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 lipoteichoic acid (Forssman antigen) is a powerful inhibitor of the homologous autolytic enzyme, an N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28, mucopeptide amidohydrolase). Low concentrations of deoxycholate can reverse the inhibition. Wall teichoic acid preparations are inactive at several hundred-fold higher concentrations. Activation of an inactive form of autolysin by in vitro incubation with choline-containing cell walls is also inhibited by lipoteichoic acid. Addition of lipoteichoic acid to the growth medium of pneumococcal cultures causes chain formation, resistance to stationary phase lysis, and penicillin tolerance. It is suggested that a physiological role of lipoteichoic acids may be in the in vivo control of autolysin activity.
Institute, National Institutes of Health, Bethesda, Md.). The activity of pneumococcal autolysin (N-acetylmuramylL-alanine amidase; EC 3.5.1.28, mucopeptide amidohydrolase) was determined in the following manner: 2.36 ,ug of isotopelabeled cell walls ([methyl-3H]choline, 1.8 ,uCi/mg) and 10 ,ul of amidase (containing about 1.5 X 108 cell equivalent units of crude amidase extract, 15 Mug of protein per 10 Mul) were mixed in a final volume of 250 Mul of 0.05 M Tris-maleate buffer (pH 6.7). After incubation at 370, 20 ,ul of 38% formaldehyde and 20 Mul of 0.5% bovine serum albumin (BSA; Armour fraction IV) solution were added and the unreacted cell wall material was removed by centrifugation (12,000 X g, 10 min;
The participation of bacterial autolytic enzymes in a variety of important physiological phenomena has been postulated by several authors. It has been suggested that replication and 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), competence 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 bactericidal and bacteriolytic action of antibiotics has been demonstrated in pneumococci (9) and in Bacillus subtilis (10). Any physiological functioning of autolysins must involve careful endogenous control of their activity. In search for potential cellular inhibitors of these enzymes we observed a powerful inhibitory effect of the pneumococcal lipoteichoic acid. Various aspects of this finding are described in this communication.
TABLE 1. Inhibition of conversion of the E-form to the C-form amidase by lipoteichoic acid Choline-cell walls + E-form amidase + lipoteichoic acid Preincubated at 00, 5 min
Deoxycholate added Incubation at 370
MATERIALS AND METHODS
The common laboratory strain of Diplococcus pneumoniae R36A was used in all studies. Several of the experimental procedures used have been described in previous publications. These include: growth of the bacteria in chemically defined medium (11) with choline or ethanolamine as the amino alcohol components; preparation and biosynthetic labeling (with radioactive isotopes) of cell walls (12); preparation of lipoteichoic acid (13) and the C- and E-forms of pneumococcal autolytic enzyme (14). Formamide extraction of wall teichoic acid (15); the preparation of C-polysaccharide (16); periodate oxidation and nitrous acid degradation (12); and disaggregation of the lipoteichoic acid by sodium dodecyl sulfate (17) were all done by published procedures. The myeloma protein (TEPC-15) was a gift of Dr. M. Potter (National Cancer
Concentration of lipoteichoic acid
Amidase activity
(Jsg/ml)
(% of control)
0.63 1.26 3.15 6.30 15.75 31.50
102 99 68 40 31 18
Inactive form (E-form) of amidase was obtained from pneumococci grown on ethanolamine-containing medium (14). A preparation of E enzyme (10 ul; containing about 1.5 X 108 of cell equivalents of bacterial lysate) was mixed with choline-containing cell walls (236 jug in 1 ml of 0.15 M saline containing 0.01 M potassium phosphate buffer, pH 8.0) and various amounts of lipoteichoic acid (LTA); these components were added in rapid succession in the order indicated. "Conversion" of the E-form to the C-form amidase was allowed to proceed for 5 min at 00 (14). Deoxycholate (25 Mul of 2% solution) was added to each reaction mixture (in order to annul the effect of lipoteichoic acid on the assay of amidase activity) and the samples were incubated at 370 in order to determine the activity of amidase, as described in Materials and Methods. No enzyme activity could be detected without "conversion."
Abbreviation: LTA, lipoteichoic acid.
1690
Proc. Nat. Acad.'Sci. USA 72
Lipoteichoic Acid: Inhibitor of Autolysin
(1975)
1691
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hi hi 0 0 -i hi IC
o
3.5
7.0
10.5 17.5 14.0 LIPOTEICHOIC ACID (1&g/ml)
21.0
35.0
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 solution was determined by pipetting 200 Ml portions into 5 ml of "Ready-Solv" scintillator (Beckman) and counting the samples in a Mark II (Nuclear Chicago) scintillation spectrometer. RESULTS Effect of Lipoteichoic Acid on Amidase Activity and on the In Vitro Activation of Amidase. 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 caused 50% inhibition; 2-3 pg/ml was sufficient to inhibit 80% of the enzyme activity. The somewhat less than complete inhibition of enzyme activity (see Fig. 1) may be more apparent than real, since 10-20% of the choline residues may be removed from the cell walls by the choline esterase (18) that is known to be present in the crude amidase preparations. Fig. 2 shows that the addition of low concentration (0.2%) of deoxycholate can completely reverse the inhibitory effect of LTA. Pneumococci grown on ethanolamine-containing medium contain an abnormal form of autolytic amidase ("E-form," low-molecular-weight and low-specific-activity) that can be "converted" to the high-molecular-weight and catalytically 0 100
active "C-form" enzyme by in vitro incubation with cholinecontaining cell walls (14). Table 1 shows that the pneumococcal LTA preparations can also inhibit this enzyme activation process.
100 o loo 50 zk
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-
1
5
10
15
FRACTION NUMBER
0 W
8 0
50
co50-
\
0.1% DEOXYCHOLATE
cC 10_ 085
1.75
425
8.50
LPOTECHOIC ACMD (mg /l)
FIG. 2. Deoxycholate reverses the inhibition of amidase reaction by lipoteichoic acid. Enzyme was added to the reaction mixtures last.
FIG. 3. Sucrose density gradient centrifugation of a lipoteichoic acid preparation in the presence of absence of sodium dodecyl 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 performed 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. Fractions (250 ,d) were collected through a pinhole pierced through the bottom of the tubes. Fraction 1 represents the bottom of the gradient. Detergent was removed by precipitation at 0° (17). Solid line: lipoteichoic acid (radioactivity) in 100 Ad fractions. Dashed line: inhibitory effect; 10 ,A fractions were tested.
1692
Biochemistry: Holtje and Tomasz
Proc. Nat. Acad. Sci. USA 72
(1975)
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7
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10 11
12 13 14
6
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12
13
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FRACTION NUMBER
FIG. 4. Fragmentation of lipoteichoic acid by nitrous acid and periodate treatments. Lipoteichoie acid (LTA) labeled with [methyl-3H1choline 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 degraded 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 LTA Preparations. The results of a series of experimentssummarized in Figs. 3 and 4 and Tables 2 and 3-indicate that the factor responsible for the inhibitory effects is the pneumococcal LTA itself. Fig. 3 demonstrates that the inhibitory activity co-migrates with the LTA during sedimentation in detergent-containing and detergent-free sucrose gradients (17). TABLE 2. Precipitation of lipoteichoic acid by a
phosphocholine-specific myeloma protein (TEPC-15)
Dilution of antiserum 1:0 1:2 1:5 1:10 No TEPC-15 (maximal lipoteichoic acid effect)
Lipoteichoic acid in supernatant
Amidase activity in presence of supernatant
(Cpm/10 IA)
(epm/150 ,A)
131 3191 6126
2935 2876 1760 1072
9692
579
622
A 50 Ml portion of a lipoteichoic acid preparation labeled with [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 precipitation with 100 ,l of saturated ammonium sulfate solution (40, 12 hr), the precipitates were removed by centrifugation (12,000 X g, 10 min). The supernatant solutions were assayed for lipoteichoic acid (radioactivity in 10 jM1 aliquots) and for inhibitory activity in the amidase reaction.
Periodate oxidation and incubation with nitrous acid cause degradation of LTA and elimination of the inhibitory activities of the preparations (Fig. 4). Table 2 shows that a phosphocholine-specific myeloma protein (19) known to precipitate the pneumococcal LTA (17) also precipitates the factor responsible for amidase inhibition. Specificity. The inhibitory activity seems to be specific for the LTA, since the structurally related wall teichoic acid (12, 20) showed no effect on the amidase activity even at several TABLE 3. Effect of choline-containing wall teichoic acid preparations on the amidase activity
Compound tested Wall-teichoic acid (Formamide) Prep I Prep II
Concentration
Amidase activity
(Mg/mi)
(% of control)
89 179
100 108
45
104
89
110
C-carbohydrate
84
Lipoteichoic acid
336 0.5 4.0
103 102 50 10
Formamide-extracted pneumococcal wall teichoic acid and Ccarbohydrate were prepared by procedures described in Materials and Methods.
Proc. Nat. Acad. Sci. USA 72
Lipoteichoic Acid: Inhibitor of Autolysin
(1975)
MEMBRANE
CYTOPLASM
1693
WALL
(2-CHOLINE ESTERASE Lipotoichoic Acid 300 RI
|(I)-HOI
®D-CHOLINE 200 /
K
;
ESTERASE
PENICILLIN +
LIPOTEICHOIC ACID \0
100 _
FIG. 6. A hypothetical scheme for the regulation of the activity of pneumococcal amidase.
50
PENICILLIN
__
1
2
3
4
5
6
7
HOURS
FIG. 5. Prevention of penicillin-induced lysis of pneumococci by lipoteichoic acid. Pneumococcal cultures at a concentration of 3.75 X 107 viable units/ml were given penicillin (0.1 unit/ml) (dash and dot line) or penicillin (0.1 unit/ml) plus lipoteichoic acid (4 mg/ml) (dashed line). A control culture was also grown for comparison. Bacterial lysis and growth were measured by monitoring the light scattering of the culture with a Coleman nephelometer (N: nephelos units).
hundred-fold higher concentrations (Table 3). High concentrations (140 ,g/ml) of pneumococcal LTA had no inhibitory effect on the hydrolytic activity of egg white lysozyme or on the autolytic activity(s) of crude B. subtilis 168 extracts. Mechanism. The mechanism of inhibitory action of LTA does not seem to involve association with the cell wall material, since there was no demonstrable attachment of radioactive LTA to pneumococcal cell walls (Table 4).
In Vivo Effects of LTA. Fig. 5 shows the protective effect of LTA (added to the growth medium of a growing pneumococcal culture) against the bacteriolytic activity of penicillin G. Under the same conditions, LTA also gave protection against vancomycin (30 Mg/ml). In addition, bacteria growing in the presence of LTA showed chain formation and resistance to spontaneous culture lysis in the stationary phase of growth. DISCUSSION
In spite of their widespread occurrence, the physiological function(s) of lipoteichoic acids are not well understood. A possible role in the binding of Mg++ ions at the cell surface has been proposed (20). More recently, Glaser and his associates have presented evidence for the participation of lipoteichoic acids in the biosynthesis of cell wall teichoic acid of Staphylococcus aureus (22). The observations described in this paper suggest an additional and novel physiological function for a lipoteichoic acid-as a natural inhibitor of autolysin activity.
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 inhibitor(s) has been reported in Streptococcus faecalis (24). At least two facts make the pneumococcal lipoteichoic acid a good candidate for the role of endogenous autolysin inhibitor. One is the cellular localization at the outer surface of the plasma membrane (17), i.e., in an area where autolysin molecules engaged in their postulated physiological activity are likely to reside. The other advantageous feature is the structural similarity to the wall teichoic acid (17, 23). The ability of the pneumococcal autolysin to specifically recognize the homologous choline-containing wall teichoic acid has already been well documented (12). The observations with autolysin-defective pneumococci illustrate clearly the potentially suicidal consequences of uncontrolled autolysin action (9). The existence of a variety of regulatory mechanisms is alsQ suggested by the postulated role of autolysins in the complex processes of cell wall enlargement and cell division. Studies on the pneumococcal system suggest several possible levels of regulation and these are depicted in the scheme in Fig. 6. Small amounts of the lowmolecular-weight and inactive E-form autolysin (typical of TABLE 4. Binding studies of [3H]choline-labeled lipoteichoic acid to pneumococcal cell walls Lipoteichoic acid concentration
C totg/165 w)
.a radioactivity: 2 X 104
5.6 5.6 5.6
cpm
Cel wal concentration
Radioactivity in 100 ,l of supernatant (cpm)
(MAg/i65 Il)
Trial I
Trial II
0 30
14,450 14,130 13,590
12,190 12,640 12,200
75
Various amounts of cell walls were incubated with lipoteichoic acid (in 0.15 M NaCl solutions containing 0.01 M potassium phosphate buffer, pH 8) at 00 for 2 hr. After centrifugation (12,000 X g, 10 min) the amount of lipoteichoic acid remaining in the supernatant solutions was determined by measuring radioactivity in 100 Ml aliquots.
1694
Biochemistry: H6ltje and Tomasz
ethanolamine-grown pneumococci) (14) have been detected in choline-grown cells also (unpublished observation). Thus, the E-form autolysin may represent the precursor (proenzyme) for this enzyme, synthesized on the ribosomes. By analogy to the in vitro conversion of E-form to C-form autolysin, the activation of this proenzyme may occur after transport through the plasma membrane by interaction with choline residues in the wall teichoic acid. The existence of inactive autolysin precursor and its conversion to a catalytically active enzyme by proteolysis has been described by Shockman and his associates in the case of the Strep. faecalis muramidase (24). The pneumococcal enzyme activation as well as the catalytic activity of the "converted" autolysin are both inhibited by lipoteichoic acid-as the data presented in this paper indicate. The recently described teichoic acid phosphocholine esterase (18) offers an additional potential regulatory mechanism through the removal of a critical set of phosphocholine residues from either the cell wall or from the lipoteichoic acid (which is also a substrate for this enzyme). Since the catalytic activity of autolysin seems to have an absolute requirement for choline moieties in the substrate (28), the removal of a fraction of choline residues by the esterase is likely to create autolysinresistant zones in the cell wall. Similarly, the inhibitory power of lipoteichoic acid may be affected by the removal of phosphocholine residues. While the in vivo relevance of this scheme is entirely hypothetical, several of its predictions are experimentally testable. The most striking evidence suggesting the in vivo inhibition of autolysin by lipoteichoic acid is provided by the physiological effects of lipoteichoic acid on growing cultures. One of the physiological effects-the inhibition of stationary phase lysis-was observed at a concentration as low as 30 Ag/ml. Protection against antibiotic action and chaining required higher concentrations (3-4 mg/ml). Nevertheless, the agent causing these effects still seems to be the LTA, since it fractionates with the Forssman antigen; it is resistant to proteolysis, nucleases and lipid solvents; it contains no detectable cell wall components and it is destroyed by nitrous acid. The high concentrations of extracellular LTA used may be necessary for several reasons: the LTA molecules may have to "coat" the cell surface in order to reach the sites of endogenous autolysin activity; in addition, live cells may have a mechanism for degrading the LTA. The relatively high concentrations of LTA had no effect on the bacterial growth rate. The LTA-induced inhibition of cell separation (chain formation), penicillin resistance, and resistance to stationary phase lysis are the same phenomena that have been observed in pneumococci with defective autolytic systems, such as ethanolaminegrown cultures (14) and the amidase-defective mutant (25). It seems, therefore, that LTA molecules can penetrate the bacterial surface to some degree and can inhibit the autolytic system. A further observation of possible in vivo relevance is the reversal of lipoteichoic acid inhibition by detergents. Deoxycholate is known to induce lysis of pneumococci by the induction of an endogenous autolytic process (12). A deoxycholateresistant autolysin-defective mutant of pneumococcus requires for cellular lysis the combined addition of both de-
Proc. Nat. Acad. Sci. USA 72
(1975)
oxycholate and wild-type autolysin (26). It is conceivable that the mechanism of induction of bacterial lysis by deoxycholate and other detergents may involve a specific dissociation of inhibited autolysin-lipoteichoic acid complexes, rather than a nonspecific removal of some membrane barrier that may separate autolysin molecules from their endogenous substrate in vivo. It is not clear at the present time to what extent these conclusions might also apply to other species of bacteria. However, the ubiquitous occurrence of lipoteichQic acids among most Gram-positive species (27) and the potential general importance of our observations for antibiotic sensitivity would seem to warrant the testing of other bacterial species too for the type of lipoteichoic acid effects described in this paper for pneumococci. This investigation has been supported by a grant from the
U.S. National Institutes of Health. 1. Weidel, W. & Pelzer, H. (1964) Advan. Enzymol. 26, 193228. 2. Rogers, H. J. (1970) Bacteriol. Rev. 34, 194-214. 3. Higgins, M. L. & Shockman, G. D. (1971) CRC Crit. Rev. Microbiol. 1, 29-72. 4. Shockman, G. D., Daneo-Moore, L. & Higgins, M. L. (1974) Ann. N.Y. Acad. Sci. 235, 161-197. 5. Young, F. E., Tipper, D. J. & Strominger, J. L. (1964) J. Biol. Chem. 239, 3660-3662. 6. Ranhand, J. M., Leonard, C. G. & Cole, R. G. (1971) J. Bacteriol. 106, 257-268. 7. Seto, H. & Tomasz, A. (1975) J. Bacteriol. 121, 344-353. 8. McDonnell, M., Ronda-Lain, C. & Tomasz, A. (1975) Virology 63, 577-582. 9. Tomasz, A., Albino, A. & Zanati, E. (1970) Nature 227, 138-140. 10. Rogers, H. J. & Forsberg, C. W. (1971) J. Bacteriol. 108, 1235-1243. 11. Tomasz, A. (1970) J. Bacteriol. 101, 860-871. 12. Mosser, J. L. & Tomasz, A. (1970) J. Biol. Chem. 245, 287298. 13. Goebel, W. F., Shedlovsky, T., Lavin, G. I. & Adams, M. H. (1943) J. Biol. Chem. 148, 1-15. 14. Tomasz, A. & Westphal, M. (1971) Proc. Nat. Acad. Sci. USA 68, 2627-2630. 15. Krause, R. M. & McCarty, M. (1961) J. Exp. Med. 114, 127-140. 16. Liu, T. Y. & Gotschlich, E. C. (1963) J. Biol. Chem. 238, 1928-1939. 17. Briles, E. B. & Tomasz, A. (1973) J. Biol. Chem. 248, 63946397. 18. H6ltje, J.-V. & Tomasz, A. (1974) J. Biol. Chem. 249, 70327034. 19. Potter, M. & Lieberman, R. (1970) J. Exp. Med. 132, 737751. 20. Brundish, D. E. & Baddiley, J. (1968) Biochem. J. 110, 573-582. 21. Heptinstall, S., Archibald, A. R. & Baddiley, J. (1970) Nature 225, 519-521. 22. Fiedler, F. & Glaser, L. (1974) J. Biol. Chem. 249, 26842689. 23. Fujiwara, M. (1967) Jap. J. Exp. Med. 37, 581-597. 24. Shockman, G. D., Thompson, J. S. & Conover, M. J. (1965) J. Bacteriol. 90, 575-588. 25. Tomasz, A. (1974) Ann. N.Y. Acad. Sci. 235, 439-447. 26. Lacks, S. (1970) J. Bacteriol. 101, 373-383. 27. Knox, K. W. & Wicken,'J. (1973) Bacteriol. Rev. 37, 215257. 28. Holtje, J.-V. and Tomasz, A. (1975) J. Biol. Chem., in press.
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 lipoteichoic acid (Forssman antigen) is a powerful inhibitor of the homologous autolytic enzyme, an N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28, mucopeptide amidohydrolase). Low concentrations of deoxycholate can reverse the inhibition. Wall teichoic acid preparations are inactive at several hundred-fold higher concentrations. Activation of an inactive form of autolysin by in vitro incubation with choline-containing cell walls is also inhibited by lipoteichoic acid. Addition of lipoteichoic acid to the growth medium of pneumococcal cultures causes chain formation, resistance to stationary phase lysis, and penicillin tolerance. It is suggested that a physiological role of lipoteichoic acids may be in the in vivo control of autolysin activity.
Institute, National Institutes of Health, Bethesda, Md.). The activity of pneumococcal autolysin (N-acetylmuramylL-alanine amidase; EC 3.5.1.28, mucopeptide amidohydrolase) was determined in the following manner: 2.36 ,ug of isotopelabeled cell walls ([methyl-3H]choline, 1.8 ,uCi/mg) and 10 ,ul of amidase (containing about 1.5 X 108 cell equivalent units of crude amidase extract, 15 Mug of protein per 10 Mul) were mixed in a final volume of 250 Mul of 0.05 M Tris-maleate buffer (pH 6.7). After incubation at 370, 20 ,ul of 38% formaldehyde and 20 Mul of 0.5% bovine serum albumin (BSA; Armour fraction IV) solution were added and the unreacted cell wall material was removed by centrifugation (12,000 X g, 10 min;
The participation of bacterial autolytic enzymes in a variety of important physiological phenomena has been postulated by several authors. It has been suggested that replication and 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), competence 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 bactericidal and bacteriolytic action of antibiotics has been demonstrated in pneumococci (9) and in Bacillus subtilis (10). Any physiological functioning of autolysins must involve careful endogenous control of their activity. In search for potential cellular inhibitors of these enzymes we observed a powerful inhibitory effect of the pneumococcal lipoteichoic acid. Various aspects of this finding are described in this communication.
TABLE 1. Inhibition of conversion of the E-form to the C-form amidase by lipoteichoic acid Choline-cell walls + E-form amidase + lipoteichoic acid Preincubated at 00, 5 min
Deoxycholate added Incubation at 370
MATERIALS AND METHODS
The common laboratory strain of Diplococcus pneumoniae R36A was used in all studies. Several of the experimental procedures used have been described in previous publications. These include: growth of the bacteria in chemically defined medium (11) with choline or ethanolamine as the amino alcohol components; preparation and biosynthetic labeling (with radioactive isotopes) of cell walls (12); preparation of lipoteichoic acid (13) and the C- and E-forms of pneumococcal autolytic enzyme (14). Formamide extraction of wall teichoic acid (15); the preparation of C-polysaccharide (16); periodate oxidation and nitrous acid degradation (12); and disaggregation of the lipoteichoic acid by sodium dodecyl sulfate (17) were all done by published procedures. The myeloma protein (TEPC-15) was a gift of Dr. M. Potter (National Cancer
Concentration of lipoteichoic acid
Amidase activity
(Jsg/ml)
(% of control)
0.63 1.26 3.15 6.30 15.75 31.50
102 99 68 40 31 18
Inactive form (E-form) of amidase was obtained from pneumococci grown on ethanolamine-containing medium (14). A preparation of E enzyme (10 ul; containing about 1.5 X 108 of cell equivalents of bacterial lysate) was mixed with choline-containing cell walls (236 jug in 1 ml of 0.15 M saline containing 0.01 M potassium phosphate buffer, pH 8.0) and various amounts of lipoteichoic acid (LTA); these components were added in rapid succession in the order indicated. "Conversion" of the E-form to the C-form amidase was allowed to proceed for 5 min at 00 (14). Deoxycholate (25 Mul of 2% solution) was added to each reaction mixture (in order to annul the effect of lipoteichoic acid on the assay of amidase activity) and the samples were incubated at 370 in order to determine the activity of amidase, as described in Materials and Methods. No enzyme activity could be detected without "conversion."
Abbreviation: LTA, lipoteichoic acid.
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Proc. Nat. Acad.'Sci. USA 72
Lipoteichoic Acid: Inhibitor of Autolysin
(1975)
1691
-j
rz0
0 h.
0 z -I
J
hi hi 0 0 -i hi IC
o
3.5
7.0
10.5 17.5 14.0 LIPOTEICHOIC ACID (1&g/ml)
21.0
35.0
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 solution was determined by pipetting 200 Ml portions into 5 ml of "Ready-Solv" scintillator (Beckman) and counting the samples in a Mark II (Nuclear Chicago) scintillation spectrometer. RESULTS Effect of Lipoteichoic Acid on Amidase Activity and on the In Vitro Activation of Amidase. 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 caused 50% inhibition; 2-3 pg/ml was sufficient to inhibit 80% of the enzyme activity. The somewhat less than complete inhibition of enzyme activity (see Fig. 1) may be more apparent than real, since 10-20% of the choline residues may be removed from the cell walls by the choline esterase (18) that is known to be present in the crude amidase preparations. Fig. 2 shows that the addition of low concentration (0.2%) of deoxycholate can completely reverse the inhibitory effect of LTA. Pneumococci grown on ethanolamine-containing medium contain an abnormal form of autolytic amidase ("E-form," low-molecular-weight and low-specific-activity) that can be "converted" to the high-molecular-weight and catalytically 0 100
active "C-form" enzyme by in vitro incubation with cholinecontaining cell walls (14). Table 1 shows that the pneumococcal LTA preparations can also inhibit this enzyme activation process.
100 o loo 50 zk
-
x
2.
0
II
F
I.)
02%DEOXYCHOLATE
-
1
5
10
15
FRACTION NUMBER
0 W
8 0
50
co50-
\
0.1% DEOXYCHOLATE
cC 10_ 085
1.75
425
8.50
LPOTECHOIC ACMD (mg /l)
FIG. 2. Deoxycholate reverses the inhibition of amidase reaction by lipoteichoic acid. Enzyme was added to the reaction mixtures last.
FIG. 3. Sucrose density gradient centrifugation of a lipoteichoic acid preparation in the presence of absence of sodium dodecyl 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 performed 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. Fractions (250 ,d) were collected through a pinhole pierced through the bottom of the tubes. Fraction 1 represents the bottom of the gradient. Detergent was removed by precipitation at 0° (17). Solid line: lipoteichoic acid (radioactivity) in 100 Ad fractions. Dashed line: inhibitory effect; 10 ,A fractions were tested.
1692
Biochemistry: Holtje and Tomasz
Proc. Nat. Acad. Sci. USA 72
(1975)
-i
z
0 I.
0
8It
f
U ) or
o
i
4c UA.
C.)
I>-
x
0
a.
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-3H1choline 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 degraded 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 LTA Preparations. The results of a series of experimentssummarized in Figs. 3 and 4 and Tables 2 and 3-indicate that the factor responsible for the inhibitory effects is the pneumococcal LTA itself. Fig. 3 demonstrates that the inhibitory activity co-migrates with the LTA during sedimentation in detergent-containing and detergent-free sucrose gradients (17). TABLE 2. Precipitation of lipoteichoic acid by a
phosphocholine-specific myeloma protein (TEPC-15)
Dilution of antiserum 1:0 1:2 1:5 1:10 No TEPC-15 (maximal lipoteichoic acid effect)
Lipoteichoic acid in supernatant
Amidase activity in presence of supernatant
(Cpm/10 IA)
(epm/150 ,A)
131 3191 6126
2935 2876 1760 1072
9692
579
622
A 50 Ml portion of a lipoteichoic acid preparation labeled with [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 precipitation with 100 ,l of saturated ammonium sulfate solution (40, 12 hr), the precipitates were removed by centrifugation (12,000 X g, 10 min). The supernatant solutions were assayed for lipoteichoic acid (radioactivity in 10 jM1 aliquots) and for inhibitory activity in the amidase reaction.
Periodate oxidation and incubation with nitrous acid cause degradation of LTA and elimination of the inhibitory activities of the preparations (Fig. 4). Table 2 shows that a phosphocholine-specific myeloma protein (19) known to precipitate the pneumococcal LTA (17) also precipitates the factor responsible for amidase inhibition. Specificity. The inhibitory activity seems to be specific for the LTA, since the structurally related wall teichoic acid (12, 20) showed no effect on the amidase activity even at several TABLE 3. Effect of choline-containing wall teichoic acid preparations on the amidase activity
Compound tested Wall-teichoic acid (Formamide) Prep I Prep II
Concentration
Amidase activity
(Mg/mi)
(% of control)
89 179
100 108
45
104
89
110
C-carbohydrate
84
Lipoteichoic acid
336 0.5 4.0
103 102 50 10
Formamide-extracted pneumococcal wall teichoic acid and Ccarbohydrate were prepared by procedures described in Materials and Methods.
Proc. Nat. Acad. Sci. USA 72
Lipoteichoic Acid: Inhibitor of Autolysin
(1975)
MEMBRANE
CYTOPLASM
1693
WALL
(2-CHOLINE ESTERASE Lipotoichoic Acid 300 RI
|(I)-HOI
®D-CHOLINE 200 /
K
;
ESTERASE
PENICILLIN +
LIPOTEICHOIC ACID \0
100 _
FIG. 6. A hypothetical scheme for the regulation of the activity of pneumococcal amidase.
50
PENICILLIN
__
1
2
3
4
5
6
7
HOURS
FIG. 5. Prevention of penicillin-induced lysis of pneumococci by lipoteichoic acid. Pneumococcal cultures at a concentration of 3.75 X 107 viable units/ml were given penicillin (0.1 unit/ml) (dash and dot line) or penicillin (0.1 unit/ml) plus lipoteichoic acid (4 mg/ml) (dashed line). A control culture was also grown for comparison. Bacterial lysis and growth were measured by monitoring the light scattering of the culture with a Coleman nephelometer (N: nephelos units).
hundred-fold higher concentrations (Table 3). High concentrations (140 ,g/ml) of pneumococcal LTA had no inhibitory effect on the hydrolytic activity of egg white lysozyme or on the autolytic activity(s) of crude B. subtilis 168 extracts. Mechanism. The mechanism of inhibitory action of LTA does not seem to involve association with the cell wall material, since there was no demonstrable attachment of radioactive LTA to pneumococcal cell walls (Table 4).
In Vivo Effects of LTA. Fig. 5 shows the protective effect of LTA (added to the growth medium of a growing pneumococcal culture) against the bacteriolytic activity of penicillin G. Under the same conditions, LTA also gave protection against vancomycin (30 Mg/ml). In addition, bacteria growing in the presence of LTA showed chain formation and resistance to spontaneous culture lysis in the stationary phase of growth. DISCUSSION
In spite of their widespread occurrence, the physiological function(s) of lipoteichoic acids are not well understood. A possible role in the binding of Mg++ ions at the cell surface has been proposed (20). More recently, Glaser and his associates have presented evidence for the participation of lipoteichoic acids in the biosynthesis of cell wall teichoic acid of Staphylococcus aureus (22). The observations described in this paper suggest an additional and novel physiological function for a lipoteichoic acid-as a natural inhibitor of autolysin activity.
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 inhibitor(s) has been reported in Streptococcus faecalis (24). At least two facts make the pneumococcal lipoteichoic acid a good candidate for the role of endogenous autolysin inhibitor. One is the cellular localization at the outer surface of the plasma membrane (17), i.e., in an area where autolysin molecules engaged in their postulated physiological activity are likely to reside. The other advantageous feature is the structural similarity to the wall teichoic acid (17, 23). The ability of the pneumococcal autolysin to specifically recognize the homologous choline-containing wall teichoic acid has already been well documented (12). The observations with autolysin-defective pneumococci illustrate clearly the potentially suicidal consequences of uncontrolled autolysin action (9). The existence of a variety of regulatory mechanisms is alsQ suggested by the postulated role of autolysins in the complex processes of cell wall enlargement and cell division. Studies on the pneumococcal system suggest several possible levels of regulation and these are depicted in the scheme in Fig. 6. Small amounts of the lowmolecular-weight and inactive E-form autolysin (typical of TABLE 4. Binding studies of [3H]choline-labeled lipoteichoic acid to pneumococcal cell walls Lipoteichoic acid concentration
C totg/165 w)
.a radioactivity: 2 X 104
5.6 5.6 5.6
cpm
Cel wal concentration
Radioactivity in 100 ,l of supernatant (cpm)
(MAg/i65 Il)
Trial I
Trial II
0 30
14,450 14,130 13,590
12,190 12,640 12,200
75
Various amounts of cell walls were incubated with lipoteichoic acid (in 0.15 M NaCl solutions containing 0.01 M potassium phosphate buffer, pH 8) at 00 for 2 hr. After centrifugation (12,000 X g, 10 min) the amount of lipoteichoic acid remaining in the supernatant solutions was determined by measuring radioactivity in 100 Ml aliquots.
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Biochemistry: H6ltje and Tomasz
ethanolamine-grown pneumococci) (14) have been detected in choline-grown cells also (unpublished observation). Thus, the E-form autolysin may represent the precursor (proenzyme) for this enzyme, synthesized on the ribosomes. By analogy to the in vitro conversion of E-form to C-form autolysin, the activation of this proenzyme may occur after transport through the plasma membrane by interaction with choline residues in the wall teichoic acid. The existence of inactive autolysin precursor and its conversion to a catalytically active enzyme by proteolysis has been described by Shockman and his associates in the case of the Strep. faecalis muramidase (24). The pneumococcal enzyme activation as well as the catalytic activity of the "converted" autolysin are both inhibited by lipoteichoic acid-as the data presented in this paper indicate. The recently described teichoic acid phosphocholine esterase (18) offers an additional potential regulatory mechanism through the removal of a critical set of phosphocholine residues from either the cell wall or from the lipoteichoic acid (which is also a substrate for this enzyme). Since the catalytic activity of autolysin seems to have an absolute requirement for choline moieties in the substrate (28), the removal of a fraction of choline residues by the esterase is likely to create autolysinresistant zones in the cell wall. Similarly, the inhibitory power of lipoteichoic acid may be affected by the removal of phosphocholine residues. While the in vivo relevance of this scheme is entirely hypothetical, several of its predictions are experimentally testable. The most striking evidence suggesting the in vivo inhibition of autolysin by lipoteichoic acid is provided by the physiological effects of lipoteichoic acid on growing cultures. One of the physiological effects-the inhibition of stationary phase lysis-was observed at a concentration as low as 30 Ag/ml. Protection against antibiotic action and chaining required higher concentrations (3-4 mg/ml). Nevertheless, the agent causing these effects still seems to be the LTA, since it fractionates with the Forssman antigen; it is resistant to proteolysis, nucleases and lipid solvents; it contains no detectable cell wall components and it is destroyed by nitrous acid. The high concentrations of extracellular LTA used may be necessary for several reasons: the LTA molecules may have to "coat" the cell surface in order to reach the sites of endogenous autolysin activity; in addition, live cells may have a mechanism for degrading the LTA. The relatively high concentrations of LTA had no effect on the bacterial growth rate. The LTA-induced inhibition of cell separation (chain formation), penicillin resistance, and resistance to stationary phase lysis are the same phenomena that have been observed in pneumococci with defective autolytic systems, such as ethanolaminegrown cultures (14) and the amidase-defective mutant (25). It seems, therefore, that LTA molecules can penetrate the bacterial surface to some degree and can inhibit the autolytic system. A further observation of possible in vivo relevance is the reversal of lipoteichoic acid inhibition by detergents. Deoxycholate is known to induce lysis of pneumococci by the induction of an endogenous autolytic process (12). A deoxycholateresistant autolysin-defective mutant of pneumococcus requires for cellular lysis the combined addition of both de-
Proc. Nat. Acad. Sci. USA 72
(1975)
oxycholate and wild-type autolysin (26). It is conceivable that the mechanism of induction of bacterial lysis by deoxycholate and other detergents may involve a specific dissociation of inhibited autolysin-lipoteichoic acid complexes, rather than a nonspecific removal of some membrane barrier that may separate autolysin molecules from their endogenous substrate in vivo. It is not clear at the present time to what extent these conclusions might also apply to other species of bacteria. However, the ubiquitous occurrence of lipoteichQic acids among most Gram-positive species (27) and the potential general importance of our observations for antibiotic sensitivity would seem to warrant the testing of other bacterial species too for the type of lipoteichoic acid effects described in this paper for pneumococci. This investigation has been supported by a grant from the
U.S. National Institutes of Health. 1. Weidel, W. & Pelzer, H. (1964) Advan. Enzymol. 26, 193228. 2. Rogers, H. J. (1970) Bacteriol. Rev. 34, 194-214. 3. Higgins, M. L. & Shockman, G. D. (1971) CRC Crit. Rev. Microbiol. 1, 29-72. 4. Shockman, G. D., Daneo-Moore, L. & Higgins, M. L. (1974) Ann. N.Y. Acad. Sci. 235, 161-197. 5. Young, F. E., Tipper, D. J. & Strominger, J. L. (1964) J. Biol. Chem. 239, 3660-3662. 6. Ranhand, J. M., Leonard, C. G. & Cole, R. G. (1971) J. Bacteriol. 106, 257-268. 7. Seto, H. & Tomasz, A. (1975) J. Bacteriol. 121, 344-353. 8. McDonnell, M., Ronda-Lain, C. & Tomasz, A. (1975) Virology 63, 577-582. 9. Tomasz, A., Albino, A. & Zanati, E. (1970) Nature 227, 138-140. 10. Rogers, H. J. & Forsberg, C. W. (1971) J. Bacteriol. 108, 1235-1243. 11. Tomasz, A. (1970) J. Bacteriol. 101, 860-871. 12. Mosser, J. L. & Tomasz, A. (1970) J. Biol. Chem. 245, 287298. 13. Goebel, W. F., Shedlovsky, T., Lavin, G. I. & Adams, M. H. (1943) J. Biol. Chem. 148, 1-15. 14. Tomasz, A. & Westphal, M. (1971) Proc. Nat. Acad. Sci. USA 68, 2627-2630. 15. Krause, R. M. & McCarty, M. (1961) J. Exp. Med. 114, 127-140. 16. Liu, T. Y. & Gotschlich, E. C. (1963) J. Biol. Chem. 238, 1928-1939. 17. Briles, E. B. & Tomasz, A. (1973) J. Biol. Chem. 248, 63946397. 18. H6ltje, J.-V. & Tomasz, A. (1974) J. Biol. Chem. 249, 70327034. 19. Potter, M. & Lieberman, R. (1970) J. Exp. Med. 132, 737751. 20. Brundish, D. E. & Baddiley, J. (1968) Biochem. J. 110, 573-582. 21. Heptinstall, S., Archibald, A. R. & Baddiley, J. (1970) Nature 225, 519-521. 22. Fiedler, F. & Glaser, L. (1974) J. Biol. Chem. 249, 26842689. 23. Fujiwara, M. (1967) Jap. J. Exp. Med. 37, 581-597. 24. Shockman, G. D., Thompson, J. S. & Conover, M. J. (1965) J. Bacteriol. 90, 575-588. 25. Tomasz, A. (1974) Ann. N.Y. Acad. Sci. 235, 439-447. 26. Lacks, S. (1970) J. Bacteriol. 101, 373-383. 27. Knox, K. W. & Wicken,'J. (1973) Bacteriol. Rev. 37, 215257. 28. Holtje, J.-V. and Tomasz, A. (1975) J. Biol. Chem., in press.
