FEMS Microbiology Letters 22 (1984) 301-305 301 Published by Elsevier

FEM 01722

Synthesis of penicillin-binding proteins in penicillin-treated Streptococcus pneumoniae

(Key words not submitted)

Russel l Wi l l i amson * and Alexande r T o m a s z **

Laboratory of Microbiology, The Rockefeller Unwersitv, New York, NY 10021, U.S.A.

1. SUMMARY

(Not submitted)

2. I N T R O D U C T I O N

The inhibition of growth of bacteria by fl-lactam antibiotics is dependent on the concentration of the antibiotic and the incubation time. After addi- tion of the antibiotic at low concentrations (in terms of low multiples of the minimum inhibitory concentration, MIC) pneumococcal cultures con- tinue to grow before the onset of inhibition; the length of their residual growth period is roughly inversely proportional to the antibiotic concentra- tion. One factor that may contribute to this delay in the inhibitory effect may be the continued synthesis of the penicillin binding proteins (PBPs). There have been several reports on the 'physio- logical' aspects of PBPs in growing bacteria or in organisms treated with a variety of different in- hibitors [1-6], and at least some reports suggested that penicillin may interfere with the synthesis of PBPs [4,5].

In this study we have examined the PBPs of S. pneumoniae growing in inhibitory concentrations

* Present address: Laboratoire de Microbiologie, Institut Bio- medical des Cordeliers, Paris 75270, France.

** To whom correspondence should be addressed.

of [3H]benzylpenicillin to detect possible changes in the amounts of PBPs. Our results indicate that PBPs make up a constant fraction of total pneu- mococcal protein. No change in the total or indi- vidual PBPs could be observed in different phases of growth nor in cultures exposed to various doses of penicillin.

3. MATERIALS A N D METHODS

S. pneumoniae lyt 4-4, an autolytic-deficient transformant of R6, was isolated by R.Z. Jiang of this laboratory. Pneumococci were grown without aeration at 37°C in a chemically defined medium [7] with glucose (0.2%, w / v ) but without lysine or phenylalanine, and growth was monitored with a Coleman nephelometer.

The degree of saturation and total amount of individual PBPs in S. pneumoniae exposed to vari- ous concentrations of [3H]benzylpenicillin (26 Ci /mmol , Merck, Sharp and Dohme, Rahway, N J) were determined essentially as described [15]. Samples of S. pneumoniae were saturated with an additional 1 jag of [3H]benzylpenicillin after addi- tion of Triton X-100. The bacteria were lysed by incubation with 5 /tl of a crude pneumococcal autolytic enzyme preparation [8] (4.4 mg /ml , 12 uni ts /mg).

The amounts of [3H]benzylpenicillin bound to S. pneumoniae undergoing residual growth in the

0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies

302

presence of the antibiotic were also determined in duplicate samples (0.5 ml) which were mixed with ice-cold trichloroacetic acid (TCA, 10% w/v , final concentration). The bacteria were collected on glass-fibre filters (Whatman, G F / A ) , washed ex- tensively with 5% TCA, distilled water, 95% ethanol, and dried. The total amount of radioactiv- ity on the filters was measured in a toluene-based scintillation fluid [9], and counted in a Nuclear- Chicago Mark II spectrometer, with a counting efficiency of 10.4%.

The number of PBP molecules per cell of S. pneumoniae were calculated for each of the nine time points at each antibiotic concentration. Ini- tially the total pmoles of antibiotic bound per sample (in the TCA-precipitated bacteria) were converted to molecules per sample (using Avogadro's Number), divided by the number of bacteria in the sample (1 nephelometric unit equalled 2.5-105 viable organisms and 7 .5 - l 0 s total cells/ml) and adjusting for the average % saturation for the total amount of PBPs in each sample based on the densitometric data from the fluorograms of duplicate samples (amount of [3 H]benzylpenicillin bound during growth divided by the total amount able to bind after addition of the saturating concentration of [3H]benzylpenicil- lin).

4. RESULTS

Increasing concentrations of [3H]benzylpenicil- lin caused a progressively faster onset in growth inhibition of S. pneumoniae cultures (Fig. 1A), although at 1 x MIC the exponential growth rate continued for about 1.5 generations before a de- crease in the rate of growth was observed. Mea- surement of the total amount of [3H]benzylpenicil- lin able to bind under saturating conditions to the PBPs of bacteria growing without antibiotic, showed that the rate of increase in the amounts of PBPs paralleled the increase in cellular mass (see control curves in Fig. 1, A and B). This correlation was retained even in cultures exposed to different concentrations of [3H]benzylpenicillin (Fig. 1, A and B). Thus, the total quantity of PBPs per turbidity unit of S. pneumoniae remained essen-

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/ ~- /o

100 i i ~ i 100 , ! n n I i 0 3 0 6 0 g O 120 150

T ime (rain)

/o ~0

i i i i i i [ i f i 30 60 90 120 150

Fig. 1. Effect of [3H]benzylpenicillin on the growth and total amount of penicillin-binding proteins in S. pneumoniae. The concentrations of antibiotic (MIC 0.008 /zg/ml) used were: e , Control; O, l x M I C ; *, 2 x M I C ; z~, 3 x M I C ; , , 4 x M I C ; and ~ , 6 x MIC. The total amounts of PBPs shown in B were determined after saturation of the organisms with an additional 1 ~g of [3H]benzylpenicillin, and densitometry of fhiorograms.

tially constant throughout the experiment, and was independent of the presence or absence of antibio- tic, and the length of incubation (Fig. 2, Table 1, Exp. I). The overall mean value of 108 observa- tions was 8.97 + 0.55, and there were no signifi- cant differences between the means of each anti- biotic-treated culture and the control culture. Simi- lar results were obtained in a repeat experiment with a greater range of antibiotic concentrations (Table 1, Exp. II). In addition to the total amounts

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Fig. 2. Quantity of penicillin-binding proteins per turbidity unit in S. pneumoniae. The data were derived from Fig. 1, and the symbols represent the same concentrations of [3H]benzylpeni- cillin used for the growth inhibition. The solid line indicates the mean value of all the observations, and the dashed lines indi- cate the standard deviation.

Table 1

Total amounts of penicillin-binding proteins in S. pneumoniae grown without or with [3 H]benzylpenicillin

303

Experiment Control Concentration of antibiotic ( × MIC)"

1 2 3 4 6 8 16

1 9.04 b 9.24 9.40 8.88 8.91 8.43 (0.39) c (0.49) (0.50) (0.48) (0.30) (0.44)

II 10.86 b 11.07 11.19 11.90 11.40 10.67 (1.63) (1.65) (1.36) (1.61) (1.72) (1.42)

II 19950 d 20250 21850 21 100 21 300 single cocci (3 050) (1 950) (1 550) (550) (1 900) chain size = 3 [6650] [6750] [7283] [7033] [7100]

MIC was 0.008 ~tg/ml. b Data calculated from densitometry of fluorograms (arbitrary units/nephelometric unit), and given as means of duplicate samples at

nine time points during growth. Numbers in parentheses are standard deviations.

d Numbers of PBP molecules per cell calculated from TCA precipitated bacteria and densitometric data for PBPs examined in samples taken at the same time points. The higher numbers were calculated assuming that the physical and viable units of pneumococci were identical (i.e., single cocci). The same data calculated for a chain size of 3 are shown in the square brackets.

o f [ 3 H ] b e n z y l p e n i c i l l i n b o u n d u n d e r s a t u r a t i n g

c o n d i t i o n s , we a l so d e t e r m i n e d the a m o u n t s of

a n t i b i o t i c b o u n d d u r i n g r e s i d u a l g r o w t h in the

p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f [ 3 H ] b e n z y l -

p e n i c i l l i n f r o m the a m o u n t o f t o t a l [ 3 H ] p e n i c i l l i n

b o u n d ( T C A - p r e c i p i t a t e d s a m p l e s ) a n d f r o m t he

a v e r a g e % s a t u r a t i o n of t he P B P s (as d e t e r m i n e d

in s e p a r a t e s a m p l e s f r o m d e n s i t o m e t r y of f l uo ro -

g r a m s ) we c o u l d e s t i m a t e t he t o t a l n u m b e r o f P B P

m o l e c u l e s p e r cel l ( T a b l e 1, Exp . II). T h e va lues

o b t a i n e d d i d n o t d i f f e r s i g n i f i c a n t l y e i t h e r w i t h t he

l e n g t h of i n c u b a t i o n o r the c o n c e n t r a t i o n of a n t i -

b i o t i c in t h e m e d i u m , a n d the ove ra l l m e a n o f 90

o b s e r v a t i o n s was 20 800 ___ 2150 m o l e c u l e s p e r cell.

M e a s u r e m e n t of t he r e l a t ive p r o p o r t i o n s of e a c h

P B P u n d e r s a t u r a t i n g c o n d i t i o n s , d e t e r m i n e d as

t h e % d i s t r i b u t i o n of the [ 3 H ] b e n z y l p e n i c i l l i n ,

s h o w e d t h a t t h e r e we re n o s t a t i s t i ca l ly s igni f i -

c a n t l y se lec t ive c h a n g e s in t he ce l lu l a r c o n c e n t r a -

t i o n s of v a r i o u s P B P s e i t h e r d u r i n g e x p o n e n t i a l -

Table 2

Percentage distribution under saturating conditions of [ 3 H]benzylpenicillin to penicillin-binding proteins

Experiment PBP Control Concentration of antibiotic ( x MIC) a

1 2 3 4 6 8 16

la 24.7 b (1.6) c 24.8 (1.5) 26.1 (2.9) 24.7 (3.1) 25.5 (2.5) lb 5.1 (0.4) 5.0 (0.4) 5.0 (0.4) 5.0 (0.4) 5.2 (0.6) 2a 27.5 (1.4) 28.3 (1.5) 26.7 (1.3) 27.0 (1.1) 27.0 (0.8) 2b 16.6 (1.7) 16.4 (1.6) 17.6 (2.5) 17.3 (2.9) 16.7 (2.9) 3 26.1 (2.2) 25.5 (2.2) 24.6 (1.2) 26.0 (2.1) 25.6 (1.8)

la 27.6 (1.7) 27.3 (1.7) 26.1 (1.9) 25.4 (3.5) lb 5.3 (0.8) 5.3 (1.3) 5.3 (1.0) 5.6 (1.0) 2a 27.8 (2.8) 28.9 (2.1) 29.2 (2.2) 29.6 (1.9) 2b 13.6 (2.2) 14.4 (2.5) 14.5 (2.4) 14.8 (2.5) 3 25.7 (3.1) 24.1 (3.5) 24.9 (3.3) 24.6 (2.7)

24.0 (2.8) 5.3 (0.5)

28.2 (1.5) 16.2 (1.2) 26.3 (1.5)

25.1 (2.6) 5.5 (0.7)

29.8 (2.3) 15.1 (3.1) 24.5 (1.6)

25.2 (2.4) 5.7 (0.8)

29.0 (2.0) 15.5 (2.8) 24.6 (2.3)

a MIC was 0.008/~g/ml. b Means of 18 values (duplicate samples at nine time points). c Numbers in parentheses are standard deviations.

304

phase growth of control cultures or during growth and subsequent inhibition with the antibiotic (Ta- ble 2). The overall means of percentage distribu- tion (with standard deviation) for PBPs la, lb, 2a, 2b, and 3 were 25.0 (2.6), 5.1 (0.4), 27.4 (1.4), 16.8 (2.3), and 25.6 (1.8), respectively, for Exp. I, and 26.1 (2.6), 5.5 (0.9), 29.1 (2.1), 14.7 (2.7), and 24.7 (2.8) for Exp. II. These values thus allowed an estimation of the number of molecules of each PBP per cell as follows: PBP la, 5,200; PBPlb, 1,050; PBP2a, 5,700; PBP2b, 3,500; and PBP3, 5,300.

5. DISCUSSION

The present study has shown that the rate of synthesis of PBPs in S. pneumoniae is proportional to the rate of growth of untreated bacteria, and this has remained true even in bacteria undergoing residual growth after the addition of various con- centrations of penicillin. Thus, since the total quantity of PBPs per turbidity unit remained con- stant in the benzylpenicillin-treated cultures, the interaction of the bacteria with the antibiotic was apparently not affecting the formation of new PBPs. This is in contrast to the suggested effect of penicillin on the synthesis of PBPs in Bacillus subti#s [4]. Chase et al. [2] have presented results that the total amount of PBPs remained constant per unit weight of Escherichia coli growing without antibiotics. However, growth of these bacteria with a radiolabeled cephalosporin, which caused fila- mentation of the organisms, apparently resulted in an increase in the cellular amount of PBP 3. There is also additional evidence in E. coli that the quantity or proportion of PBPs within the organism does not vary throughout the cell cycle in synchro- nized cultures (F.B. Wientjes, A.J.M. Olijhoek, N. Nanninga and U. Schwarz, Abstract PV4, The Murein Sacculus of Bacterial Cell Walls, FEMS Int. Symp., Berlin, 1983).

It is unlikely that any significant quantity of PBPs were lost from S. pneumoniae during incuba- tion with the [3H]benzylpenicillin, since there was a good correlation between the amount of radioac-

tivity measured in the TCA precipitates (which would contain any released protein-associated [3H]benzylpenicillin) and the amount determined by fluorography which had become bound to the PBPs in duplicate samples (not shown). Martin et al. (Abstract PIII10, The Murein Sacculus of Bacterial Cell Walls, FEMS Int. Symp., Berlin, 1983) have recently shown that incubation of an autolytic-deficient S. pneumoniae with 10 x MIC of benzylpenicillin for 40 min only caused less than a 2% release into the supernatant of either new or old PBPs. In contrast, incubation of group A streptococci with 50 x MIC of benzylpenicillin for 60 min caused an apparent release of about 17% of the high M r PBPs [3].

The relative proportions of each PBP have re- mained the same during inhibition of growth of pneumococci and these proportions were in good agreement with previously obtained data for this organism [10], indicating no selective effects on particular PBPs during inhibition. The estimation of about 21000 total PBP molecules per cell of S. pneumoniae (or about 7000 molecules per physical cell unit) is substantially greater than the estimate of 2800 for E. coli [11]. Gram-positive bacteria have been shown to bind 5- to 10-fold more radio- active benzylpenicillin than E. coli [12 14].

The conclusion that the number of PBPs per cell of pneumococci remains essentially constant during inhibition of growth with benzylpenicillin has bearing on the results of experiments in which the correlation between antibiotic affinity for a particular PBP (measured over a period of a few minutes) and the MIC value of the same antibiotic (measured after at least 18 h of incubation) is studied (see, e.g., [15]). In such studies it is gener- ally assumed that the interactions between the antibiotics and the particular PBP are essentially the same in the short term labeling experiments and in the prolonged treatment during MIC de- termination. Clearly, if a particular PBP were either lost from the bacteria (by secretion or degrada- tion) or was synthesized at a different rate during exposure to the antibiotic, the MIC of the antibio- tic could not be expected to correlate with the measurement of affinity.

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