipper, D. J. & Strominger, J. L. (1964) J. phocholine residues. While the in vivo relevance of this scheme Biol. Chem. 239, 3660-3662. is entirely hypothetical, several of its predictions are experi- 6. Ranhand, J. M., Leonard, C. G. & Cole, R. G. (1971) J. mentally testable. Bacteriol. 106, 257-268. The most striking evidence suggesting the in vivo inhibition 7. Seto, H. & Tomasz, A. (1975) J. Bacteriol. 121, 344-353. 8. McDonnell, M., Ronda-Lain, C. & Tomasz, A. (1975) Virol- of autolysin by lipoteichoic acid is provided by the physio- ogy 63, 577-582. logical effects of lipoteichoic acid on growing cultures. One of 9. Tomasz, A., Albino, A. & Zanati, E. (1970) Nature 227, the physiological effects-the inhibition of stationary phase 138-140. lysis-was observed at a concentration as low as 30 Ag/ml. 10. Rogers, H. J. & Forsberg, C. W. (1971) J. Bacteriol. 108, Protection against antibiotic action and chaining required 1235-1243. 11. Tomasz, A. (1970) J. Bacteriol. 101, 860-871. higher concentrations (3-4 mg/ml). Nevertheless, the agent 12. Mosser, J. L. & Tomasz, A. (1970) J. Biol. Chem. 245, 287- causing these effects still seems to be the LTA, since it frac- 298. tionates with the Forssman antigen; it is resistant to pro- 13. Goebel, W. F., Shedlovsky, T., Lavin, G. I. & Adams, M. H. teolysis, nucleases and lipid solvents; it contains no detectable (1943) J. Biol. Chem. 148, 1-15. 14. Tomasz, A. & Westphal, M. (1971) Proc. Nat. Acad. Sci. cell wall components and it is destroyed by nitrous acid. The USA 68, 2627-2630. high concentrations of extracellular LTA used may be neces- 15. Krause, R. M. & McCarty, M. (1961) J. Exp. Med. 114, sary for several reasons: the LTA molecules may have to 127-140. "coat" the cell surface in order to reach the sites of endogenous 16. Liu, T. Y. & Gotschlich, E. C. (1963) J. Biol. Chem. 238, autolysin activity; in addition, live cells may have a mech- 1928-1939. 17. Briles, E. B. & Tomasz, A. (1973) J. Biol. Chem. 248, 6394- anism for degrading the LTA. The relatively high concentra- 6397. tions of LTA had no effect on the bacterial growth rate. The 18. H6ltje, J.-V. & Tomasz, A. (1974) J. Biol. Chem. 249, 7032- LTA-induced inhibition of cell separation (chain formation), 7034. penicillin resistance, and resistance to stationary phase lysis 19. Potter, M. & Lieberman, R. (1970) J. Exp. Med. 132, 737- 751. are the same phenomena that have been observed in pneumo- 20. Brundish, D. E. & Baddiley, J. (1968) Biochem. J. 110, cocci with defective autolytic systems, such as ethanolamine- 573-582. grown cultures (14) and the amidase-defective mutant (25). 21. Heptinstall, S., Archibald, A. R. & Baddiley, J. (1970) Na- It seems, therefore, that LTA molecules can penetrate the ture 225, 519-521. bacterial surface to some and can inhibit the 22. Fiedler, F. & Glaser, L. (1974) J. Biol. Chem. 249, 2684- degree autolytic 2689. system. 23. Fujiwara, M. (1967) Jap. J. Exp. Med. 37, 581-597. A further observation of possible in vivo relevance is the 24. Shockman, G. D., Thompson, J. S. & Conover, M. J. (1965) reversal of lipoteichoic acid inhibition by detergents. Deoxy- J. Bacteriol. 90, 575-588. cholate is known to induce lysis of pneumococci by the induc- 25. Tomasz, A. (1974) Ann. N.Y. Acad. Sci. 235, 439-447. 26. Lacks, S. (1970) J. Bacteriol. 101, 373-383. tion of an endogenous autolytic process (12). A deoxycholate- 27. Knox, K. W. & Wicken,'J. (1973) Bacteriol. Rev. 37, 215- resistant autolysin-defective mutant of pneumococcus re- 257. quires for cellular lysis the combined addition of both de- 28. Holtje, J.-V. and Tomasz, A. (1975) J. Biol. Chem., in press. Downloaded by guest on September 27, 2021