The Role of Penicillin-Binding Proteins in the Action of Cephalosporins against Escherichia coli and Salmonella typhimurium

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1 Eur. J. Biochem. 117, (1981) FEBS 1981 The Role of Penicillin-Binding Proteins in the Action of Cephalosporins against Escherichia coli and Salmonella typhimurium Howard A. CHASE, Christopher FULLER, and Peter E. REYNOLDS Sub-Department of Chemical Microbiology, Department of Biochemistry, University of Cambridge (Received February 9, 1981) The binding of two radioactively-labelled cephalosporins, LY97962 and LY121998, to the penicillin-binding proteins of Escherichia coli and Salmonella typhimurium has been studied in detail. The concentrations of the cephalosporins that resulted in 50 saturation of the penicillin-binding proteins were measured in growing cells as well as in preparations of disrupted cell envelopes. This technique has allowed the crypticity of an organism towards a particular p-lactani to be measured directly, i.e. the extent to which a penicillin-binding protein is accessible in intact cells. The cephalosporin LY appeared to have no difficulty in penetrating the outer layers of the cell whereas access of cephalosporin LY to the penicillin-binding proteins appeared to be somewhat restricted. At concentrations below the minimum growth inhibitory concentration the two cephalosporins inhibited cell division and caused the cells to grow as long filaments. When growing cells were incubated with the radioactive fl-lactam antibiotics at these concentrations, the B-lactams bound almost exclusively to penicillin-binding protein 3. The degree of binding of the cephalosporins to protein 1A in cells growing in the presence of the minimum growth inhibitory concentrations of antibiotics varied with the duration of the incubation. After exposure for 5 min, very little binding to protein 1A was apparent, whereas substantial binding was observed after min. It was only at the end of this period of growth in the presence of the cephalosporins that lysis of the cells became apparent. This provided evidence that the binding of p-lactams to protein 1 A of gramnegative organisms is an important step in the bactericidal action of certain cephalosporins. Protein 1 B was excluded as a target for the lethal action of these cephalosporins because binding to this protein was observed only at concentrations greater than those needed to inhibit growth of the cells. Microscopical examination of the growth of S. typhimurium in the presence of cefotaxime at sub-lytic concentrations showed that cells grew as filaments for at least six generations without lysis. Observations of the division of the filament into individual cells after removal of the antibiotic showed that the entire length of the filament was viable. It is concluded that inhibition of cell division as a result of the binding of p-lactams to protein 3 of gram-negative organisms is not in itself a lethal event for the cells, but requires concomitant binding of antibiotic to protein 1A before lysis is initiated. The action of some 0-lactam antibiotics against gramnegative bacteria not only results in a cessation of cell growth and a loss of cell viability but can, at certain concentrations, result in the production of dramatic morphological effects. The wide range of morphological effects produced by benzylpenicillin, including filamentation, rabbit ears and bulge formation have been known for a number of years [I -31. More recently, some of the semi-synthetic p-lactams have been shown to induce the formation of a single type of morphological response. For example, the amidino-penicillin, mecillinam, induces the formation of large spherical cells [4], whereas cephalexin, cefotaxime and a number of other cephalosporins can, at concentrations below those that inhibit growth, induce the formation of long filaments [5]. Some /J-lactams such as cephaloridine and cephalosporin C do not produce any specific morphological effects other than cell lysis that follows the inhibition of cell growth. The initial stages of the actions of p-lactam antibiotics are believed to involve binding of these compounds to the penicillin-binding proteins found in the cytoplasmic mem- Enzyme7 D,D-Carboxypeptidahe (EC ); 8-lactamase (EC ). brane of the bacterial cell 161. All bacteria contain multiple penicillin-binding proteins and the binding of p-lactams to these proteins has been studied in atlempts to correlate the initiation of the effects produced by the []-lactams with selective binding to a particular protein [7]. Indeed, it has been shown in Escherichia coli that those P-lactams which cause the formation of large spherical cells bind preferentially to protein 2 in isolated cell envelope preparations; those that cause filamentation bind to protein 3 and those whose action results in lysis without any intermediate morphological effect bind to the components of protein 1 [S]. These results were confirmed by genetic studies in which it was shown that mutants of E. coli lacking protein 2 grew as round cells [4] and mutants with a temperature-sensitive protein 3 grew as filaments at the restrictive temperature [9]. It has been proposed that protein 3 is a peptidoglycan transpeptidase on the basis of the peptidoglycan synthetic activity of strains containing increased levels of the protein [lo] but direct evidence has not yet been obtained as the purified enzyme shows no activity in peptidoglycan synthesis [I I]. The exact relationship between and the relative importance of the components of protein 1 (1 A and 1 B) is complicated. Mutants lacking either protein 1A or protein 1B have been

2 302 isolated [12], but mutants with defects in both components show thermosensitive growth and lyse at the restrictive temperature. Hence it has been proposed that protein 1A and protein 1 B may both catalyse peptidoglycan synthesis and that protein 1A fulfills the function of protein 1B in cells that have lost the activity of the latter protein. Both components of protein 1 have recently been purified from the membrane of E. coli [I 11. Protein 1 B catalysed both peptidoglycan transglycosylase and peptidoglycan transpeptidase activities [13], although purified protein 1A failed to show either activity. The morphological effects that can be observed with fl-lactams are often shown only at low concentrations below the minimum growth inhibitory concentration; at higher concentrations, a lytic response may rapidly be observed [7]. With some antibiotics, even at low concentrations, if growth of filaments resulting from selective binding of p-lactams to protein 3 is followed for several generations, lysis can eventually be observed. It is possible that the latter effect is due to the prolonged inhibition of the activity of protein 3, which eventually results in destabilisation of the integrity of the cell wall or the triggering of lysis. Another possibility is that the fl-lactams bind to penicillin-binding proteins in addition to protein 3 after a prolonged period of growth in the presence of the p-lactams and it is inhibition of the function of these other proteins that results in cessation of cell growth and the onset of lysis. It is doubtful whether binding of /3-lactams to protein 3 and the inhibition of its function results per se in a lethal event for the cell, as mutants with a temperaturesensitive protein 3 grow as long filaments without lysing at the non-permissive temperature [9]. The aim of the work reported here was to investigate the binding of cephalosporins, whose primary morphological effect on gram-negative organisms was to cause the formation of filaments, to the penicillin-binding proteins of these organisms. It was hoped that this would enable us to determine whether binding of P-lactams to protein 3 was by itself a lethal event in the mechanism of action of the antibiotics. Furthermore, it was envisaged that the results obtained would provide information as to the ease with which the cephalosporins could penetrate the outer layers of the cell. MATERIALS AND METHODS Bacterial Strains and Conditions of Growth Escherichia coli K12, strain 44 [14] and Salmonella typhimurium SW 1061 [15] were used throughout this study. The organisms were grown as described by Chase [16] for Bacillus megaterium except that growth was followed by measuring the absorbance of the culture at 600 nm in a Zeiss PMQ3 spectrophotometer. Preparation of Disrupted Cell Envelope Fractions Batches of cells were harvested in the mid-exponential phase of growth at a culture density of 0.4 mg dry weight ml-', by centrifugation [17]. The cell pellet was resuspended in 10 mm sodium phosphate buffer, ph 7.0 at a density of 20 mg dry weight ml-' and cells were broken by sonication. A Dawe 'Soniprobe' type 7532A fitted with a Branson Converter was used with the output control set to position5. The cell suspension was kept at 4 C and 30-s bursts of sonication were carried out. A total of 3 min of sonication resulted in breakage of more than 90% of the cells. Intact cells were removed by centrifugation at 7000 x g for 1 rnin and the cell envelopes were collected by centrifugation (50000 x g, 20 min, 4"C), washed twice in 10 mm sodium phosphate buffer, ph 7.0, resuspended in the same buffer at a density of 10 mg protein ml-' and stored at - 30 "C. Labelling of Penicillin-Binding Proteins in Isolated Cell Envelope Preparations with Radioactive b-lactams 45-pl samples of a suspension of disrupted cell envelopes were incubated with various concentrations of a radioactively labelled P-lactam in a final volume of 50 p1 for 15 rnin at 37 "C. Binding was terminated by the addition of unlabelled P-lactam (1 mg m1-l) and Sarkosyl NL-97 (1 % w/v, final concentration). The samples were incubated at 37 "C for 5 min to solubilize the proteins from the cytoplasmic membrane [18]. The insoluble outer membrane was pelleted by centrifugation at x g for 15 rnin at 20 "C. The supernatants were added to 15 p1 of a buffer containing 50 mm Tris/HCl, ph 7.2, 50% (w/v) glycerol, 5% (w/v) sodium dodecyl sulphate, 50 % (v/v) 2-mercaptoethanol and 0.01 % (w/v) bromophenol blue. The membrane proteins were separated by dodecyl sulphate/polyacrylamide gel electrophoresis and the radioactive p-lactam, penicillin-binding protein complexes detected by fluorography and quantified by microdensitometry as described by Shepherd et al. [15]. Labelling of Penicillin-Binding Proteins in Growing Cells with Radioactively Labelled /3-Lactams 50 % binding values were determined by incubating with shaking 10-ml samples of cultures in the mid-exponential phase of growth ( mg dry weight ml-') at 37 "C for 15 min with various concentrations of a radioactively labelled fi-lactam. At the end of the incubation, unlabelled P-lactam was added (1 mgml-l) and the organisms harvested by centrifugation (9000 x g, 4 "C, 1 min). The pellets were resuspended in 1 ml 10 mm sodium phosphate buffer, ph 7.0 and the cells were broken by sonication. A microprobe attachment was fitted to the converter of the Soniprobe and with the power output set to the minimum value, sonication for 1 min in a glass test-tube (50 x 10 mm) was sufficient to break more than 90 % of the cells. The disrupted cell envelopes were collected by centrifugation (40000 x g, 20 min, 4 "C) and the pellets resuspended in 40 pl of phosphate buffer. Cytoplasmic membrane protein was solubilised and treated as described above for the detection of penicillin-binding proteins in preparations of cell envelopes. The time course of the binding of p-lactams to the penicillinbinding proteins was determined by incubating 150 ml culture in the early exponential phase of growth with shaking at 37 C with a concentration of a radioactively labelled P-lactam close to the value of the minimum growth inhibitory concentration. The initial density of the culture was 0.15 mg dry weight ml-'. Suitable volumes of the culture containing 6 mg dry weight of cells (as determined from measurements of the absorbance of the culture) were removed at intervals and unlabelled cephalosporin was added to a final concentration of 1 mg ml-'. The samples were then centrifuged and the detection of the P-lactam. penicillin-binding protein complexes was determined as described above.

3 Microscopical Observation of the Morphological Effects A flat layer of nutrient agar (Difco Bacto) containing a suitable concentration of the particular /3-lactam under investigation was formed on the surface of a glass microscope slide (76 x 26 mm). The surface of the agar was inoculated with a small drop of an exponentially growing culture of S. typhimurium. A glass coverslip was then carefully lowered into position and the edges of the cover slip sealed with molten paraffin wax to prevent evaporation. The growth of single cells was then examined using phase-contrast optics under the 100 x objective of a Leitz 'Ortholux' microscope. The slide was incubated at 25 "C or 37 "C in position under the microscope and photographs were taken at intervals. In experiments to investigate the reversal of the morphological effects after removal of the antibiotic, S. typhimurium was grown in the presence of a suitable concentration of the antibiotic at 37 "C in liquid medium with aeration until the morphological effects produced by the antibiotic were pronounced. The cells were collected by centrifugation and washed with growth medium at 37 C. A small drop of the culture was spread onto a thin layer of nutrient agar on a microscope slide and the growth of cells at 37 "C was followed as described above. Determinations of Minimum Growth Inhibitory Concentrations Growth in Liquidfrom a Large Inoculum. Cultures of cells were grown exponentially to a density of 0.04 mg dry weight ml-'. 50-ml aliquots were added to 100-ml conical flasks containing p-lactam at various concentrations. The flasks were incubated with shaking in a water bath at 37 "C and the absorbance of 3-ml samples measured every min at 600 nm. The minimum growth inhibitory concentration is defined as the concentration at which the rate of growth is reduced by 50% over a period of three or four generations. Growth in Liquid from a Small Inoculum. 5-ml aliquots of growth medium in sterile tubes at various concentrations were inoculated with an exponentially growing culture to a final density of lo6 organisms ml-i. The tubes were incubated at 37 "C with aeration and checked for visible turbidity after 24 h. The minimum growth inhibitory concentration is defined as the minimum concentration of antibiotic at which growth was reduced to less than So/, of that of a control culture. Growth OJZ Solid Media. Various amounts of P-lactams were added to molten Isosensitest agar (Oxoid Ltd) at 45 "C which was immediately poured into petri dishes. The agar was allowed to set and the plates were dried at 37 C for 30min. A series of small inocula of cells growing in liquid medium at various cell densities was applied to each plate and the plates were incubated overnight at 37 "C. The minimum growth inhibitory concentration was defined as the minimum concentration of the antibiotic at which the appearance of colonies was not observed. Antibiotics and Chemicals ''C-labelled LY97962 (57 Ci mol-') and LY (52 Ci mol- l) and the unlabelled derivatives were the generous gift of Lilly Research Laboratories (Indianapolis, USA). ['"CI- Benzylpenicillin (potassium 6-phenyl [l-'4c]acetamidopenicillanate) was purchased from the Radiochemical Centre (Amersham, England). Cefotaxime was obtained from Roussel Laboratories (London) and benzylpenicillin sodium (Crystapen) was from Glaxo Laboratories (Greenford, England). Sarkosyl NL-97 was obtained from Ciba-Geigy (Man- Chester, England). All other reagents were of AnalaR quality if possible. RESULTS Microscopical Examination of Morphological Changes Induced In order to determine whether the induction of filamentation in cells treated with certain cephalosporins necessarily results in loss of cell viability and an onset of lysis, the growth of single cells of Salmonella typhimurium on agar in the presence of cefotaxime was observed microscopically. Treatment with cefotaxime at a concentration of 0.1 pg ml-', resulted in an inhibition of cell division, but permitted cells to grow as stable filaments for at least 3 h (Fig. 1 A). During this period, the cells had elongated to approximately 30 times the normal length as measured just after cell division and had therefore grown through six generations. The rate of cell growth was the same as in a control that had not been treated with antibiotic. At lower concentrations of cefotaxime, cell division was less effectively inhibited whereas at higher concentrations filamentation was also observed, but bulges formed in the filaments as the incubation was continued and subsequently the cells were observed to lyse. It was noted that when such a filament lysed, it appeared to lose its cytoplasmic contents from the complete length of the cell. This implied that the process being inhibited was the formation of septa in the filament rather than the separation of the individual cells that would have comprised the filament. Cells that had been allowed to form filaments in liquid culture in the presence of cefotaxime at 0.1 pg ml-' were washed free of the antibiotic, incubated on agar at 31 "C and observed microscopically. Cells of normal length were produced from the filament, initially from the two poles but eventually the whole length of the filament had divided into standard length cells (Fig. 1 B). This result indicated that the complete length of the filament was viable but it is not clear why cell division occurred first at the poles of the filament and subsequently worked towards the centre. Similar experiments have been carried out with mecillinam. When incubated in the presence of this antibiotic, the cells lost their original rod shape and formed large spherical cells (Fig. 2A). These spherical cells were viable, however, and continued to divide and replicate with a generation time very similar to that of an untreated control. On removal of the antibiotic, the spherical cells regained their rod shape and resumed normal growth (Fig.2B). None of the cells observed appeared to lose its viability upon treatment with mecillinam at this concentration although at higher concentrations, some of the spherical cells lysed. These results are a preliminary indication therefore that a change of morphology induced by a /3-lactam does not necessarily result in a loss of cell viability and an onset of lysis. Determination of Minimum Growth Inhibitory Concentrations The minimum growth inhibitory concentrations of two cephalosporins LY97962 and LY against Escherichia coli and S. typhimurium were determined using a number of different procedures (Table 1). The values determined after a long incubation period either on solid or in liquid medium

4 304 A Time (min) Fig. 1. The morphological ejyecfs of cefotaxime on S. typhimurium and recovery from these efects,following pretreatment with the antibiotic. An exponentially growing culture of S. typhimurium was incubated at 25 "C on a thin layer of agar on a microscope slide in the presence of cefotamine (0.1 pgm1-l) (as described in Materials and Methods) and observed under a microscope. Photographs were taken at the times indicated (A). To study the effect of antibiotic treatment on cell viability, S. typhimurium was grown with cefotaxime (0.1 pg m1-i) for 150 min at 37 C in liquid medium. The cells were washed free of the antibiotic (as described in Materials and Methods) and reversion to normal rod-shaped cells after incubation on a thin-layer of agar was followed microscopically. Photographs were taken at the times indicated (B) A Time (rnin) Fig. 2. The morphological eflects of mecillinam on S. typhimurium and recovery,from these effrcts following pretreaiment with the antibiotic. An exponentially growing culture of S. typhimurium was incubated at 25 C on a thin layer of agar on a microscope slide in the presence of mecillinam (0.1 pg ml-') (as described in Materials and Methods) and observed under a microscope. Photographs were taken at the times indicated (A). To study the effect of antibiotic treatment on cell viability, S. typhimuriurn was grown with mecillinam (0.1 pg ml-') for 240 min at 37 C in liquid medium. The cells were washed free of the antibiotic (as described in Materials and Methods) and reversion to normal rodshaped cells after incubation on a thin layer of agar was followed microscopically. Photographs were taken at the times indicated (B)

5 ~ -- ~.- ~~ 305 Table 1. Determinafion of minimum growth inhibitory concentrutions The effect of cephalosporins on the growth of E. coli and S. typhimurium was determined by three different methods. The details of the procedures used and the definitions of the minimum growth inhibitory concentrations are described in Materials and Methods. n.a. = not applicable. LY is not stable over a 24-h period and the values obtained were not applicable to the investigation described here Protein Method of determination LY97962 with LY with -~ -~ - b colr S typhl- E coli S typhimurrum murium pg ml-' -. ~~ ~ - ~ ~- ~~ Solid media: inoculum size: 1.4~ lo ma. n.a. inoculum size: 1.4 x lo n.a. n.a. Liquid medium in tubes n.a. n.a. Liquid medium in shaking %asks from a small inoculum were, in general, higher than those determined in short-term growth experiments in which the reduction in the rate of growth was observed. A probable explanation of this result is that the cephalosporins were somewhat unstable under these conditions and when the concentrations of active antibiotic had eventually fallen below those which would inhibit growth, any unlysed viable cells still remaining were able to grow. Because the labelling of the penicillin-binding proteins in growing cells by /?-lactams was carried out with cultures growing with aeration in thick suspension, over periods no longer than 3 h, we have decided that the values of the minimum growth inhibitory concentrations determined by studying the reduction in the rate of growth of cells are the most suitable for use in correlations with the concentrations that result in binding to the penicillinbinding proteins. Concentrations of the cephalosporins that appeared to have little or no effect on the rate of increase of the absorbance were found, however, to cause the cells to grow as filaments; with LY97962, both E. coli and S. typhimurium started to form filaments at concentrations as low as 0.06 pg ml-', and the concentrations of LY that were needed before filamentation was observed were 0.6 pg ml-' for E. coli and 0.06 pg nil- ' for S. typhimurium. Thus the cephalosporins LY97962 and LY appear to have a similar mode of action to cephalexin and cefotaxime in that they induce filamentation at sub-lethal doses. Binding of Cephalosporins to Penicillin-Binding Proteins in Envelope Preparations The binding of radioactively labelled cephalosporins to penicillin-binding proteins in envelope preparations was investigated to confirm directly our hypothesis that LY97962 and LY were inducing filamentation of gram-negative cells by binding selectively to protein 3. The concentrations of the cephalosporins that resulted in 50% of the maximal binding to each penicillin-binding protein in envelope preparations from E. coli and S. typhimurium are shown in Table 2. Apart from one additional protein (protein 4') that bound LY at high concentrations, no proteins that bound A B C D E Fig. 3. Binding ~f'~c-label/ed &lactam uiitibiotics io the pmicillin-hmdi,ig proteins o/ E. coli und S. typhimurium. 450-pg samples of isolated cell envelope preparations from E. coli (tracks A-C) and S. fyphimurium (tracks D -F) were incubated with a ''C-labelled B-lactam antibiotic (10 pg ml-') for 15 min at 37 C. Binding was terminated by the addition of unlabelled B-lactam (1 mg m1-l) and Sarkosyl NL-97 (1 x, w/v). The binding of the radioactive /l-lactams to the penicillin-binding proteins was determined by dodecylsulphate/polyacrylamide gel electrophoresis followed by fluorography as described in Materials and Methods. The fluorogram of the gel is shown. The radioactive /J-lactams used were: ['4C]benzylpenicillin (tracks A and D), [I4C]LY97962(B and E), ['"CI- LY (C and F) the cephalosporins but not ['4C]benzylpenicillin were detected (Fig. 3). With both organisms, the two cephalosporins had the highest affinity for protein 3 and it was notable that protein 4 was particularly refractory to labelling by LY97962 and LY Protein 4 of E. coli (H. A. Chase and P. E. Reynolds, unpublished observations) and S. typhimurium [I 51 has Dpcarboxypeptidase, transpeptidase and endopeptidase activities and has a high affinity for ['4C]benzylpenicillin [15,19]. Protein 5/6 which is a upcarboxypeptidase [15,20], also had a low affinity for both cephalosporins. Labelling of the Penicillin-Binding Proteins in Growing Cells with Cephalosporins We have shown previously with Bacillus megaterium [21, 221, E. coli and S. typhimurium [23,24] that it is not possible to correlate directly the concentrations of a fl-lactam that result in 50 "/, binding to the penicillin-binding proteins, when studied in isolated membrane or envelope preparations, with those concentrations that have an effect on the cell. In order to avoid difficulties in the interpretation of such results because of the outer layers of the cell influencing the access of /?-lactams to the cytoplasmic membrane, we have studied the binding of b-lactams to the penicillin-binding proteins in growing cells [ The results of experiments to investigate the binding of radioactively labelled LY97962 and LY to the penicillin-binding proteins in growing cells of E. coli and S. typhi- F

6 ~~ - ~~ ~ ~- ~ _. ~~~ ~ ~ ~~ ~~ ~ ~ 306 Table 2. Alfinities and crypticity values of the penicillin-binding proteins of E. coli and S. typhirnurium for cephalosporins The affinities of the penicillin-binding proteins of E. coli and S. typhimurium for cephalosporins were determined both in growing cells and isolated cell envelope preparations as described in Materials and Methods. The crypticity values were calculated as described in the text. The concentrations of the cephalosporins that resulted in 50% binding to the proteins are given. means no labelling could be observed at a concentration of cephalosporin of 6 pg rnl-' Protein LY97952 with LY with ~- ~ ~~ ~~~ ~ ~~ E. coli S. typhimurium 8. coli S. typhimurium _ ~- ~ ~ - ~ isolated growing crypticity isolated growing crypticity isolated growing crypticity isolated growing crypticity envelopes cells value envelopes cells value envelopes cells value envelopes cells value pg ml-' ~-~ 1A 0.1 1B > > P8 mi- -~ < 0.03 $6-10 $6 - > <0.06 >6 $6 6 >6 pg rnl-' > 30 1 > 30 - pg rnl-' _~ ~ >6 3 >2 >2 10 > $ > > murium are shown in Table 2. We have been unable to determine the 50 % binding values for penicillin-binding proteins when this value is greater than 6pgml-' as experiments using higher concentrations of the radioactive cephalosporins would have consumed large amounts of these compounds which were only available in limited quantities. However, as the minimum growth inhibitory concentrations of the cephalosporins for both organisms were considerably lower than 6 pg rnl-', it is unlikely that the insensitive proteins (proteins 4, 5/6, 7, 8) play any role in the lethal effects of these p-lactams. The results show that for LY97962, there are not significant differences between the concentrations that result in 50% binding to the penicillin-binding proteins in cell envelopes or in growing cells for either E. coli or S. typhimurium. A significant exception is that a higher concentration of antibiotic was required before protein 1 A of E. coli was 50% saturated when binding was studied in growing cells rather than cell envelope preparations. With LY , it was observed that for all penicillin-binding proteins, higher concentrations were required to achieve 50% binding in growing cells than in cell envelopes. The total amounts of proteins 7 and 8 that were available for labelling in growing cells were much greater than in envelope preparations and it is possible that the procedure by which the cell envelope fraction was obtained may have resulted in partial denaturdtion of these proteins, thus reducing their affinities for P-lactams. The term 'crypticity' has been used to define the interaction of a cell-bound P-lactamase of gram-negative organisms with the barrier function of the cell envelope [25]. Its numerical value is obtained by dividing the specific P-lactamase activity of broken cells by the specific activity of whole cells. The results presented here can be used to calculate a similar value which can also be referred to as a crypticity value, namely the ratio of the concentration of a P-lactam antibiotic required to bind to 50 % of an available penicillinbinding protein when studied in growing cells (in vivo) and in cell envelope preparations (in vitro). The direct measurement of the affinity of penicillin-binding proteins in vivo, from which the crypticity values are calculated, takes into account not only the difficulty in penetrating the outer membrane but also the ability of the 0-lactam molecule to withstand the action of a P-lactamase located in the periplasmic space. Therefore, in this paper, we define the crypticity values as follows: crypticity value of a P-lactam = concentration of P-lactam required to bind to 50% of a particular penicillinbinding protein in intact cells/concentration of 0-lactam required to bind to 50% of the same protein in isolated cell envelope preparations. Hence, if a penicillin-binding protein is freely accessible in growing cells, the crypticity value should be 1. If the outer layers restrict or otherwise prevent the free passage of the p-lactam, the crypticity value will be greater than 1. Values less than 1 indicate that a penicillinbinding protein appears to be more accessible in whole cells than in isolated envelopes. A possible explanation of such a value (e.g. the value for protein 7 of E. coli with LY97962) is that the protein is forced into a less accessible orientation in the membrane or is partially denatured during envelope preparation. It has not been possible to measure crypticity values for penicillin-binding proteins that have low affinities for p-lactams. Examination of the crypticity values for LY (Table 2) shows that apart from protein 1A of E. coli, which has a crypticity value greater than 1, the antibiotic appeared to have no difficulty in reaching and reacting with its targets in growing cells. The situation was markedly different with LY where all the measured crypticity values were greater than 1. Of particular interest was the high value observed for protein 3 of E. coli which had a crypticity value of In general, the crypticity values observed for S. typhimurium were lower than those measured for E. coli. The results suggest that LY had more difficulty than LY in penetrating to the penicillin-binding proteins and that the outer layers of the cell envelope of E. coli were more effective at preventing the access of 0-lactams to the cytoplasmic membrane than were the outer layers of S. typhimurium. A similar situation was observed when studying the binding of ['4C]benzylpenicillin to intact cells of E. coli and S. typhimurium [23,24]. Correlation of the minimum growth inhibitory concentrations with the concentrations of the cephalosporins that resulted in 50 "/, binding to the penicillin-binding proteins in

7 ~ ~ ~ ~~~ ~~ ~~ ~- ~ ~~ ~~~~~ ~ ~ Table 3. Time courses of labelling of penicillin-binding proteins of growing cells of E. coli and S. typhimurium hy cephalosporins E. coli and S. typhimurium were grown in the presence of ['4C]cephalosporins at the minimum growth inhibitory concentrations. Samples containing 6 mg dry weight of cells were removed at intervals, the cell envelopes were isolated and the binding of the cephalosporins to the penicillin-binding proteins was determined by dodecylsulphate/polyacrylamide gel electrophoresis as described in Materials and Methods. The degree of saturation of a particular protein was determined by comparison with that of the corresponding protein in a cell envelope preparation that had been treated with a saturating concentration of the same cephalosporin. The time (min) by which various degrees of labelling of the proteins was observed is shown. means no labelling of this protein was detected at this concentration of cephalosporin 307 Protein Time for E. coli with Time for S. fyphimurium with LY97962 (0.1 pg ml-') LY (1 pg m1-l) LY97962 (0.2 pg m1-l) LY (0.3 pg ml -') labelling labelling labelling labelling.~ ~ ~ trace 50% 1000/, trace 50% 100% trace 50% 100% trace 50% 100% ---. ~ min 1A >I80 1B n > >I80 >I80 < >I <5 30 _ ~ >120 >I80 >180 >180 >I >I80 >180 ~ ~ >I80 >I growing cells suggests that protein 3 is the most likely target for the growth inhibitory actions of the cephalosporins. However, this conclusion is in conflict with the results presented earlier which suggest that binding of P-lactams to protein 3 alone does not result in loss of viability and onset of lysis. Indeed, there was substantial labelling of protein 3 in whole cells at concentrations below those that affected the rate of growth and there is a good correlation between the concentrations necessary to induce filamentation and those that resulted in binding to protein 3. The protein that possesses the next highest affinity for LY97962 and LY is protein IA, but in all cases the concentrations that resulted in 50 % binding in viva were approximately 5-10-times greater than the minimum growth inhibitory concentrations. However, the time period used for the labelling of penicillin-binding proteins in growing cells was only 15 min and yet the effects on the rate of growth and the initiation of lysis were not apparent until exposure to the antibiotic had continued for at least 60 min. Therefore, we carried out a series of experiments to investigate the variation with time of the degree of binding of the radioactive cephalosporins to the penicillin-binding proteins when growing cells were incubated with the antibiotic at concentrations close to the minimum growth inhibitory concentrations. The continued synthesis and insertion of penicillinbinding proteins into the cytoplasmic membrane during the period of the growth could have resulted in apparent increases in the degree of binding of the antibiotics of the proteins. In an attempt to avoid such misleading results, samples containing a constant weight of cells (rather than a constant volume) were removed at each time point. Pilot experiments indicated that the amount of any particular penicillin-binding protein remained constant if a constant weight of cells was taken from a control culture under these conditions. However, the total amount of protein 3 that had been labelled after a min incubation of cells in the presence of radioactive cephalosporin appeared to be greater than that present in an equivalent sample from an untreated control. This observation was taken into account when Protein IA 1B 2 3 L 516 I 8 M I80 M lime lrninl Fig. 4. Time course of labelling of the penicillin-binding proteins oj'growing cells of S. typhimurium by radioactive cephalosporins. S. typhimurium was grown in the presence of [14C]LY97962 at a concentration of 0.2 pg ml-'. Samples containing 6 mg dry weight of cells were removed at the times indicated, the cell envelopes were isolated and the binding of the cephalosporin to the penicillin-binding proteins was determined by dodecylsulphate/polyacrylamide gel electrophoresis followed by fluorography as described in Materials and Methods. The fluorogram of the gel is shown. The tracks marked M are 500-pg samples of a cell envelope fraction of S. typhimurium incubated with ['4C]benzylpenicillin (10 pg m1-i) for 15 min at 37 C followed by the addition of unlabelled benzylpenicillin (1 mg ml -I). The radioactive penicillin-binding proteins were detected as described in Materials and Methods assessing the degree of binding to protein 3. The increased amount of protein 3 could have resulted from an enhanced rate of synthesis in the culture treated with cephalosporin in an attempt by the cells to overcome the inhibitory effects or to the fact that the relationship between absorbance and dry weight of short rods and filaments is different. The fluorogram of a typical experiment is shown in Fig.4 and the complete set of results obtained is given in Table 3. It is clear from such experiments that although the cephalosporins bind to protein 3 rapidly, they also bind to

8 protein 1A but at a slower rate. It was observed that, although scarcely any binding to protein 1 A was apparent after exposure for 5 min, there was a steady increase in the degree of labelling with time and that substantial binding to protein 1A had occurred after exposure for 60 niin or longer. It was only after this length of time that the growth was reduced at these concentrations and lysis became apparent. Lysis is evident at the 180-min time point (Fig.4) as all the penicillin-binding proteins normally labelled in isolated envelope preparations at this concentration of LY97962 (0.2 pg ml-l) can be seen clearly. Time courses of labelling at higher concentrations showed that protein 1 A was labelled more rapidly under these conditions and this result correlates well with the observations that the rate of growth of the cells is also affected more rapidly. Binding of Cefotaxime to the Penicillin-Binding Proteins in Growing Cells of E. coli The variation with time of the degree of binding of cefotaxime to the penicillin-binding proteins in growing cells of E. coli was also investigated. As cefotaxime was not available to us in radioactive form, cells were grown in the presence of the unlabelled antibiotic at various concentrations for either 10 min or 120 min. After rapid preparation of the cell envelope fractions, [I4C] benzylpenicillin (10 pg ml-') was added for 10 min at 37 "C to label the proteins that were not saturated with cefotaxime. Binding of cefotaxime to protein 3 after growth for 10 min could just be observed at 0.03 pg nil-' and the protein was saturated at 0.1 pg ml-'. Cefotaxime did not bind to protein 1A until the concentration was between pg m1-l. However, after exposure for 2 h, protein 3 was saturated at concentrations less than 0.01 pg m l ~ and protein 1 A was more than 80 % saturated at 0.03 pg ml-'. Hence, the degree of binding of a p-lactam at a particular concentration to a penicillin-binding protein in growing cells was again shown to increase with the duration of exposure. The minimum concentration at which filamentation was observed after 2 h was 0.03 pg ml-' and cultures where significant binding to protein 1 A was observed after 10 rnin had lysed by 120 min. Some formation of filaments was observed in the latter case before lysis occurred. Cultures growing in concentrations of cefotaxime at which binding to protein 1A could be observed after 2 h were also found to lyse upon continued growth in the presence of the antibiotic. We would suggest therefore that the binding of P-lactams to protein 1A is an important primary step in the cessation of cell growth and the induction of lysis. DISCUSSION The production of filaments in Escherichia coli and Salnzonelfa typhimurium as a result of growth in the presence of cephalosporins LY97962 and LY correlates well with the binding of these cephalosporins to protein 3 in growing cells. This result confirms directly the results of Spratt [8] which suggested that filamentation of gram-negative rodshaped cells resulted from the binding of p-lactam antibiotics to protein 3. We have shown that the production of filaments caused by cefotaxime is not, in itself, a lethal event for the cell. Similar morphological studies by Rolinson et al. [26] showed that E. coli formed filaments when incubated in the presence of ampicillin or cephalexin and that these filaments retained their viability. Our results indicate that when binding of the cephalosporins to protein 1A had occurred, a loss of cell viability and the induction of lysis became apparent. We propose, therefore, that the bactericidal action of cephalosporins that induce filamentation at low concentrations through binding to protein 3, results from binding to protein 1 A. Spratt [8] proposed that protein 1 is involved in cell elongation and that its inhibition by p-lactams results in cell lysis. After protein 1 had been resolved into at least two components [27], it was claimed that there was a good correlation between the potency of a p-lactam as an inhibitor of cell elongation and its binding to protein 1 B. However, considerable caution should be exercised in interpreting the validity of such conclusions as the experiments involved comparisons of concentrations that resulted in 50 saturation of penicillinbinding proteins in isolated envelope preparations with the concentrations that affected the growth of cells in long-term growth tests. The results presented here emphasize the importance of studying the binding of p-lactams to the penicillinbinding proteins under the same conditions and for the same periods of time as those in which the effects on growth and viability are assessed. Our recent studies of the binding of P-lactams to the penicillin-binding proteins of Bacillus megaterium have demonstrated that only under these circumstances could these concentrations be strictly compared [22]. Binding of cephalosporins to protein 1 B in growing cells occurred only at concentrations much greater than those needed to inhibit growth. These results are consistent with our previous studies of the binding of [14C] benzylpenicillin to growing cells of E. coli in which we were able to detect binding to protein 1A and not to protein 1B at concentrations that inhibited cell growth [23,24]. Although protein 1B appears to be an important enzyme in peptidoglycan synthesis [ll- 131, it is evidently not necessary for it to be saturated by p-lactams in order for the antibiotics to exert their lethal action. It appears that protein 1B either cannot deputise for the function of protein 1A when the latter has been inhibited or that binding of P-lactams to protein 1 A results in a triggering of cell lysis despite the action of protein 1B. The mechanism by which the binding of a [Mactam to certain penicillin-binding proteins can initiate cell lysis is currently under investigation [28,29]. Further confirmation of the importance of the role of protein 1 A in the lytic response to P-lactams comes from studies on mutants lacking protein 1 A. Such mutants are viable, suggesting that the activity of protein 1A may not be important for cell growth, and the mutants showed identical morphological responses to various p-lactam antibiotics at the same concentrations as the parent strain. However, the rate of lysis of these mutants in the presence of several p-lactams was much slower than that of the wild type [27]. It is not clear however, why mutants lacking protein 1 B should be super-sensitive to P-lactams [12]. It has been suggested that in these mutants protein 1 A and/or protein 2 may fulfill the function of the absent protein 1B [12]. It is a possibility that such mutants, which are known to be temperature-sensitive, have defective peptidoglycan structures which render the cells more susceptible to the lytic action of P-lactams. LY appeared to have little difficulty in penetrating the outer layers of the cell as revealed by the low crypticity values observed. LY did not apparently penetrate as easily as LY97962 and as a consequence was a less efficient inhibitor of cell growth. The affinities of both compounds for the penicillin-binding proteins in.isolated envelope prepa-

9 rations were similar. Hence, provided that a /?-lactam molecule is resistant to inactivation by /j-lactamases and has a high affinity for the important penicillin-binding proteins in the cytoplasmic membrane, /3-lactams that penetrate to their targets easily will be better inhibitors of growth than molecules that penetrate less readily. Although benzylpenicillin has a fairly high affinity for the penicillin-binding proteins of I?. coli, it has difficulty in penetrating the outer membrane and is also susceptible to /l-lactamases, thus rendering it an inefficient antibiotic against this organism. It is a possibility that the structures of the side-chains that enable a /?-lactam to penetrate readily and also to be resistant to 8-lactamases may result in a molecule that has its highest affinity for protein 3. Filamentation induced by P-lactams binding exclusively to protein 3 results in a cessation of the increase in the number of separate viable cells; any method for determining the minimum growth inhibitory concentration that measures the number of viable cells will count a filament consisting of the equivalent of many cells as a single colony forming unit. However, this apparent bacteriostatic effect on cell numbers caused by the formation of filaments is not reflected by an inhibition of cell growth. If it is assumed that the magnitude of the toxic effects of bacterial growth on mammalian cells is proportional to total cell mass rather than cell number, then the induction of filamentation by a cephalosporin per se would have no desirable therapeutic consequence although it would restrict the number of separate infective units. We are not aware of any evidence that filamentation of bacteria under clinical conditions promotes their clearance from the host. In addition to the two radioactively labelled cephalosporins used in this study, experiments were carried out with unlabelled cefotaxime. The degree of binding of cefotaxiine to a penicillin-binding protein was measured by determining the amount of that protein still available to bind [I4C]- benzylpenicillin. Cefotaxime forms complexes with penicillinbinding proteins that do not break down rapidly under the conditions used (H. A. Chase and P. E. Reynolds, unpublished observations). The results obtained again suggested that filamentation was induced by the binding of cefotaxime to protein 3 and that lysis resulted from binding to protein 1 A. These results could be confirmed directly if cefotaxime were available in a radioactive form. We are currently carrying out experiments with a series of /?-lactams to investigate their binding to penicillin-binding proteins in growing cells using the 'reverse-labelling' technique as used in the experiments with cefotaxime. Penetration of molecules with relative molecular masses up to through the outer membrane of gram-negative cells is believed to occur via passive diffusion through proteins (porins) in the outer membrane [30]. Protein 3 has a 10-fold or greater affinity than protein 1A for the cephalosporins studied in these investigations and hence the majority of the molecules that penetrated through the outer membrane to the cytoplasmic membrane would be expected to bind to protein 3 rather than protein 1A unless the former were already saturated. This argument assumes that both proteins are equally accessible in the cytoplasmic membrane. Experiments that followed the time course of labelling showed that binding to protein 1A did not occur until protein 3 was saturated [31]. The situation is further complicated by the continued synthesis and insertion of penicillin-binding proteins into the membrane that occurs during growth in the presence of a /l-lactam. The continual insertion of high- affinity targets and their binding of /l-lactams increased the amount of antibiotic that has to penetrate before there is sufficient to bind to the targets with lower affinities. The rate of penetration through the outer layers of the cell is dependent on the concentration of p-lactam in the growth medium [30]; the higher the concentration, the faster the /?-lactam will be able to penetrate, the more quickly will protein 3 be saturated and therefore the more quickly will sufficient,+lactams have penetrated for binding to occur to protein 1 A or to proteins with even lower affinities. This suggestion was confirmed by thc quicker onset of lysis that was observed in the presence of high concentrations of 8-lactams. /?-Lactams such as cephaloridine and cephalosporin C show a lytic effect without prior filamentation and this is adequately explained by the observation that these /3-lactams have a higher affinity for protein 1A than protein 3 when binding is studied in preparations of isolated cell envelopes [19]. We thank Dr D. Boyd of Eli Lilly and Co. for providing the radioactive cephalosporins and the Medical Research Council for providing financial support (grant G 978/44/SB). REFERENCES 1. Duguid, J. P. (1946) Edinburgh Med. J. 53, Cardner, A. D. (1940) Nuturc (Lond.) 146, Schwarz, U., Asmus, A. & Frank, H. (1969) J. Mol. Biol Spratt, B. G. (1977) J. Antinzicroh. Chemother. 3, Zimmerman, S. B. & Stapley, E. 0. (1976) Aniimiwoh. Agetzis Chumother. 9, Blumberg, P. M. & Strominger, J. L. (1974) Bacferiol. Rev Gale, E. F., Cundliffe, E., Reynolds, P. E., Richmond, M. 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