Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Eur. J. Biochem. 151,475-483 (1985) (c"l FEBS 1985

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin Russell WILLIAMSON and Alexander TOMASZ The Rockefeller University, New York City, New York

(Received April 2/June 13, 1985) - EJB 850349

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to ['H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2 b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.

The currently dominant idea concerning the mode of ac- tion of b-lactam antibiotics assumes that these drug molecules act as active-site-directed acylating agents forming en- zymatically inactive covalent complexes with bacterial enzymes that catalyze certain terminal reactions in cell wall biosynthesis [I]. It is assumed that the formation of such inactive complexes would reduce the rate of peptidoglycan synthesis and thereby limit bacterial growth. It would follow therefore that the effectiveness of b-lactam antibiotics as inhibitors of bacterial growth would be a function of the effectiveness of these compounds as inhibitors of cell wall synthetic enzymes. Numerous attempts have been described to establish some quantitative relationship between the minimum concentration of an antibiotic which affects bacterial growth (minimal inhibitory concentration, MIC) and the concentration needed to saturate (inactivate) some fraction (e .g 50% or 90%) of one or more of the penicillin-binding proteins (PBPs) of the same bacterium. The rate of formation and the stability of penicilloyl- enzyme complexes can be quantified with the simple technique of labelling with radioactive benzylpenicillin and fluorography of the PBPs [2]. The

Correspondence to A. Tomasz, The Rockefeller University, 1230

Ahhrcviatiarzs. MIC. minimal inhibitory concentration; PBP, York Avenue, New York City, New York, USA 10021

penicillin-binding protein ; CI,AcOH, trichloroacetic acid.

strategy followed in most of these studies was to examine, in vifro, the degree of binding of a particular p-lactam antibiotic to various PBPs in membrane preparations during short exposures at or close to the MIC.

This type of experiment has produced reasonable cor- relations between the binding of antibiotics to three of the higher molecular weight PBPs of Escherichia coli and the growth inhibitory effectiveness of the same compounds in this bacterium [3 - 51. On the other hand, studies with pneumococci and some other bacteria have failed to establish such a relationship; in these bacteria all the PBPs showed considerable antibiotic binding at the biologically effective concentrations of the drugs [6]. Thus, even in the cases were tentative evidence for a correlation exists, it is not known if one or several PBPs must become completely or partially saturated by the antibiotic before slow-down or inhibition of peptidoglycan incorporation ensue which, presumably, would lead to inhibition of growth. The failure to establish such a correlation may be a reflection of the fact that under the conditions of experimental or therapeutic use of benzylpenicillin, e. g. in the determination of MIC, growing cultures of bacteria are exposed to the antibiotic for 18 - 24 h. Even if drug stability and quick permeation of the antibiotic through the cell wall can be assumed, as is the case in pneumococci, the rate of inactivation (acylation) of PBPs would have to compete with at least two processes supplying active PBPs: the

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biosynthesis of new PBPs and the deacylation of the PBP- antibiotic complexes. With these thoughts in mind, we have performed experiments to identify a possible correlation between the extent of saturation of PBPs and the degree of inhibition of peptidoglycan synthesis, protein synthesis and bacterial growth in a more dynamic experimental design that involved exposure in vivo of growing Streptococcus pneu- moniue cultures to various concentrations of [3H]benzylpenicillin over extended periods of time.

MATERIALS AND METHODS

Bacterial strains and growth conditiom

S. pizrumoniue R6, a derivative of the Rockefeller Univer- sity strain R36A, and lyt 4-4, an autolytic deficient trans- formant of R 6 constructed by R. Z. Jiang of this laboratory, were used throughout these studies. DNA from an autolysin defective mutant of pneumococcus was used to transform the lyt marker into the lysis-prone R 6 cells and colonies showing autolytic defect were selected. Extract of the lyt 4-4 transfor- mants contained only about 0.1% of the specific autolysin activity of the recipient (R6) cells and these bacteria showed no detectable cell wall degradation during treatment with penicillin. Bacteria were grown without aeration at 37°C in a chemically defined medium [7] with glucose (0.2Y0 w/v), but without lysine or phenylalanine. Growth was monitored in a Coleman nephelometer.

Anlibiotics and reagents

Benzylpenicillin (referred to hereinafter as penicillin) was obtained from Eli Lilly & Co., Indianapolis, IN, and p[,H]- benzylpenicillin, ethylpiperidinium salt (26 Ci/mmol) was obtained from Merck, Sharp & Dohme, Rahway, NJ. Trypsin was purchased from Worthington Biochemical Corp., Freehold, NJ, and ~-['~C]lysine (321 Ci/mol), ~-[4,5-~H]lysine (76.9 Ci/mmol), ~-['~C]phenylalanine (514 Ci/mol), and [methy/-3H]choline chloride (82 Ci/mmol) were purchased from New England Nuclear Corp., Boston, MA. M I - muramidase was the generous gift of Dr K. Yokogawa (Dainippon Pharmaceutical Co., Ltd, Suita/Osaka, Japan) and was purified to homogeneity.

Determinations o j the synthesis of peptidoglycan and protein

The effects of penicillin on the rates of synthesis of peptidoglycan and protein were measured by a pulse-labelling technique. Exponential-phase organisms (1400 ml) were grown to a culture density of approximately 2 x lo7 colony- forming units/ml. At this point, 200-ml samples were dis- tributed into prewanned flasks containing different amounts of [3H]penicillin, and at various times thereafter samples were withdrawn for labelling procedures. Pairs of duplicate samples (0.5 ml) were immediately mixed with either 5 pI of [14C]lysine (0.5 pCi, 0.24 pg) and incubated for 3 min or 10 p1 of [14C]phenylalanine (1.0 pCi, 0.32 pg) and incubated for 5 min, before the addition of 125 p1 of ice-cold trichloroacetic acid (CI,AcOH, So%), followed by thorough mixing and storage in ice. Using these conditions, the incorporation of label was linear in each case over the time period used. Samples labelled with ['4C]lysine were heated at 100°C for 30 min, and then the C1,AcOH-insoluble material was collected on glass fiber filters (Whatman GF/A, 2.4 cm diam- eter), and washed extensively with ice-cold 5% C1,AcOH and distilled water. To determine the amount of label in

peptidoglycan, the filters were transferred to scintillation vials (1.3 x 4.2 cm) and 3 ml of 100 rnM sodium phosphate buffer, pH 7.8, containing 0.5 mM CaCl, and 100 pg/ml of trypsin was added to each, followed by incubation at 31' C for 18 h [S]. The buffer was removed by filtration over a second filter, and the filters washed extensively with distilled water and then 95% ethanol, before being dried at 100°C for 60 min. Samples labelled with [ ''C]phenylalanine were collected on glass fiber filters and washed extensively with ice-cold 5% Cl,AcOH, distilled water and 95% ethanol before being dried. The amounts of radioactivity on the filters were measured in 4-ml volumes of a toluene-based scintillation fluid, and counted in a Nuclear Chicago Mark 11 spectrometer.

Labelling, sepuration and detection of penicillin-binding proteins

At the same time as samples were taken to determine the rates of synthesis of peptidoglycan and protein, additional quadruplicate samples (1 .O ml) were taken and mixed with 10 pl of Triton X-100 (loo/,, w/v) to stop further synthesis of PBPs. One pair of the samples (A) also received 5 p1 of penicillin (200 mg/ml) and was placed on ice, and the other pair of samples (B) received 10 p1 of [3H]penicillin (100 pg/ ml) and was incubated for 10 min at 37°C to fully saturate the PBPs, before addition of the unlabelled penicillin. The bacteria were collected by centrifugation (4500 x g for 10 min at 2 T ) , the supernatants removed by aspiration, and the organisms resuspended in 50 pl of 50 mM sodium phosphate buffer, pH 7.0, containing 5 p1 of a crude R 6 pneumococcal autolytic enzyme preparation [9] (4.4 mg/ml, 12 units/mg), followed by incubation at 37°C which resulted in coinplcte lysis of the bacteria within 5 min. The lysates were then sub- jected to discontinuous polyacrylamide slab gel electrophoresis, stained, and the PBPs detected by fluorography, as described earlier [lo]. In quantifying band densities on the fluorograms, the results were corrected for any variation in band widths.

The percentage saturation was calculated as the amount of [3H]penicillin bound in sample A divided by the amount bound in sample B, multiplied by 100. The total amount of non-acylated (free) PBP available in a culture after a given period of antibiotic treatment was estimated as the numerical difference between the total and the bound forms (B-A), expressed in arbitrary density units. The A and B samples corresponding to the same sampling times were run next to each other on the gels.

RESULTS

Incorporation of peptidoglycun

The method used for labelling the peptidoglycan of the pneumococci gave recoveries of about 35Y0 of the [3H]lysine in cold-C1,AcOH-insoluble material as being resistant to the subsequent hot C1,AcOH extraction and trypsin treatment, and some 97% of this was solubilized by the M 1 muramidase. In contrast, the recovery of [3H]phenylalanine-containing material after the hot C13AcOH and trypsin treatments was only 3% of that insoluble in cold CI,AcOH, indicating removal of most protein. Extensive loss (> 99%) of [3H]choline-containing material was also obtained with the method utilized. The use of trypsin was necessary since pronase treatment caused almost coiiiplete solubilization of the [3H]lysine- labelled material, and this was probably due to lysozyme contamination [ l l ] . The decrease of the rate of [3H]lysine

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Fig. 1. Eiyect of /3H]henzylpenicillin on growth and the synthesis of peptidoglycan und protein. The radioactive antibiotic was added at 0 min, at concentrations expressed as multiples of the MIC (8 ngiml). Control (0 ) ; 1 x MIC (0); 2 x MIC (A); 4 x MIC ( A ) ; 8 x MIC (+); and 16 x MIC (0). The effects on turbidity, and the rate of synthesis of peptidoglycan and of protein wcrc mcasurcd. Each ratc of synthesis was determined in duplicate samples by pulse-labelling at intervals, as described in Materials and Methods

incorporation in this system by the addition of penicillin suggests that, in analogy with some other bacterial systems [12- 141, transpeptidation may be a primary mechanism for the attachment of newly synthesized material to the pre- existing peptidoglycan of pneumococci.

Analysis of pep t idogly can t urno ver during growth

Since previous examinations of wall turnover in pneumococci had utilized either [3H]choline-labelled teichoic acid or peptidoglycan labelled with either ['4C]glutamic acid or [14C]lysine during growth in ethanolamine-containing medium [IS, 16a], it was necessary to establish that lysine-labelled peptidoglycan synthesized in the usual choline-containing medium was also conserved and that release of cell wall label did not complicate the interpretation of peptidoglycan synthesis experiments. Exponential phase cultures of wild type, autolysis prone R 6 and the autolysis deficient strain lyt 4-4 were pulse labeled (3 min) with [3H]lysine (1 pCi/ml, 0.19 ng/ ml) and subsequently grown with an excess of unlabelled lysine (225 pg/ml) added immediately after the pulse. This addition of lysine had no effect on the growth rates of the cultures, and the amount added was that usually used in the defined medium. During the chase period there was no further increase in the amount of [3H]lysine incorporated into either cold-C13AcOH-insoluble material or peptidoglycan (data not shown), and there was also no loss of radioactivity from either of these fractions from both strains during the subsequent three generations of growth. Thus, there was no measurable turnover of the peptidoglycan during normal growth. There was also no release of radioactivity from similarly labelled cultures of lyt 4-4 during treatment with 10 times the MIC of penicillin for 3 h (data not shown).

EfJect of (3HJpenicillin on the synthesis of peptidoglycan und protein in growing organisms

The generation time of the control culture was calculated to be 51 min by analysis of the turbidity increase and 48 min

by analysis of the rates of incorporation of [14C]lysine into peptidoglycan (Fig. l), indicating a good correlation between these parameters. However, the doubling time calculated from the increase in rate of incorporation of ['4C]phenylalanine was 72 min (Fig. l), and the reason for this difference is not known.

Addition of penicillin at the MIC or at higher concentrations to the exponentially growing cultures of the autolysis- defective pneumococcus did not result in an instantaneous inhibition of growth. Complete cessation in the turbidity increase of the antibiotic-treated cultures occurred only after prolonged period of residual growth, the length of which was roughly inversely related to the concentration of penicillin (expressed in multiples of the MIC). Even upon the addition of 16 times the MIC of penicillin, culture turbidity increased normally for about 30min. In this culture a drop of approximately 30% in the rate of peptidoglycan synthesis was observed after 15 min, and a 20% drop in the rate of protein synthesis was noted after 30 min. At intermediate concentrations of penicillin, the inhibition of peptidoglycan synthesis always preceded inhibition of protein synthesis.

In a second experiment using a narrower range of penicillin concentrations, the onset of inhibition of peptidoglycan synthesis was also always observed before inhibition of growth measured turbidimetrically (Fig. 2).

It is important to note that in cultures treated with up to eight times the MIC of penicillin, incorporation of peptidoglycan continued for prolonged time periods with virtually the same rates as was seen at the time of onset of growth inhibition. For example, in the culture treated with six times the MIC of penicillin (Fig.2), growth stopped at about 90 - 110 min after drug addition and peptidoglycan incorporation continued for at least another hour at about 90% of the rate observed at the time of penicillin addition. On the other hand, 16 times the MIC of penicillin (Fig.1) resulted in a rapid decline in the rate of peptidoglycan incorporation dropping to about 10% of the initial rate in 150 min. I n this culture the rate of protein synthesis also declined to about 30% of the initial rate.

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Peptidoglycm synthesis 600 -/

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Fig. 2. Effzct crf'(3H]ben~~~lpeniciNin on growth and the synthesis of peptidoglycan. The details of the experiment are the same as those Fig.1. Control (0) ; 1 xMIC (0); 2 x MIC (A); 3 xMIC ( A ) ; 4 x MIC (4); and 6 x MIC (0)

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Fig. 3 . Binding oj'[3H]benz,vlpenicillin lo a PBP during growth. The amount o f radioactive antibiotic bound to PBP 1 a during growth of bacteria in the antibiotic a t 1 x MIC ( A ) and the total amount of this PBP availablc to a saturating concentration of the [3H]benzylpenicillin (A) were determined in duplicate samples of each by quantificationofthcfluorograms.Theresu1tsarefromtheexperiment shown in Fig. 2

Fig. 4. Amounts of PBPs l a (lejt panel) and 2h (righr punel) not bound to the antibiotic in organisms growing in difjierent concentrations of [3H]benzy/peni~illin. The data from the experiment shown in Fig. 2 was obtained by subtracting the amount bound during growth from the total amount bound under saturating conditions. 1 x MIC (0 ) ; 2 x M I C (0); 3 x M I C (A); 4 x M I C ( A ) ; and 6 x M I C (4 )

Interact ion of' ['H]pcnicillin with P RPs during growth

The cultures incubated with various concentrations of [3H]penicillin in Figs 1 and 2 werc also evaluated to determine the rates and degrees of acylation of the five pneumococcal PBPs as the cultures approached inhibition of growth. Two kinds of determinations were made a t each time point. One pair of samples of the bacterial culture was treated with Triton X-100, to stop synthesis ofPBPs instantly. These samples were then exposed to a saturating dose of [3H]penicillin to label and estimate the total amounts of PBP present in the cultures at various time points throughout the experiment (sample B, see Materials and Methods). The presence of the detergent had no effect on the total amount of radioactive antibiotic that was able to bind to the PBPs under saturating conditions.

At each experimental time point, a second pair of samples was also removed from the cultures and was processed directly Tor dodecyl sulfate/polyacrylainide gel electrophoresis and fluorography, in order to quantify the amount of the various PBPs that had become acylated during incubation of the growing pneumococci with [3H]penicillin (sample A, see Ma-

terials and Methods). The quantitative results of this proce- dure for PBP 1 a are shown in Fig. 3. The total amounts of PBP l a (solid triangles) and the amounts of PBP l a that had become acylated during the exposure of the culture to penicillin at the MIC (open triangles) are plotted against sampling times. The total amounts of PBP l a closely parallel the growth curve (see Fig. 2). This was true for all the cultures shown in Figs 1 and 2 [17] and not only for PBP l a but for the total amounts of all the other PBPs as well (not documented). The second curve (open triangles) in Fig. 3 shows the rate of accuinulation of acylated PBP 1 a in the same experiment. It took at least 90 min for this PBP to reach maximum saturation under these conditions. The difference between the densitometric values obtained in assays B and A (B ~ A) represents the amount of free PBP 1 a, i . e. the fraction of the protein not bound by the antibiotic and, thus, presumably available for the catalysis of some reaction in wall synthesis. Comparison of the curve for the total amounts of PBP 1 a (solid triangles in Fig. 3) with the curve showing the amounts of free PBP 1 a in the same culture (solid circles, left panel of Fig.4), clearly shows that most of PBP 1 a synthesized

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’-1 lXMIC

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Fig. 5. Satirrution ~ M T V P S qf PBPs in orgunisms growing in wrious concentrations of [3HJhenz~lpenicillin. The data was calculated from the experiment shown in Fig.2 as the fraction (%) of total PBPs bound during growth in thc presence of the particular concentration of [3H]penicillin. The values for each PBP are shown a t the indicated MIC multiples

Table 1 . Saturution of PBPs ut vurious levels yf’inhibition ojpeptidoglycun synthesis Inhibition is expressed as the rate of synthesis as a percentage of that in untreated control bacteria at the same time points. The data was obtained from Figs 2 and 5. The length of incubation is the time point at which the given level of inhibition was estimated to occur, and thc numbcr of entries under this column represents the number of sets of individual PBP assays. Means _+ SD for saturation were calculated from the valucs for each PBP a t the various time points at each MIC multiplc for the defined levels of inhibition

Inhibition Length ofincubation with MIC multiple of Mean saturation of PBP

1 2 3 4 6 l a I h 2a 2 b 3

S 10 20 2s 33 so

16 52 21 11 6 9 k 5 4 5 + 2 3 5 k 6 2 5 + 4 1 1 & 4 92 66 32 20 1 2 k 4 51 + 2 41 i 5 2 S + 6 7 9 k 4

126 91 50 33 1 8 + 2 5 9 k 3 4 9 + 5 2 8 k S 8 5 + 1 - 101 58 39 19k1 6 2 + 3 S 4 k S 3 1 k 4 8 1 + 1 - 126 13 51 81 + I 66,4 5 9 k 4 3 4 k 2 9 0 k 1 - - 104 13 8 5 & 1 7 4 + 1 6 8 + 4 3 8 k 4 9 3 + 1

by the cells was quickly trapped by the low concentration ( x MIC) of [3H]penicillin present in the medium. In contrast, considerable amounts of the low affinity PBP 2 b produced by cultures incubated with [3H]penicillin at the MIC remained free and accumulated in the culture (solid circles, right panel of Fig. 4). It required exposure to six times the MIC to prevent accumulation of the non-acylated form of this PBP. The different behavior of PBPs 1 a and 2 b parallels the very different affinities of these two binding proteins for the antibiotic.

The data generated by the double sampling of PBPs was also used to calculate the percentage saturation of each PBP in the bacterial cultures and Fig. 5 shows the compilation of such data for each PBP in cultures treated with one, four and six times the MIC of penicillin for various time periods (data from experiment shown in Fig. 2 ) . The amount of penicillin bound to all PBPs increased throughout the experiment, and the extent of saturation corresponded to the order of relative affinities of the individual PBPs for penicillin: 3 > 1 a > 1 b > 2a > 2b. This result is in agreement with previous results [6, 101.

Comparison of the kinetics of penicillin binding (Fig. 5 ) with the data on the minimum antibiotic dose needed to bring about cessation of culture growth (Fig.2) did not make it possible to identify a single PBP as a likely ‘killing’ target. For example, in the culture treated with six times the MIC of

penicillin, all the PBPs reached at least 80% saturation by the time the culture stopped growing (90- 110 min). On the other hand, in the culture incubating with four times the MIC of the antibiotic, only the PBPs with the highest affinity (PBPs l a and 3) reached over 80% saturation, at the time when growth stopped (120- IS0 min); about 70% of PBPs 1 b and 2a and only about 30% of PBP 2 b were found to be acylated at the same time. In the culture that was exposed to penicillin at the MIC for 2 h (at which time the culture had just barely begun showing signs of slower growth), about 80% of PBP 3, 70% of PBP 1 a, and 20% of PBP 2 b were already acylated.

Table 1 compares in another way the PBP saturation data generated in the experiments in Fig. 2. The table shows what fraction of each PBP was bound by the antibiotic at defined levels of inhibition (5 - 50%) of peptidoglycan incorporation. As with the inhibition of growth, the incubation times needed to reach a certain level of inhibition were roughly inversely related to the antibiotic concentration. For example, to reduce the rate of peptidoglycan incorporation by 20% required about 120min of treatment with two times the MIC and 90 min, SO min and 30 min incubation with three, four and six times the MIC of penicillin, respectively. When the penicillin- bound (acylated) fraction of the various PBPs was determined in such cultures, each PBP exhibited a characteristic degree of saturation that was remarkably constant for a given degree

Table 2. Interaction c?f/3H]benzylpeni~iilin with the PBPs ut the onset qf‘inhibition of peptidoglycan synthesi.v The time of threshold dose is the time at which the inhibition of synthesis of peptidoglycan was first apparent. The data was obtained from Figs 2 and 5. The inhibition of synthesis is expressed as a percentage of synthesis in the untreated control culture at thc same time point

I

~

Anti biotic Time of Inhibition Saturation of PBP ~~ ~ -~ threshold _____

concentration MIC dose l a I b 2a 2 b 3 multiple

8 I 110 4 7 71 37 43 22 77 16 2 75 4.8 76 40 35 23 82 24 3 45 3.5 66 41 35 29 78 32 4 15 2.5 54 36 23 21 64 49 6 1s 5 7 60 42 45 19 58

Mean 2 SD 4.2 & 1.2 65 + 9 39 f 3 36 f 9 2 3 + 4 72 f 10 _ _ _ _ _ _ _ _ _ _ ~~ ~~~

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Fig. 6. l?ffi>c.r of long-ferm expos~trc~ of’ growing bacteria fo /3H]henzq.lpenicillin. The radioactive antibiotic was added a t the M IC (8 ngiml) and at three limes the MIC (24 ng/mlj at 0 min. The effccts on turbidity (Aj, and the perccntage saturation of PBl’s at the MIC (R) and (hi-ee times the MIC (C) WCI-e measured, and the arrows indicate the times of cessation of growth. PBPs 2a and 2 b were not sufficicntly rcsolved in this expcriment, and PRP 1 b was too faint for accurate measurement

of peptidoglycan inhibition. Within this ( 5 - 50%) rangc of inhibition ofpeptidoglycan incorporation, only the saturation of PBP 3 approached 100% (93% saturation at 50% peptidoglycan inhibition); the rest of the PBPs were acylated by penicillin in proportion to their relative affinities for the antibiotic. The data did not show a direct correlation between the extent of inhibition and the degree of acylation of aiiy single PBP or group of PBPs.

Further examination of the data for Fig.2 showed that for each concentration of penicillin there was an ‘inhibition point’, which was when the onset of inhibition of peptidoglycan synthesis occurred. At these ‘inhibition points’ each PBP had reached a characteristic percentage saturation, and this was essentially independent of the antibiotic concentration used (Table 2). The average amount of inhibition of peptidoglycan synthesis was only about 4% at these time points, but it was clear that each PBP had bound relative- ly substantial amounts of antibiotic, which until these time points had not been sufficient to causc the inhibition of incor- pora tion.

In the experiments illustrated in Fig. 6 the degrees of PBP saturation were also determined after exposure of cultures to one and three times the MIC of penicillin for prolonged incubation times (30 h), similar to the incubation times used in the MIC determinations. At both of these pcnicillin concentrations the percentage saturation of PBPs continued to increase beyond the time of complete growth inhibition (see arrows in Fig. 6). Moreover, none of the PBPs reached 100% saturation even after 30 h of incubation with the [‘HI- penicillin.

The bacterial cultures incubated with various concentrations of penicillin (Fig. 2) continued to produce peptidoglycan at various rates, depending on the concentration and length of exposure to the antibiotic. This production of peptidoglycan must be the product of the catalytic activity of one or more of the PBPs that remained free (i.e. unbound) by the antibiotic. The data generated by our experiments allowed us to test if there might by a correlation between the amount of peptidoglycan produced by these bacteria and the amounts of unacylated PBPs. The amount of Il4C]lysine

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Fig. I. Correlation qf’pc~ptidoglycan synthesis with the availability of’ unbound (antibiotic-free) PBP 1 u , 2u, und 2h. The symbols arc thc same as those in Fig.4

incorporated into peptidoglycan per unit of unbound (antibiotic-free) PBP was calculated for each PBP at the various time points throughout the experiment with the five concentrations of penicillin. As shown in Fig.7, the ratio of the amount of synthesis per arbitrary unit of available PBP increased (up to sixfold) both with time and antibiotic concentration for PBPs 1 a and 2a, and the ratio increased up to eightfold with PBPs 1 b and 3 (data not shown). However, an almost constant ratio was found for PBP 2b; the mean value at all time points at each concentration of antibiotic was 185 dpm per arbitrary unit of PBP unbound, with a standard deviation of 29.

DISCUSSION

Most of the data relating to the binding of /r-lactam antibiotics to PBPs has been presented as the concentration of the antibiotic required to half-saturate any particular PBP, measured cither directly or by the inhibition of subsequent binding by a radioactive antibiotic [3 - 51. Using this method some bacterial species were found [2, 18 - 201 in which only one of the several PBPs had sufficient affinity to bind the antibiotic at the MIC and this PBP was therefore considered as the ‘killing target’ of the antibiotic. In some other bacteria (e. g. the methicillin resistant Staphylococcus uureus 1211 and S. .faeciur?z [221), the converse situation was encountered: all PBPs, except one, were completely saturated by the antibiotic far below the MIC, suggesting that the physiologically important target in this case may be the PBP with the lowest drug affinity. In other bacterial species, including pneumococci, this approach did not allow the identification of any single PBP as the physiologically important target [6].

Even in the cases in which the antibiotic binding studies point to a single PBP as a killing target, one should use caution in the interpretation of data since the short (10min) drug exposures used in the PBP titrations may not reveal the inter- actions between I-lactam molecules and PBPs during prolonged (18-24 h) exposure to the antibiotics, as is done in the course of MIC determinations. In the growing cultures used in the physiological experiments, the amounts of active PBPs do not remain constant and it is possible, for instance, that a particular PBP may remain at 50% saturation during exposure to an antibiotic, but because the total amount of the PBP increases with growth, the physiological activity of the

PBP would also parallel bacterial growth. It should also be remembered that 50% saturation of a PBP need not necessar- ily involve a similar reduction in activity 1231. For these reasons, any apparent relationships observed between PBP half-saturation and MIC values may be fortuitous. Indeed, little correlation of antibiotic binding values with measures of concomitant inhibition of enzyme function in growing bacteria has been found [24-2261.

In order to avoid at least some of these problems we designed experiments in which growing cultures of lysis- defective pneumococci were incubated with various concentrations of [3H]penicillin for various time periods and determined the amounts of free and acylated PBPs as well as the effects of the antibiotic on bacterial growth and the rates of cell wall incorporation and protein synthesis. We are not aware of similar experiments in the published literature. Growing organisms [12, 24, 27, 281, chloramphenicol-treated bacteria [8, 29, 301, ether-treated cells [31 -341, wall-membrane preparations [16, 35, 361, and particulate fractions [25, 37, 381 have all been demonstrated to be capable of b-lactam antibiotic-sensitive synthetic reactions. However, in none of these cases were experiments reported on the correlation of inhibition of synthetic activity with direct measurement of the binding of the inhibitor to the enzymes (PBPs) involved in the process during the inhibition. The profound influence of continued PBP production during penicillin treatment on the course of the inhibitory effects of the antibiotic is clearly shown by the experiments described. Since in pneumococci penicillin does not selectively interfere with the synthesis of PBPs [17], the net rate of acylation of the various PBPs was primarily the function of their relative affinities for penicillin. In the case of the high affinity PBP 1 a, even brief exposure to the minimal growth inhibitory concentration of penicillin resulted in the rapid saturation of much of the newly made PBPs so that, for example. no net accumulation of free, unacylated PBP 1 a accompanied the almost eightfold increase in cellular mass during the residual growth in the presence of this concentration of penicillin (see Fig.4, left panel). In contrast, a substantial amount of the newly made low affinity PBP 2 b remained free and accumulated during the same time period of incubation (see Fig. 4, right panel). It has been estimated that pneumococci contain about 21 000 molecules of PBPs per cell, out of which the PBPs 1 a, 1 b, 2a, 2 b and 3 represent about 5200. 1050, 5700, 3500 and 5300 molecules, respectively [I71 and in the absence of penicillin presum-

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ably all of these molecules are available to catalyse steps in wall synthesis. In the pneumococcal culture exposed to penicillin at the MIC for 2.5 h, the number of free PBP molecules per cell differed greatly depending on the individual PBP. For PBPs 1 a - 3, the number of free PBP molecules per cell was estimated at 1300, 600, 3100,2700 and 950 molecules, respectively. Not only was more than half (60%) of all the PBPs trapped in penicilloyl complexes in such cells, but the relative amounts of free PBPs were completely different from those of the normally growing cell: over 80% of PBPs 1 a and 3 were acylated and thus, presumably, nonfunctional while most (80%) of PBP 2b remained free and in terms of number of free protein molecules, PBP 2b has become one of the major penicillin-binding proteins in the antibiotic-treated cell. Such a distortion in the relative amounts of cell wall synthetic enzymes (rather than the inhibition of a single PBP) may be the ultimate cause of the inhibition of cellular growth. In our studies we found no good correlation between saturation of any single PBP and inhibition of wall synthesis (measured by incorporation of lysine into peptidoglycan) or growth (measured as turbidity). In fact, the results clearly showed that defined levels of inhibition of synthesis did not correspond to similar percentages of saturation of any particular PBPs. Certainly. complete saturation of any or all PBPs was not required for the cessation of culture growth. Saturation of PBPs was observed to continue long past the time when growth has come to a halt. Conversely, pneumococcal cultures incubated with penicillin at the MIC showed normal rates of cell wall synthesis (and growth) for at least 2 h in spite of the fact that 20% of PBP 2b and as much as 75-85% of PBP 3 became bound by the antibiotic. Assuming that non-bound PBPs cannot increase their catalytic efficiencies, this result suggests that PBPs may be present in excess of the minimum required to maintain peptidoglycan synthesis at the normal rate. This may be yet another factor contributing to the lack of correlation between saturation data and the inhibition of peptidoglycan synthesis and growth.

During incubation with penicillin at various multiples of the MIC the growth of pneumococcal cultures began to slow down after exposures to approximately equal antibiotic doses, i. e. after time periods that were roughly inversely proportional to the concentration of the antibiotic (see Figs 1 and 2). We determined the degree of saturation of each PBP at these times of ‘threshold antibiotic dose’, i. e. when the antibiotic-treated cultures started showing the first symptoms of growth inhibition and found that each PBP has reached a characteristic degree of saturation which was not related to antibiotic concentration but to the antibiotic dose (i. e. concentration- x length of exposure).

We assume that upon exposure to the ‘threshold antibiotic dose’ PBPs reach a critical degree of saturation at which their individual or concerted function can no longer sustain the normal rate of cell wall synthesis. It is conceivable that in pneumococci several, if not all, PBPs perform functions essential for normal growth, and the antibacterial effects of penicillin may arise from a summation of the inhibition of several PBPs and/or from the abnormal relative concentrations of free PBPs in the penicillin-treated cells. This may be what is reflected by the consistent pattern of percentage PBP saturation detected at the ‘threshold dose points’ of our experiments. In support of this suggestion is the finding that acquisition of high level resistance to penicillin involves modification of four of the five pneumococcal PBPs [39]. It should be realized that the existence of a single ‘killing target’ has remained an unproven assumption in the field.

The intriguing correlation observed between the amount of PBP 2 b that was not acylated by penicillin (and therefore presumably enzymically active) and the amount of peptidoglycan synthesized during incubation with the antibiotic requires comment. The contribution of PBP 2b to the incorporation of new peptidoglycan in normal pneumococci is not known. However, it is conceivable that in the presence of penicillin, as the acylation of this PBP surpasses the critical threshold degree of saturation (about 25%), the cellular concentration of antibiotic-free PBP 2 b may become the rate- limiting step for peptidoglycan synthesis in penicillin-treated organisms.

As the pneumococcal cultures exposed to different concentrations of penicillin started to slow down in growth, the first observable effect was always on the rate of cell wall incorporation which was followed by a slow-down in protein synthesis. This order of effects is similar to those observed in S. vnutuns treated with penicillin [27, 401.

While exposure of pneumococci to high concentrations of penicillin (16 times the MIC) caused a rapid decline in the rate of peptidoglycan incorporation, cultures that have come to a complete halt in turbidity increase after exposure to penicillin at one to eight times the MIC have continued to incorporate peptidoglycan and produce protein at considerable rates (see Figs 1 and 2) for long time periods. The nature and site of incorporation of this material is not known. Some of the peptidoglycan made may be secreted into the surrounding medium [41]. Under these conditions inhibition of the growth of the autolysis-defective pneumococcus by penicillin cannot be explained by the decline in the cellular capacity to synthesize and incorporate peptidoglycan. Inhibition of cellular multiplication may be the consequence of a regulatory signal or it may be related to some structural anomaly of the cell wall produced by the abnormal complement of synthetic enzymes (PBPs) present in the penicillin-treated cells.

We are grateful to Drs T. J. Dougherty, L. Gutniann, M. Hitchcock, and S. Zighclboim-Daum, and A. Koller, for essential assistance. The stimulating discussions with Drs E. Tuomanen, T. J . Dougherty and D. Tipper are gratefully acknowledged. These in- vestigations have been supported by a grant (A1 16794) from the National Institutes of Health. US Public Health Service.

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Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin