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 in defective mutant." Most of the methods used in these in- defective mutant pneumococcus we have observed three vestigations have been described in detail in earlier publica- novel phenomena: (z) Growth of the mutant cultures is inhib- ited by the same concentrations of penicillin that induce tions. These include the composition of growth media and lysis in the wild type. (ii) Mutant bacteria treated with the monitoring growth by nephelometry (4), preparation of cell minimum growth inhibitory concentration of penicillin will walls (5), autolytic enzyme (6), and pneumococcal F-antigen lyse upon the addition of wild-type autolysin to the growth (7), and the assay of autolysin activity (8), "Loading" of the medium. Chloramphenicol and other inhibitors of protein mutant cells with wild-type autolysin was performed in the synthesis protect the cells against lysis by exogenous enzyme. following manner: Sensitivity of the cells to exogenous autolysin requires treat- ment with penicillin or other inhibitors of cell wall synthesis Fifty microliters of highly purified autolysin (containing (e.g., D-cycloserine or fosfonomycin) since exogenous autolys- 3.5 jg of protein) was added to 1 ml of autolysin defective in alone has no effect on bacterial growth. (ii) Treatment pneumococci (2 X 107 to 2 X 108 viable units/ml) and incu- with penicillin (or other inhibitors of cell wal synthesis) bated at 370 for 30 min. The cells were next filtered (Milli- causes the escape into the medium of a choline-containing pore, 0.45 Mm), washed-on the filter-three times with 2 macromolecule that has properties suggesting that it contains ml of warm (370) growth medium, and resuspended in pneumococcal lipoteichoic acid (Forssman antigen). Each one of these three phenomena (growth inhibition, sensitiza- growth medium. No significant amounts of enzyme seem to tion to exogenous autolysin, and leakage of lipoteichoic acid) escape to the medium during subsequent exponential shows the same dose response as that of the penicillin-in- growth of the enzyme-loaded bacteria. Detailed studies of duced lysis of wild-type pneumococci. this system will be described elsewhere*. On the basis of these findings we propose a new hypothe- All chemicals were of reagent grade. 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 RESULTS the endogenous complex of an autolysin inhibitor (lipo Effects of penicillin on autolysin defective teichoic acid) and autolytic enzyme. Escape of lipoteichoic pneumococci acid-like material to the growth medium is a consequence of this labilization. Chloramphenicol protects bacteria against The use of a mutant pneumococcus defective in the major penicillin-induced lysis by interfering with the activity of the cellular autolytic enzyme (an N-acetyl-muramyl-L-alanine autolytic enzyme, rather than by depleting the concentration amidase) has allowed us to identify in the drug-treated bac- of the enzyme at the cell surface. teria three penicillin-induced effects that appear to be of general significance for the mode of action of bacteriolytic The rapid loss of viability and cellular lysis of pneumococci antibiotics. treated with penicillin and other cell wall inhibitory bacteri- Bacteriostatic Effect. Unlike the wild-type cells, which cidal drugs is known to require the activity of the pneumo- are lysed by penicillin, growing cultures of the mutant bac- coccal autolytic enzyme (an N-acetylmuramyl-L-alanine teria respond to the addition of penicillin by inhibition of amidase) (1, 2). However, the mechanism by which inhibi- growth (1). This bacteriostatic response has exactly the same tion of wall synthesis is coupled to the "suicidal" activity of drug concentration dependence as the lytic response of wild- this enzyme is not known. In this communication we report type cells (Fig. 1). observations that bear on this question and we propose a Sensitization to Externally Added Autolytic Enzyme. new hypothesis for the mechanism by which penicillin and Addition of wild-type autolysin preparation to the growth other lytic antibiotics cause destruction of the bacterial cell. medium of penicillin-inhibited mutant bacteria makes these cells lyse (Fig. 2). Several details of this observation are im- MATERIALS AND METHODS portant. (i) Penicillin treatment is essential for the sensitiza- An autolysin defective mutant derivative of the R36A strain tion of the cells to the enzyme, since autolysin without anti- of Diplococcus pneumoniae (3) was used as the source of biotic has no effect on the bacterial growth. (ii) Fig. 2 shows DNA with which the autolysin defect was introduced into that the doses of penicillin treatment needed for sensitiza- competent cells of the parent (wild-type) strain via genetic tion are the same as the doses required for inhibition of transformation. A culture of the transformant was kindly growth (in the mutant) or for lysis (in the wild type). (ii) given to us by Dr. S. Lacks (Brookhaven National Laborato- Autolysin and penicillin need not be present at the same ry), and cultures of these cells will be referred to as "autolys- * A. Tomasz and S. Waks, Biochem. Biophys. Res. Commun. Abbreviations: CAP, chloramphenicol; LTA, lipoteichoic acid. (1975), in press. 4162 Downloaded by guest on September 28, 2021 Microbiology: Tomasz and Waks Proc. Nat, Avad. Sci. USA 72 (1975) 4163 2 e . -* * 0.03 a100 0.04 CD 001 50 I 30_ 10 1 2 3 4 5 HOURS FIG. 1. Bacteriostatic effect of penicillin in an autolysin defec- 400 tive pneumococcus. Exponentially growing cultures received peni- MINTES cillin G at the concentrations indicated; growth was monitored by 2. of nephelometry (Coleman FIG. Sensitlzution autolysin defective pneumococci to ex- Nephelometer). Numbers indicate the ogenous wild-type autolysin. Exponentially growing cultures re- concentrations of penicillin (in U/ml). ceived penicillin at the 60th min of growth and autolysin (109 cell equivalents per ml of medium) at the times lndicated by the ar- time. The bacteria could be sensitized to the enzyme by the rows. Growth was followed by nephelometry. Numbers indicate following sequence of treatments: the penicillin concentrationst 0.1 (1), 0.05 (2), 0.04 (3), 0.03 (4), exposure to penicillin (0.1 and 0.02 (5) U/ml. Tube 6 received no drug; tube 7 received only unit/ml for 60 min), treatment with penicillinase (Neutra- autolysin. pen, Rikers Labs; 10 units/ml for 5 min) followed by the ad- dition of autolysin to the medium. Such bacteria lyse (Fig. parallel with the addition of penicillin prevents lysis. Lysis is 3). (iv) Sensitization is transient: after removal of penicillin also prevented by the addition of pneumococcal lipoteichoic (by penicillinase) bacteria recover their normal autolysin re- acid (Forssman antigen) to the medium (Fig. 4), 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: first CAP, 5 min later penicillin. (vi) Penicillin as a sensitizing 1000 7 agent can be replaced by other drugs, such as: vancomycin (50 jig/ml), cycloserine (50 jig/ml) (1), /3-chloro-D-alanine 2 (500 jig/ml), fosfonomycin (50 ug/ml) (1), cephalothin (1 tg/ml), and oxacyllin (1 ,ug/ml) (2, *). The common feature of these treatments is that they all interfere with cell wall biosynthesis. 3 Mechanism of sensitization to autolysin It 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 bacterial surface. However, this does not seem to be the case, since, by appropriate techniques, the autolysin molecules can be "ad- sorbed" into the surface of live, mutant bacteria prior to the treatment of the cells with the sensitizing agents. The auto- lysin, experimentally introduced into the cells, is retained in cell-bound form through several cell generations. 10 1 (The success of this "enzyme replacement therapy" is O 1 2 3 4 5 6 clearly indicated by the fact that these reconstituted mutant HOURS cells "revert" immediately to the wild phenotype in proper- FIG. 3. Sequential treatment of autolysin defective pneumo- ties that are associated with the in vivo activity of the auto- cocci with penicillin and autolysin. An exponentially growing cul- lytic amidase. ture was treated~~~~~~~~~~4with penicillin (0.1 U/ml, added at the time indi- Details of this system will be described else- where*.) Addition of penicillin (either at the minimum in- cated by the solid arrow) for 60 min. After this treatment, the cul- hibitory concentration, i.e., ture was divided into three portions: one was left to incubate with at 0.02 unit/ml, or at higher penicillin (3), the other two (2 and 4) received penicillinase (10 concentrations) to such "enzyme-loaded" bacteria (either units/ml for 5 min; time of addition indicated by solid arrow). Cul- immediately after enzyme adsorption or even after growth ture 4 received autolysin (109 cell equivalent units/ml of medium) for one generation) results in cellular lysis with a kinetics immediately after the penicillinase addition, while culture 2 was comparable to that of the wild type. Other bacteriolytic an- allowed to incubate for about 2.5 hr before addition of autolysin. tibiotics (oxacyllin, 1 gg/ml; cephalothin, 1 sg/ml; vancom- (Addition of autolysin is indicated by the winged arrows.) A con- ycin, 50 trol culture (1) received no additions at all.