Proc. Nat. Acad. Sci. USA Vol. 72, No. 10, pp. 4162-4166, October 1975 Microbiology Mechanism of action of penicillin: Triggering of the pneumococcal autolytic enzyme by inhibitors of cell wall synthesis (release of lipoteichoic acid/autolysin) ALEXANDER TOMASZ AND SUSAN WAKS The Rockefeller University, New York, N.Y. Communicated by William Trager, July 9,1975 ABSTRACT During penicillin treatment of an autolysin defective mutant pneumococcus we have observed three novel phenomena: (z) Growth of the mutant cultures is inhib- ited by the same concentrations of penicillin that induce lysis in the wild type. (ii) Mutant bacteria treated with the minimum growth inhibitory concentration of penicillin will lyse upon the addition of wild-type autolysin to the growth medium. Chloramphenicol and other inhibitors of protein synthesis protect the cells against lysis by exogenous enzyme. Sensitivity of the cells to exogenous autolysin requires treat- ment with penicillin or other inhibitors of cell wall synthesis (e.g., D-cycloserine or fosfonomycin) since exogenous autolys- in alone has no effect on bacterial growth. (ii) Treatment with penicillin (or other inhibitors of cell wal synthesis) causes the escape into the medium of a choline-containing macromolecule that has properties suggesting that it contains pneumococcal lipoteichoic acid (Forssman antigen). Each one of these three phenomena (growth inhibition, sensitiza- tion to exogenous autolysin, and leakage of lipoteichoic acid) shows the same dose response as that of the penicillin-in- duced lysis of wild-type pneumococci. On the basis of these findings we propose a new hypothe- sis for the mechanism of penicillin-induced lysis of bacteria. It is suggested that inhibition of cell wall synthesis by any means triggers bacterial autolytic enzymes by destabilizing the endogenous complex of an autolysin inhibitor (lipo teichoic acid) and autolytic enzyme. Escape of lipoteichoic acid-like material to the growth medium is a consequence of this labilization. Chloramphenicol protects bacteria against penicillin-induced lysis by interfering with the activity of the autolytic enzyme, rather than by depleting the concentration of the enzyme at the cell surface. The rapid loss of viability and cellular lysis of pneumococci treated with penicillin and other cell wall inhibitory bacteri- cidal drugs is known to require the activity of the pneumo- coccal autolytic enzyme (an N-acetylmuramyl-L-alanine amidase) (1, 2). However, the mechanism by which inhibi- tion of wall synthesis is coupled to the "suicidal" activity of this enzyme is not known. In this communication we report observations that bear on this question and we propose a new hypothesis for the mechanism by which penicillin and other lytic antibiotics cause destruction of the bacterial cell. MATERIALS AND METHODS An autolysin defective mutant derivative of the R36A strain of Diplococcus pneumoniae (3) was used as the source of DNA with which the autolysin defect was introduced into competent cells of the parent (wild-type) strain via genetic transformation. A culture of the transformant was kindly given to us by Dr. S. Lacks (Brookhaven National Laborato- ry), and cultures of these cells will be referred to as "autolys- in defective mutant." Most of the methods used in these in- vestigations have been described in detail in earlier publica- tions. These include the composition of growth media and monitoring growth by nephelometry (4), preparation of cell walls (5), autolytic enzyme (6), and pneumococcal F-antigen (7), and the assay of autolysin activity (8), "Loading" of the mutant cells with wild-type autolysin was performed in the following manner: Fifty microliters of highly purified autolysin (containing 3.5 jg of protein) was added to 1 ml of autolysin defective pneumococci (2 X 107 to 2 X 108 viable units/ml) and incu- bated at 370 for 30 min. The cells were next filtered (Milli- pore, 0.45 Mm), washed-on the filter-three times with 2 ml of warm (370) growth medium, and resuspended in growth medium. No significant amounts of enzyme seem to escape to the medium during subsequent exponential growth of the enzyme-loaded bacteria. Detailed studies of this system will be described elsewhere*. All chemicals were of reagent grade. RESULTS Effects of penicillin on autolysin defective pneumococci The use of a mutant pneumococcus defective in the major cellular autolytic enzyme (an N-acetyl-muramyl-L-alanine amidase) has allowed us to identify in the drug-treated bac- teria three penicillin-induced effects that appear to be of general significance for the mode of action of bacteriolytic antibiotics. Bacteriostatic Effect. Unlike the wild-type cells, which are lysed by penicillin, growing cultures of the mutant bac- teria respond to the addition of penicillin by inhibition of growth (1). This bacteriostatic response has exactly the same drug concentration dependence as the lytic response of wild- type cells (Fig. 1). Sensitization to Externally Added Autolytic Enzyme. Addition of wild-type autolysin preparation to the growth medium of penicillin-inhibited mutant bacteria makes these cells lyse (Fig. 2). Several details of this observation are im- portant. (i) Penicillin treatment is essential for the sensitiza- tion of the cells to the enzyme, since autolysin without anti- biotic has no effect on the bacterial growth. (ii) Fig. 2 shows that the doses of penicillin treatment needed for sensitiza- tion are the same as the doses required for inhibition of growth (in the mutant) or for lysis (in the wild type). (ii) Autolysin and penicillin need not be present at the same * A. Tomasz and S. Waks, Biochem. Biophys. Res. Commun. (1975), in press. 4162 Abbreviations: CAP, chloramphenicol; LTA, lipoteichoic acid. Downloaded by guest on March 21, 2020
Transcript
Proc. Nat. Acad. Sci. USAVol. 72, No. 10, pp. 4162-4166, October 1975Microbiology
Mechanism of action of penicillin: Triggering of the pneumococcalautolytic enzyme by inhibitors of cell wall synthesis
(release of lipoteichoic acid/autolysin)
ALEXANDER TOMASZ AND SUSAN WAKSThe Rockefeller University, New York, N.Y.
Communicated by William Trager, July 9,1975
ABSTRACT During penicillin treatment of an autolysindefective mutant pneumococcus we have observed threenovel phenomena: (z) Growth of the mutant cultures is inhib-ited by the same concentrations of penicillin that inducelysis in the wild type. (ii) Mutant bacteria treated with theminimum growth inhibitory concentration of penicillin willlyse upon the addition of wild-type autolysin to the growthmedium. Chloramphenicol and other inhibitors of proteinsynthesis protect the cells against lysis by exogenous enzyme.Sensitivity of the cells to exogenous autolysin requires treat-ment with penicillin or other inhibitors of cell wall synthesis(e.g., D-cycloserine or fosfonomycin) since exogenous autolys-in alone has no effect on bacterial growth. (ii) Treatmentwith penicillin (or other inhibitors of cell wal synthesis)causes the escape into the medium of a choline-containingmacromolecule that has properties suggesting that it containspneumococcal lipoteichoic acid (Forssman antigen). Eachone of these three phenomena (growth inhibition, sensitiza-tion to exogenous autolysin, and leakage of lipoteichoic acid)shows the same dose response as that of the penicillin-in-duced lysis of wild-type pneumococci.On the basis of these findings we propose a new hypothe-
sis for the mechanism of penicillin-induced lysis of bacteria.It is suggested that inhibition of cell wall synthesis by anymeans triggers bacterial autolytic enzymes by destabilizingthe endogenous complex of an autolysin inhibitor (lipoteichoic acid) and autolytic enzyme. Escape of lipoteichoicacid-like material to the growth medium is a consequence ofthis labilization. Chloramphenicol protects bacteria againstpenicillin-induced lysis by interfering with the activity of theautolytic enzyme, rather than by depleting the concentrationof the enzyme at the cell surface.
The rapid loss of viability and cellular lysis of pneumococcitreated with penicillin and other cell wall inhibitory bacteri-cidal drugs is known to require the activity of the pneumo-coccal autolytic enzyme (an N-acetylmuramyl-L-alanineamidase) (1, 2). However, the mechanism by which inhibi-tion of wall synthesis is coupled to the "suicidal" activity ofthis enzyme is not known. In this communication we reportobservations that bear on this question and we propose anew hypothesis for the mechanism by which penicillin andother lytic antibiotics cause destruction of the bacterial cell.
MATERIALS AND METHODSAn autolysin defective mutant derivative of the R36A strainof Diplococcus pneumoniae (3) was used as the source ofDNA with which the autolysin defect was introduced intocompetent cells of the parent (wild-type) strain via genetictransformation. A culture of the transformant was kindlygiven to us by Dr. S. Lacks (Brookhaven National Laborato-ry), and cultures of these cells will be referred to as "autolys-
in defective mutant." Most of the methods used in these in-vestigations have been described in detail in earlier publica-tions. These include the composition of growth media andmonitoring growth by nephelometry (4), preparation of cellwalls (5), autolytic enzyme (6), and pneumococcal F-antigen(7), and the assay of autolysin activity (8), "Loading" of themutant cells with wild-type autolysin was performed in thefollowing manner:
Fifty microliters of highly purified autolysin (containing3.5 jg of protein) was added to 1 ml of autolysin defectivepneumococci (2 X 107 to 2 X 108 viable units/ml) and incu-bated at 370 for 30 min. The cells were next filtered (Milli-pore, 0.45 Mm), washed-on the filter-three times with 2ml of warm (370) growth medium, and resuspended ingrowth medium. No significant amounts of enzyme seem toescape to the medium during subsequent exponentialgrowth of the enzyme-loaded bacteria. Detailed studies ofthis system will be described elsewhere*.
All chemicals were of reagent grade.
RESULTSEffects of penicillin on autolysin defectivepneumococciThe use of a mutant pneumococcus defective in the majorcellular autolytic enzyme (an N-acetyl-muramyl-L-alanineamidase) has allowed us to identify in the drug-treated bac-teria three penicillin-induced effects that appear to be ofgeneral significance for the mode of action of bacteriolyticantibiotics.
Bacteriostatic Effect. Unlike the wild-type cells, whichare lysed by penicillin, growing cultures of the mutant bac-teria respond to the addition of penicillin by inhibition ofgrowth (1). This bacteriostatic response has exactly the samedrug concentration dependence as the lytic response of wild-type cells (Fig. 1).
Sensitization to Externally Added Autolytic Enzyme.Addition of wild-type autolysin preparation to the growthmedium of penicillin-inhibited mutant bacteria makes thesecells lyse (Fig. 2). Several details of this observation are im-portant. (i) Penicillin treatment is essential for the sensitiza-tion of the cells to the enzyme, since autolysin without anti-biotic has no effect on the bacterial growth. (ii) Fig. 2 showsthat the doses of penicillin treatment needed for sensitiza-tion are the same as the doses required for inhibition ofgrowth (in the mutant) or for lysis (in the wild type). (ii)Autolysin and penicillin need not be present at the same
* A. Tomasz and S. Waks, Biochem. Biophys. Res. Commun.(1975), in press.
FIG. 1. Bacteriostatic effect of penicillin in an autolysin defec-tive pneumococcus. Exponentially growing cultures received peni-cillin G at the concentrations indicated; growth was monitored bynephelometry (Coleman Nephelometer). Numbers indicate theconcentrations of penicillin (in U/ml).
time. The bacteria could be sensitized to the enzyme by thefollowing sequence of treatments: exposure to penicillin (0.1unit/ml for 60 min), treatment with penicillinase (Neutra-pen, Rikers Labs; 10 units/ml for 5 min) followed by the ad-dition of autolysin to the medium. Such bacteria lyse (Fig.3). (iv) Sensitization is transient: after removal of penicillin(by penicillinase) bacteria recover their normal autolysin re-sistance in parallel with the resumption of growth (see Fig.3). (v) Chloramphenicol (CAP; 100,gg/ml) can block sensiti-zation by penicillin if added in the following order: firstCAP, 5 min later penicillin. (vi) Penicillin as a sensitizingagent can be replaced by other drugs, such as: vancomycin(50 jig/ml), cycloserine (50 jig/ml) (1), /3-chloro-D-alanine(500 jig/ml), fosfonomycin (50 ug/ml) (1), cephalothin (1tg/ml), and oxacyllin (1 ,ug/ml) (2, *). The common feature
of these treatments is that they all interfere with cell wallbiosynthesis.Mechanism of sensitization to autolysinIt appeared possible that the mechanism of sensitization in-volves some nonspecific "damage" to the cell surface(caused by the inhibitor) which is needed to allow the at-tachment of extracellular enzyme molecules to the bacterialsurface. However, this does not seem to be the case, since, byappropriate techniques, the autolysin molecules can be "ad-sorbed" into the surface of live, mutant bacteria prior to thetreatment of the cells with the sensitizing agents. The auto-lysin, experimentally introduced into the cells, is retained incell-bound form through several cell generations.
(The success of this "enzyme replacement therapy" isclearly indicated by the fact that these reconstituted mutantcells "revert" immediately to the wild phenotype in proper-ties that are associated with the in vivo activity of the auto-lytic amidase. Details of this system will be described else-where*.) Addition of penicillin (either at the minimum in-hibitory concentration, i.e., at 0.02 unit/ml, or at higherconcentrations) to such "enzyme-loaded" bacteria (eitherimmediately after enzyme adsorption or even after growthfor one generation) results in cellular lysis with a kineticscomparable to that of the wild type. Other bacteriolytic an-tibiotics (oxacyllin, 1 gg/ml; cephalothin, 1 sg/ml; vancom-ycin, 50 gg/ml; D-cycloserine, 50 gg/ml) cause lysis, where-as inhibition of protein and/or ribonucleic acid synthesis
I
400MINTES
FIG. 2. Sensitlzution of autolysin defective pneumococci to ex-ogenous wild-type autolysin. Exponentially growing cultures re-ceived penicillin at the 60th min of growth and autolysin (109 cellequivalents per ml of medium) at the times lndicated by the ar-rows. Growth was followed by nephelometry. Numbers indicatethe penicillin concentrationst 0.1 (1), 0.05 (2), 0.04 (3), 0.03 (4),and 0.02 (5) U/ml. Tube 6 received no drug; tube 7 received onlyautolysin.
parallel with the addition of penicillin prevents lysis. Lysis isalso prevented by the addition of pneumococcal lipoteichoicacid (Forssman antigen) to the medium (Fig. 4),
1000 7
2
3~~~~~~~~~~410 1O 1 2 3 4 5 6
HOURS
FIG. 3. Sequential treatment of autolysin defective pneumo-cocci with penicillin and autolysin. An exponentially growing cul-ture was treated with penicillin (0.1 U/ml, added at the time indi-cated by the solid arrow) for 60 min. After this treatment, the cul-ture was divided into three portions: one was left to incubate withpenicillin (3), the other two (2 and 4) received penicillinase (10units/ml for 5 min; time of addition indicated by solid arrow). Cul-ture 4 received autolysin (109 cell equivalent units/ml of medium)immediately after the penicillinase addition, while culture 2 wasallowed to incubate for about 2.5 hr before addition of autolysin.(Addition of autolysin is indicated by the winged arrows.) A con-trol culture (1) received no additions at all. Growth (and lysis)were monitored by following the turbidity of the cultures (OD660)with a Zeiss spectrophotometer.
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FIG. 4. Protection of "autolysin-loaded" mutant pneumococciagainst penicillin induced lysis by chloramphenicol and lipo-teichoic acid. Autolysin defective pneumococci were "loaded" withwild-type autolysin (see Materials and Methods). After resuspen-sion in growth medium the bacteria were distributed into threetest tubes, one of which received chloramphenicol (100 ug/ml; tubeA); pneumococcal lipoteichoic acid (200 pg/ml) was added to thesecond tube (B), the third tube received no additions (control, notshown C). After 30 min of incubation at 370, penicillin G (0.1U/ml) was added to each of the three cultures and incubation con-
tinued for 3 hr. At this time samples were examined by phase con-
trast microscopy (Zeiss R.A. microscope equipped with 100/1.25planachromat oil immersion lens). Photographs were made on
Panatomic X film. Upper photo, tube A; lower photo, tube B. Con-trol showed only amorphous debris (not documented).
Inhibition of DNA synthesis under similar conditions (bymitomycin C, 5-fluorodeoxyuridine, or 5-hydroxyphenyla-zauracil) had no effect (inhibitory or stimulatory) on thepenicillin-induced lysis*.
Penicillin-Induced "Leakage" of a Lipoteichoic Acid-Like Substance from the Cells. Table 1 demonstrates the
Table 1. Release of a choline-containing macromolecularmaterial from penicillin-treated autolysin
Bacteria were labeled by growth in [3H]choline-containing medi-um as described in the legend to Fig. 4. The cells were distributedinto tubes containing various concentrations of penicillin with orwithout simultaneously added chloramphenicol (100 jg/ml). InExp. 1, the prelabeled bacteria were allowed to grow for 60 min inisotope-free medium before the addition of drugs; in Exp. 2, thisperiod was shorter, about 30 min. The cells were removed from themedium by centrifugation (10,000 x g, 10 min), and 100-pl por-tions of the supernatant were pipetted into 1 ml of cold, 10% tri-chloroacetic acid solution. The precipitates were collected ontoglass fiber filter (GFA) and dried in an oven (1000, 10 min). Radio-activity was determined using a toluene base scintillation cocktail.Total, acid-precipitable radioactivity was determined by pipetting100-ul portions of whole bacterial suspensions into trichloroaceticacid and measuring radioactivity in the precipitates, as describedabove.
penicillin-induced escape of a radioactive choline-labeledmacromolecular component to the growth medium. Lowconcentrations of penicillin (near the minimal inhibitoryconcentration) were sufficient to induce release. It should beemphasized that this release occurs without added autolysin,i.e., in the complete absence of cellular disintegration. Fig. 5shows the sedimentation of this component during centrifu-gation in sucrose density gradients. The shift in the apparentmolecular size (disaggregation) of the material in the deter-gent (sodium dodecyl sulfate)-containing gradient is typicalof the behavior of the pneumococcal lipoteichoic acid (LTA)(7).The autolysin inhibitory activity of the penicillin-treated
culture filtrate is shown in Table 2. It can be seen that theinhibitory factor is nondialyzable, it is resistant to heating at1000 for 30 min, and is sensitive to periodate. Deoxycholateadded together with the filtrate has reversed the inhibitionof autolysin activity.
Another observation suggesting the escape of LTA intothe growth medium is illustrated in Table 3, which showsthat after exposure of the cells to a relatively small dose ofpenicillin (0.1 U/ml for 1.5 hr) the bacteria seem to have lostthe powerful autolysin inhibitory activity that can be nor-mally found in the lipoteichoic acid fraction of pneumococci(9). In fact, some stimulatory activity may be present. Thenature of this activity is presently being investigated.Two other inhibitors of cell wall synthesis, D-cycloserine
(50 gsg/ml) and fosfonomycin (50 jug/ml), also caused re-lease of choline-labeled material. On the other hand, inhibi-tion of protein synthesis (by chloramphenicol) has substan-tially reduced the penicillin induced release of LTA-likematerial (Table 1).
FIG. 5. Sedimentation behavior of the choline-labeled macromolecular material leaked into the growth medium during penicillin treat-ment. A culture of the autolysin defective pneumococci was labeled with radioactive choline ([methyl-3H]choline; 0.1 ,Ci and 5 Mg/ml) bythree generations of growth in 10 ml of isotope-containing medium. The cells were washed free of extracellular radioactivity (Millipore filtra-tion), resuspended in isotope-free growth medium and (after 30 min of growth at 370) penicillin was added to the culture (0.1 U/ml). Afterfurther incubation for 1.5 hr the cells were removed by centrifugation (10,000 X g, 15 min) and the supernatant medium was collected, di-alyzed (overnight) against water, and concentrated to a small volume (0.5 ml) by embedding the dialysis bag in Aquacide II. Portions (100 Ml)of the solution were layered on sucrose gradients [5-25% sucrose, in 0.15 M NaCl solution with (--- -) and without (-) 2% sodium dodecylsulfate], and centrifuged at 35,000 rpm at 180 for 16 hr in a Spinco model L3-50 ultracentrifuge. A swinging bucket (SW 50.1) rotor and po-lyallomer tubes were used. The gradients were analyzed for radioactivity; 8-drop fractions were collected through a pinhole pierced throughthe bottom of the tubes, and the radioactivity was measured using the "Readysolv" scintillation liquid (Beckman) and a Mark II scintilla-tion spectrometer (Nuclear Chicago Corp.).
Table 2. Appearance of autolysin inhibitory activityin the culture filtrate of penicillin-treated pneumococci
A culture of exponentially growing wild-type pneumococci (100ml; 2.5 x 107 cells per ml) received penicillin (0.1 unit/ml). After1.5 hr of incubation, both cultures were harvested (centrifugationat 10,000 x g, 20 min). The supernatant fluid was dialyzed againstseveral changes of distilled water and lyophilized to dryness; theresidue was dissolved in water (500 d). Before testing for autolysininhibitory activity the solution was heated at 1000 for 30 min to in-activate possible residual autolysin. A 100-Al portion of the solutionwas treated with periodate (0.025 M paraperiodic acid in 0.05 Msodium acetate buffer, pH 4.8) at 00 in the dark for 24 hr. Excessreagent was destroyed by the addition of 10 Ml of glycerol (0.1 M)and incubation at 370 for 60 min.The inhibitory activity was tested in the following manner: 5-Ml
portions of the filtrate were added to the following reaction mix-ture: 200 Al of buffer (Tris-maleate, 0.05 M, pH 6.9), 10 ,l of [3H]-choline-labeled cell wall suspension (containing 200 Mg dry weightand 108 cpm/ml), and 10 Al of autolysin (109 cell equivalents perml). The enzyme reaction was allowed to run at 370 for 10 min.Each reaction tube received 25 Ml of formaldehyde (38% solution)and 25 Ml of carrier bovine serum albumin (4% solution), and wascentrifuged at 10,000 x g for 15 min in an Eppendorff centrifuge.Portions (150 Ml) of the supernatant solutions were assayed for thedetermination of solubilized radioactivity as described in thelegend to Table 1.
DISCUSSIONSuppression of the endogenous autolytic activity greatly re-duced the rate with which pneumococci lose their viabilityduring penicillin treatment and virtually abolished cellularlysis by this antibiotic (1, 2). These findings indicate that thepneumococcal autolysin-an N-acetylmuramyl-L-alanineamidase-plays an essential role in the penicillin-induceddisintegration of these bacteria.
However, it is not clear how the inhibition of bacterial cellwall synthesis by penicillin brings about the deranged activi-ty of the autolytic enzyme. The observations described inthis paper suggest a mechanism by which the primary (in-hibitory) activity of penicillin may lead to the autolytic de-
Table 3. Loss of autolysin inhibitory activity from theForssman antigen (lipoteichoic acid) fraction of
Two cultures of wild-type pneumococci (100 ml each) were usedfor the preparation of F antigen (7). One of the cultures was treatedwith penicillin (0.1 unit/ml) for 1.5 hr before the preparation. TheF antigen fraction of each preparation was dissolved in 0.5 ml ofwater. This fraction contained 1.3 x 1010 cell equivalents of mate-rial in the case of the penicillin-treated culture and 6.9 X 109 cellequivalents in the control. Portions (100 Ml) of the extracts weretested for their effect on the activity of the autolysin by the methoddescribed in the legend to Table 2.
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struction of the cell. We propose that the key event in thismechanism is the labilization or inactivation of an endoge-nous autolysin inhibitor that (under normal conditions)keeps the activity of this enzyme suppressed. The existenceof a powerful and specific autolysin inhibitor in pneumococ-ci has recently been demonstrated and identified as thecomplex, choline-containing LTA (Forssman antigen) of thisbacterium (9). The results summarized in Table 2 indicatethat this inhibitory activity is not detectable in penicillin-treated cells. The escape of choline-containing macromole-cules (resembling the Forssman antigen) from penicillin-treated pneumococci may represent the release of someform of the LTA into the medium. Similar leakage can beobserved in both autolysin defective (mutant) and the wild-type cells (undocumented). We suggest, therefore, that thesensitization of autolysin defective pneumococci to exog-enous enzyme and the triggering of autolysin activity in thewild-type cells are analogous phenomena: they are both theconsequences of the removal of an amidase inhibitor.
It should be emphasized that the exact chemical nature ofthe choline-containing material released to the medium isnot known at present, and we shall refer to it in this discus-sion as "lipoteichoic acid-like material" (LTA-1 for brevity'ssake).How penicillin induces the release of LTA-1 material is
not clear at the moment. It is important to note, however,that all cell wall inhibitors tested-irrespective of their exactsite of action-cause release of LTA-1. Since inhibition ofeven the earliest step in cell wall synthesis (by fosfonomycin)causes LTA-1 release, it seems unlikely that biosynthetic in-termediates of the Park-peptide type are mediators in thiseffect. Possibly, the interruption of the flow of biosyntheticprecursors might lead to the accumulation of Glycosyl Car-rier Lipid (10) and/or the lipoteichoic acid-like carrier de-scribed by Glaser and his associates (11). Such carrier, "un-charged" with the biosynthetic building blocks of cell wall,may play a regulatory role in the labilization of the inhibi-tor-autolysin complex. This possibility is presently beingtested. Alternatively, autolysin triggering by cell wall inhibi-tors may have a more complex mechanism. Whatever themechanism of this effect is, it seems clear that the site atwhich a particular inhibitor interrupts cell wall synthesis isrelatively unimportant from the point of view of the eventu-al lysis of the cells.The experiments described in this paper also suggest a
new interpretation for the role of protein synthesis in peni-cillin-induced lysis (12-14). The penicillin-induced autolysinsensitivity of enzyme-defective pneumococci is blocked bychloramphenicol in spite of the ample supply of exogenousautolytic enzyme in the medium. Furthermore, enzyme-loaded mutant pneumococci (i.e., mutant cells into whichthe missing autolysin was reintroduced experimentally) areprotected against penicillin lysis by inhibition of protein syn-thesis*. These results indicate that the currently widespreadbelief, namely, that chloramphenicol protects bacteria fromlysis by lowering the concentration of autolysin at the cell
surface, is incorrect.,The experiments described here and inan accompanying communication* clearly show that inhibi-tion of protein synthesis interferes with the in situ activityof the autolysin. Inhibition of the activity of the streptococ-cal muramidase by chloramphenicol has been suggested ear-lier by several lines of evidence (15). Our results fully con-firm this suggestion. The mechanism by which CAP inter-feres with autolysin activity is not known at present. How-ever, the substantial reduction by CAP of the penicillin-in-duced LTA-1 release hints that inhibitors of protein synthe-sis may inhibit in situ autolytic activity via modulation ofthe metabolism of LTA.The three penicillin-induced phenomena described in this
paper (growth inhibition, sensitization to exogenous autolys-in, and leakage of LTA-like material) occur even at the low-est dose of drug treatment that causes lysis in the wild-typecells. Furthermore, these phenomena are all transient, sinceupon addition of penicillinase the penicillin-treated mutantcultures can resume growth (Fig. 3). These facts indicatethat penicillin inhibits the same enzymatic target reaction inboth the wild type and in the autolysin defective cells andthat this inhibition itself is not sufficient to cause cellulardisintegration. The irreversible effects of penicillin (lysis-and, to some extent, loss of viability) seem to be the conse-quences of the triggering of the activity of autolytic ami-dase.
It will be important to test the validity of these suggestionsin other species of bacteria. it is possible that a better under-standing of the in vio controls of autolytic enzymes mayyield ways to improve the chemotherapeutic efficacy ofbacteriolytic antibiotics.
This investigation has been supported by a grant from the Na-tional Institutes of Health.
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