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In Vivo Interaction of Fb-Lactam Antibiotics with The

In Vivo Interaction of Fb-Lactam Antibiotics with The

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1980, p. 629-637 Vol. 18, No. 4 0066-4804/80/10-0629/09$02.00/0

In Vivo Interaction of fB-Lactam with the -Binding Proteins of Streptococcus pneumoniae RUSSELL WILLIAMSON, REGINE HAKENBECK,t AND ALEXANDER TOMASZ* The Rockefeller University, New York, New York 10021 The interactions of several ,B-lactam antibiotics with the penicillin-binding proteins (PBPs) of Streptococcus pneumoniae have been studied using whole organisms treated with such antibiotics and subsequently with [3H]benzylpenicil- lin. Differences in chemical structure were shown to cause major and selective changes in the affinities ofthe f,-lactams for the PBPs. Only 4 ofthe 18 compounds tested induced a specific morphological effect (enlargement of the equatorial region) under the particular conditions tested. In 12 of the 18 ,8-lactams studied, a close correlation was found between the minimal inhibitory concentrations and the concentrations required to half-saturate PBP2b. However, such a correlation was no longer apparent when the bacteria were treated with the antibiotics at their minimal inhibitory concentrations. These findings are discussed in the context of various approaches that have been used to identify the growth- inhibitory targets of f8-lactam antibiotics in bacteria.

The interactions of,-lactam antibiotics with recently been described for several organisms by the penicillin-binding proteins (PBPs) of several Chase and Reynolds (1). bacteria lead to a variety of biological conse- quences. Thus, at suitable concentrations, ben- MATERIALS AND METHODS zylpenicillin or , , and Bacterial strains and growth conditions. S. cephalexin or cause cell death (22), pneumoniae strains R6, a derivative of the Rockefeller growth as round forms (24), or growth as fila- University strain R36A, and CW1, an autolytic defi- ments (6, 22), respectively, when added to Esch- cient transformant of the wild type (27), were used erichia coli. Moreover, similar effects have been throughout these studies. Bacteria were grown with- out aeration at 37°C in C medium (9) at an initial pH described with a variety of Enterobacteriaceae of 8.0 and supplemented with yeast extract (0.1%; (3) and (3, 13). Each Difco). Growth was monitored with a Coleman neph- of these antibiotics has been shown to specifi- elometer. cally interact with one (or more) of the multiple Susceptibility tests. The MICs of the various ,B- PBPs of these organisms when tested using iso- lactam antibiotics were determined by twofold serial lated membranes from the bacteria. Therefore dilution in C medium. Exponential-phase organisms these specific PBPs are presumably involved in (approximately 6 x 104 colony-forming units per ml) processes or functions essential to the normal were inoculated into tubes containing the medium (1 growth of the organisms. ml) and incubated for 16 h at 37°C. The lowest con- centration to inhibit growth of the bacteria was re- In many of these and similar studies, the corded as the MIC. correlation between the observed effect and in- Morphology studies. Exponential-phase orga- teraction with a PBP has been measured in nisms (1 ml, about 6 x 104 colony-forming units per terms of the amount of the required ml) ofstrain CW1 were incubated at 37°C with various to produce the effect and that required to half- concentrations (from 100 times less than the MIC up saturate the particular PBP, although the exact to the MIC) of the different ,B-lactam antibiotics. The relevance of such values may obviously depend morphologies of the bacteria were examined by phase- on the accessibility of the PBPs in whole bac- contrast microscopy after 4 and 20 h of incubation. teria. We have therefore studied the interaction The autolytic deficient mutant was used because the wild type autolysed in stationary phase. of several f-lactam antibiotics with exponential- Antibiotics. was obtained from Bee- phase Streptococcus pneumoniae. The correla- cham Laboratories, Piscataway, N.J.; , ce- tions between the minimal growth inhibitory fadroxil (BL-S578), cephalexin, and were ob- concentrations (MICs) of each compound and tained from Bristol Laboratories, Syracuse, N.Y.; and the morphogenic effect induced by four of these (HR756) was obtained from Hoechst- antibiotics are discussed. A similar approach has Roussel Pharmaceuticals Inc., Somerville, N.J. Mecil- linam was supplied by Hoffmann-La Roche Inc., Nut- t Present address: Max-Planck-Instut fiir Molekulare Ge- ley, N.J.; piperacillin came from Lederle Laboratories netik, D-1000 Berlin 33, Germany. Div., American Cyanamid Co., Pearl River, N.Y.; ben- 629 630 WILLIAMSON, HAKENBECK, AND TOMASZ ANTIMICROB. AGENTS CHEMOTHER. zylpenicillin, cephaloridine, and cephalothin were pro- for each antibiotic and the effects on the mor- vided by Eli Lilly & Co., Indianapolis, Ind.; and cefox- phology of the organisms. itin was supplied by Merck, Sharp & Dohme, Rahway, Only 4 of the 18 /)-lactams caused a morpho- N.J. (SCE-129) and sulbenicillin were ob- logical change under the particular conditions tained from Takeda Chemical Industries Ltd., Osaka, Japan, and , and 6-aminopenicil- tested (i.e., exposure of a lyt mutant to a maxi- lanic acid (6-APA) came from Wyeth Laboratories mum of 1 x the MIC). The single type of mor- Inc., Philadelphia, Pa. para-[3H] , phological change observed was swelling of the ethylpiperidinium salt (31 Ci/mmol) was the generous central zone of the bacteria (Fig. 3). gift of E. 0. Stapley, Merck, Sharp & Dohme, Rahway, Several observations were apparent in the N.J. data summarized in Tables 1 and 2. (i) First, for Labeling, separation, and detection of PBPs. each ,B-lactam tested, there appeared to be sig- Exponential-phase organisms (1 ml, about 8 x 107 cells nificant differences in the antibiotic concentra- per ml) were incubated with various amounts of the tions needed to half-saturate one PBP or an- fB-lactam antibiotics (sub- and supra-MIC values) at 37°C for 10 min, and then with an amount of [3H]- other. Different ,8-lactams differed in the degrees benzylpenicillin sufficient to saturate the PBPs of of this type of variation. For instance, the differ- untreated organisms (1 nmol) for a further 10 min (28). ence between the concentrations of benzylpeni- The control samples were preincubated with no anti- cillin needed to half-saturate the PBPs showing biotic before exposure to the radiolabeled benzylpen- the lowest (PBP2b) versus the highest (PBP3) icillin. An excess of unlabeled benzylpenicillin (1.7 affinity for this drug was only about sixfold. In mM) was then added, and the samples were immedi- contrast, this difference was almost 100-fold in ately chilled in ice. The bacteria were recovered by the case of (see 50% benzylpenicillin centrifugation (1,100 x g for 2 min at 2°C), suspended binding inhibition [I50] values for PBP2b versus in 50 pl of 50 mM sodium phosphate buffer (pH 7.0) containing 1.7 mM benzylpenicillin and 1% Sarkosyl PBP3). NL-97, and incubated for 5 min at 37°C. This treat- (ii) Another type of drug-to-drug variation ment resulted in complete lysis of the organisms and was also clear in that fB-lactams differed widely inactivation of the PBPs. The lysates were then pre- in the fractions or multiples of the MICs needed pared for slab gel polyacrylamide electrophoresis. The to half-saturate the various PBPs. In the case of techniques used for discontinuous gel electrophoresis, penicillin, half-saturation of even the most pen- staining, and detection of the PBPs by fluorography icillin-sensitive PBP (PBP3) required as much have been described earlier (28). Binding proteins were as 2x the MIC. However, as little as 0.04x the labeled PBP1 through 3, as introduced in previous MIC of could half-saturate PBPlb. communications (5, 28, 30). Particularly noteworthy was the case of cepha- losporin C, a relatively poor inhibitor of pneu- RESULTS mococcal growth (MIC, 114 nmol/ml). This ,8- The evaluation of 18 different ,B-lactams for lactam was able to half-saturate even the least their effectiveness as inhibitors of the pneumo- sensitive PBPs (PBP2a and -2b) at 0.2x the coccal PBPs was performed with an assay in MIC, whereas the PBP most sensitive to this which exponentially growing bacteria were first B8-lactam (PBPlb) reached half-saturation at a exposed to various ,8-lactams (at concentrations small (0.01x) fraction of the MIC. representing fractions or multiples of their cor- (iii) The antibiotics also differed in their order responding MICs and subsequently to a saturat- of affinities to the various PBPs. PBP3 had the ing dose of [3H]benzylpenicillin. The bacteria highest affinity for 9 of the 18 antibiotics (see were then lysed, and the PBPs were separated values in bold face in Table 1), and, with the by gel electrophoresis. Figure 1 illustrates the exception ofbenzylpencillin, the other 9 ofthese results of this type of assay. The fluorograms could achieve half-saturation of this PBP at demonstrate binding of the [3H]benzylpenicillin concentrations that were fractions of the corre- to the pneumococcal PBPs after pretreatment sponding MICs, e.g., 0.01X, 0.03x, or 0.07x the of the live cells with cefsulodin and cefoxitin. MIC in the cases of cefoxitin, cefadroxil, or ceph- Figure 2 is a graphical representation of the alosporin C, respectively. same results. The relative affinities of the PBPs (iv) For a given PBP, ,B-lactams showed great for the unlabeled benzylpenicillin (3 > la > lb variation in their specific inhibitory activities > 2a > 2b) were the same as those obtained with (compared on the basis of their molar concen- the [3H]benzylpenicillin alone when tested in trations). For instance, in the case of PBP3, of whole organisms, thereby suggesting that the the 18 /3-lactams compared, cephaloridine and procedure was not giving anomalous results. The cefotaxime had the highest 150 values, 0.0066 and effects of 10 and 8 on 0.0086 nmol/ml, respectively. Mecillinam (150, 50 the subsequent binding of [3H]benzylpenicillin nmol/ml) and 6-APA (I50, 160 nmol/ml) had the were evaluated in a similar manner; the results lowest specific inhibitory activities. Figure 4 are summarized in Table 1 along with the MICs shows the relationships between the MICs and VOL. 18,1-LACTAMS1980 AND S. PNEUMONIAE PBPs 631 A nmoles/mI B nmoles/mI

MIC MIC 2.2 7.4 22 111 3331110 Con .12 .37 1.2 3.7 18.4 55 184 ,% mI "*a 10 __w _00m~~~~~~~"Mw ,feee WaON*goo got ==

FIG. 1. Competition of cefsulodin and cefoxitin for the PBPs of S. pneumoniae. The PBPs were detected with [3H]benzylpenicillin after pretreatment of whole organisms with various concentrations of either (A) cefsulodin or (B) cefoxitin.

A Benzylpenicillin, ampicillin, and sulbenicillin 10 3 have the same structure except for substitution on the benzylic carbon of the side chain. The MICs for ampicillin and sulbenicillin were some 4- and 51-fold greater than that of benzylpeni- cillin, but the affinities of ampicillin for the PBPs, particularly lb and 2a, were generally greater, whereas those of sulbenicillin were much lower, although not in proportion to the increase in MIC (Table 1). The small differences in structure caused marked changes in the rela- tive order of affinities of the PBPs (benzylpeni- nmoles cefaulodin/ml cillin, 3 > la > lb > 2a > 2b; ampicillin, lb > 2a B > 3 > la > 2b; sulbenicillin, 3 > lb > 2b > la 10 > 2a). Dicloxacillin and oxacillin differ only in the ~8 disubstitution of the benzyl ring, which results in the former having a sixfold-lower MIC. The - >b affinities of four of the PBPs (la, lb, 2a, and 2b) were also lower for oxacillin than dicloxacillin. -= 4- However, the interaction with PBP3 was some sixfold greater with oxacillin than dicloxacillin; c 2- m 3 in contrast, that of PBPla was reduced by a lb ^ factor of five. These differences produced a ma- jor change in the relative order of affinities of 0-01 0-03 0-1 0 3 1 3 10 30 100 dicloxacillin (la > 2a > lb > 2b > 3) and oxacillin nmoles cefoxitin/mi (3> 2a> 2b> lb> la). FIG. 2. Saturation curves of PBPs after pretreat- Cefoxitin and cephalothin are similar except ment with (A) cefsulodin or (B) cefoxitin. The relative a densities of the PBP bands in Fig. I were measured for the substitution of 7-a-methoxy group and 6by microdensitometry. an amino group on the Z side chain of cefoxitin, and this caused a sevenfold decrease in the MIC. There was also a dramatic decrease of some 425- the corresponding L5o values of each antibiotic fold in the affinity of PBPla for cefoxitin, the listed in Table 1 for PBP2b and 3. Twelve of the affinities of PBPs lb, 2a, and 2b were some ten- 18 antibiotics had a surprisingly good correlation fold less, and that of PBP3 was about twofold between these values in the case of PBP2b, but more. Thus, the greatest change in the relative not for any of the other PBPs. affinities of the PBPs for cephalothin (la, lb > (v) Some of the ,B-lactam antibiotics used in 3 > 2a > 2b) and cefoxitin (3 > lb> 2a> la> this study were structurally similar, and an at- 2b) was in the interaction with PBPla. tempt was made to correlate chemical structure Cefadroxil and cephalexin differ in the addi- with the MICs and PBP affinity patterns. tion of a hydroxyl group at the 4-position of the 632 WILLIAMSON, HAKENBECK, AND TOMASZ ANTIMICROB. AGENTS CHEMOTHER.

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FIG. 3. Scanning electron micrographs of S. pneumoniae. (A) Control organisms; (B and C) organisms grown in the presence ofhalfthe MIC ofpiperacillin for 4 h. Magnification, x30,000. The arrows indicate cell poles. benzyl ring in the former. However, this ap- cephalexin, indicating increased affinity, in line peared to have little effect on either the MIC or with a decreased MIC. The exception was affinities of these antibiotics. All the values for PBPlb, which had a twofold decrease in affinity cefadroxil were some twofold less than those of for cefadroxil. Thus, the relative affinities of 634 WILLIAMSON, HAKENBECK, AND TOMASZ ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Inhibition of binding of/5H]benzylpenicillin to the PBPs ofS. pneumoniae by ,8-lactam antibiotics at their MICs % Inhibitiona of binding to PBP: antibiotic la lb 2a 2b 3 Benzylpenicillin 27 25 23 12 38 Cefotaxime 58 91 90 40 83 Piperacillin 0 8 27 48 51 Cephaloridine 55 82 63 8 89 Nafcillin 9 59 20 41 17 Ampicillin 53 61 63 42 61 Dicloxacillin 12 18 18 11 17 Methicillin 28 39 49 37 29 Cephalothin 100 100 84 41 89 Oxacillin 24 15 56 20 82 Sulbenicillin 26 37 26 35 61 Cefoxitin 31 79 68 11 100 Cefadroxil 48 32 77 31 89 Cephalexin 52 59 73 30 86 Meciinam 11 5 31 9 25 Cefsulodin 48 35 65 40 92 6-APA 20 19 59 13 41 C 98 100 90 93 90 a Percentage of decrease in the amount of [3H]benzylpencillin bound after pretreatment with the particular antibiotic compared with the amount bound in the absence of the aptibiotic. each compound were similar (cefadroxil, 3 > 2a only 4 of the 18 antibiotics tested under the > la > lb > 2b; cephalexin, 3 > 2a > lb > la conditions used, but there was no. obvious cor- > 2b). relation to inhibition of any particular PBP. For Two of the antibiotics tested had extremely instance, for four of the PBPs (lb, 2a, 2b, and 3) high MICs, 105 nmol/ml for 6-APA and 114.4 to which the morphogenic antibiotics had strong mnol/ml for cephalosporin C, yet their interac- affinities, there were examples of other antibi- tions with the PBPs were significantly different. otics (e.g., ampicillin) that bound to each of Whereas 6-APA appeared to have relatively low these PBPs at similar or even lower concentra- affinities for most of the PBPs, cephalosporin C tions without apparent effect. had extremely high affinities in terms ofits MIC, Several attempts have been made to correlate although in molar proportions, even cephalospo- the antibacterial effects of,-lactam antibiotics rin C had low affinity as compared with other (inhibition of growth, killing, and lysis) with ,f-lactam antibiotics with much lower MICs. inhibition of one or more PBPs. Such studies have indicated PBPlb as the major function in DISCUSSION E. coli, the inhibition of which leads to rapid A major purpose ofthese studies was to estab- killing and lysis (22, 23) and induction of uncon- lish a correlation between the biological re- trolled murein hydrolase activity (8). Genetic sponses to ,-lactam antibiotics and the biochem- studies with mutants of E. coli with a thermo- ical event of binding to PBPs in a gram-positive sensitive PBPlb and defective in PBPla sup- organism. Similar studies using membrane prep- ported this notion (26). Additional studies have arations have been reported for a variety of implicated PBP3 of E. coli also as an important gram-negative bacteria (3, 13, 16, 20-22). One of antibacterial target (22, 23). Investigations with the approaches used in such studies was based mutants of E. coli defective in PBPs 4, 5, and 6 on the known selective morphological effect of (7, 10, 11, 25), which retained the same sensitiv- certain /8-lactams in E. coli and the analysis of ity to ,B-lactam antibiotics as the parental strain, the biochemical basis of this selectivity on the indicated that these PBPs did not represent interaction with the PBPs. Thus, the selective physiologically important functions. effect of mecillinam was correlated with binding The binding proteins are presumably proteins to PBP2 (24), and the less selective effects of involved in synthesis of , and it cephalexin and piperacillin were correlated with has been suggested that PBPs lb and 3 of E. binding to PBP3 (6, 22). coli may represent the penicillin-sensitive In pneumococci, only one morphological re- transpeptidases (22). However, several studies sponse has been observed (enlargement of the have failed to establish a consistent correlation equatorial region). Such a change was caused by between the antimicrobial effectiveness (MIC) VOL.V-LACTAMS18, 1980 AND S. PNEUMONIAE PBPs 635 3/ other PBPs. However, such a relationship was A 1.5 not so evident in data from the later and more 100: extensive studies of Curtis et al. (2, 4). In the present studies a similar evaluation of the affin- ities and the MICs the 18 antibiotics indicated roo 10: that only one of the PBPs (2b) exhibited pro- I 1t portionality between these values for the major- o1 ity of the ,B-lactams tested. 4 In an extension of the notion that the PBP with the highest in vitro (membrane) affinity for II B8-lactams would be the likely candidate for the physiologically important target (22), Reynolds et al. (19) studied the affinities of PBPs for benzylpenicillin in growing Bacillus megate- rium. Only one (PBP1) of the five PBPs was 10. 0.1 1 10 100 labeled during exposure of the organisms to the MIC (nmobs/ml) MIC of the antibiotic for 5 min. On the basis of this concept, PBP3 of S. pneumoniae would 100: B "17 fulfill this role, as 9 of the 18 antibiotics had their highest affinities for this PBP. However, the affinities in molar terms for this PBP and lo:^ the corresponding MICs did not show the ex- pected correlation (Fig. 4) since most of the ,8- 8 lactams inhibited this PBP at sub-MIC values. 7 It is also relevant that PBP 2b had the lowest affinity for 10 ofthe antibiotics, 5 and the affinities _s for each antibiotic except cephalosporin C were

0.1 13 all above the MIC values. PBPla was also found 3 6 to have relatively low affinity for most of the 1 antibiotics, PBP2a was generally high, and PBPlb was intermediate. Furthermore, a com- 0.01 2 4 pletely different approach for the identification of physiologically important PBPs was against PBP3 being this target. This approach involved 0.01 0.1 the identification of the changes in PBPs that MIC (nmohs/ml) accompanied acquisition of different levels of FIG. 4. Correlation between MICs of the antibi- intrinsic penicillin resistance in genetic trans- otics tested and their affinities (15;o values) for (A) formants of pneumococci (29, 30) and also in PBP2b and (B) PBP3. The data uvere obtained di- various clinical isolates of pneumococci (5). The rectly from Table 1, and the line for IPBP3 wasplotted analysis of the affinities for benzylpenicillin of after linear regression analysis. the PBPs in the transformants of various resis- tance levels clearly implicated each PBP except of a 83-lactam antibiotic and the specific inhibi- PBP3 as physiologically important targets at tory activity of these compounds in assays of specific levels of resistance. This is in clear con- either model transpeptidase actiivity (14, 17, 18) trast with the behavior of PBPs in the benzyl- or synthesis of peptidoglycan using whole orga- penicillin-susceptible bacteria, in which only the nisms (12). Several authors hasve similarly at- /8-lactam affinity of PBP2b showed a quantita- tempted to establish a quantitative correlation tive relationship with the MIC of the particular between the MICs of ,B-lactam antibiotics and antibiotic. Thus, these findings apparently con- their affinity for the various P]BPs of E. coli, tradict the notion that the most drug-sensitive assayed as the ability ofeach antiibiotic to inhibit PBP should be the antimicrobial target and are the subsequent binding of radiioactive benzyl- also inconsistent with the approach as applied penicillin by 50% (Io value) in isolated mem-. in Fig. 4, correlating MIC and I50 values. branes (2, 4, 15, 23). Using four cephalosporins, The examination of degrees of inhibition of Nozaki et al. (15) obtained a straiightline plotting the various PBPs in growing bacteria after ex- the logarithm of the MIC of ieach antibiotic posure to the antibiotics at their MICs (Table 2) tested against the logarithm of tkie respective I50 indicated that there was no obvious correlation value for PBPlb and 3 of E. coli; similar rela- to a given amount of inhibition. This approach tionships were not obtained from data for the was used to eliminate potential problems that 636 WILLIAMSON, HAKENBECK, AND TOMASZ ANTIMICROB. AGENTS CHEMOTHER. might have arisen from the interpretation of penicillin-binding proteins. Antimicrob. Agents Chem- other. 15:332-336. similar experiments performed in vitro with 3. Curtis, N. A. C., D. Orr, G. W. Ross, and M. G. membrane preparations due to possible differ- Boulton. 1979. Competition of,B-lactam antibiotics for ences in accessibility to the PBPs. The great the penicillin-binding proteins of Pseudomonas aerugi- differences in MICs (6,700-fold) ofthe antibiotics nosa, Enterobacter cloacae, Klebsiella aerogenes, Pro- teus rettgeri, and Escherichia coli: comparison with tested are most likely to reflect differences in antibacterial activity and effects upon bacterial mor- the affinities and inhibitory power of the com- phology. Antimicrob. Agents Chemother. 16:325-328. pounds for the PBPs of pneumococci since pre- 4. Curtis, N. A. C., D. Orr, G. W. Ross, and M. G. liminary experiments have indicated no appar- Boulton. 1979. Affinities of penicillins and cephalospo- rins for the penicillin-binding proteins of Escherichia ent selective permeability barrier in this orga- coli K-12 and their antibacterial activity. Antimicrob. nism. Agents Chemother. 16:533-539. None of the varied approaches discussed 5. Hakenbeck, R., M. Tarpay, and A. Tomasz. 1980. above yielded a consistent, satisfactory interpre- Multiple changes of penicillin-binding proteins in peni- cillin-resistant clinical isolates of Streptococcus pneu- tation of the quantitative relationship of the moniae. Antimicrob. Agents Chemother. 17:364-371. MIC and PBP pattern of a given ,8-lactam. One 6. lida, K., S. Hirata, S. Nakamuta, and M. Koike. 1978. major factor may have contributed to the diffi- Inhibition of cell division of Escherichia coli by a new culty in establishing this correlation using stud- synthetic penicillin, piperacillin. Antimicrob. Agents and in other bacterial Chemother. 14:257-266. ies of the type used here 7. Iwaya, M., and J. L Strominger. 1977. Simultaneous systems. The problem is illustrated in Fig. 2, deletion of D-alanine carboxypeptidase lB-C and pen- which demonstrates that the saturation curves icillin-binding component IV ina mutant of Escherichia of cefoxitin and cefsulodin for the individual coli K-12. Proc. Natl. Acad. Sci. U.S.A. 74:2980-2984. PBPs have distinctly different shapes. Thus, 8. Kitano, K., and A. Tomasz. 1979. Triggering of autolytic degradation in Escherichia coli by #l-lactam although the plot of I5o against MIC of some antibiotics. Antimicrob. Agents Chemother. 16:838- antibiotics for PBP2b gave a good correlation, 848. the more important relationship would seem to 9. Lacks, S., and R. D. Hotchkiss. 1960. A study of the genetic material determining an enzyme activity in be the degree of saturation of PBP2b by these Pneumococcus. Biochim. Biophys. Acta 39:508-517. same antibiotics added to growing bacteria at 10. Matsuhashi, M., L. N. Maruyama, Y. Takagaki, S. their MICs. The data in Table 2 indicated that Tamaki, Y. Nishimura, and Y. Hirota. 1978. Isola- in fact under this condition, the different anti- tion of a mutant of Escherichia coli lacking penicillin- for sensitive D-alanine carboxypeptidase IA. Proc. Natl. biotics with a good 150-MIC correlation Acad. Sci. U.S.A. 75:2631-2635. PBP2b (Fig. 4) gave from 11 to 48% inhibition. 11. Matsuhashi, M., Y. Takagaki, N. Maruyama, S. Ta- However, nafcillin, which did not have a good maki, Y. Nishimura, H. Suzuki, U. Ogino, and Y. Iso-MIC correlation (Fig. 4), gave 44% inhibition. Hirota. 1977. Mutants of Escherichia coli lacking in in these studies indicate highly penicillin-sensitive D-alanine carboxypeptidase The data reported activity. Proc. Natl. Acad. Sci. U.S.A. 74:2976-2979. that affinities of the ft-lactam antibiotics for the 12. Moore, B. A., S. Jevons, and K. W. Brammar. 1979. pneumococcal PBPs are greatly modified by the Peptidoglycan transpeptidase inhibition in Pseudomo- chemical structure and site of modification of nas aeruginosa and Escherichia coli by penicillins and of the cephalosporins. Antimicrob. Agents Chemother. 16: the substituents. Further examination 513-517. interactions of other,8-lactam analogs with the 13. Noguchi, H., M. Matsuhashi, and S. Mitsuhashi. 1979. approach described in this report could yield an Comparative studies of penicillin-binding proteins in establishment of important structure-function Pseudomonas aeruginosa and Escherichia coli. Eur. J. relationships. Biochem. 100:4149. 14. Noguchi, H., M. Matauhashi, M. Takaoka, and S. ACKNOWLEDGMENTS Mitsuhashi. 1978. New antipseudomonal penicillin, PC-904: affinity to penicillin-binding proteins and inhi- These investigations have been supported by the National bition of the enzyme cross-linking peptidoglycan. Anti- Institute of Health (Public Health Service grant AI-16170) microb. Agents Chemother. 14:617-624. and the National Science Foundation (grant PCM 7812770). 15. Nozaki, Y., A. Imada, and M. Yoneda 1979. SCE-963, Additional support has been provided by the Deutsche For- a new potent cephalosporin with high affinity for peni- schungsgemeinschaft (to R.H.) and a Merck Research Fellow- cillin-binding proteins 1 and 3 of Escherichia coli. Anti- ship (to R.W.). R.W. was also the recipient of a Travelling microb. Agents Chemother. 16:20-27. Fellowship from the Wellcome Trust. 16. Ohya, S., M. Yamazaki, S. Sugawara, and M. Mat- We also thank D. Phillips ofthis University forthe scanning suhashi. 1979. Penicillin-binding proteins in Proteus electron micrographs. species. J. Bacteriol. 137:474479. 17. Ohya, S., M. Yamazaki, S. Sugawara, S. Tamaki, and LITERATURE CITED M. Matsuhashi. 1978. New antibiotic, CS- 1. Chase, H. A., and P. E. Reynolds. 1980. The interaction 1170: binding affinity to penicillin-binding proteins and of,B-lactam antibiotics with penicillin-binding proteins inhibition of peptidoglycan cross-linking reactions in studied in vivo. Soc. Gen. Microbiol. Q. 7:78. Escherichia coli. Antimicrob. Agents Chemother. 14: 2. Curtis, N. A. C., C. Brown, M. Boxall, and M. G. 780-785. Boulton. 1979. Inhibition of Escherichia coli K-12 by 18. Oka, T., and H. Fujita. 1978. Effect of fi-lactam antibi- B6-lactam antibiotics with poor antibacterial activity: otics on in vivo peptidoglycan cross-linking by a partic- interaction ofpermeability and intrinsic activity against ulate fraction from Escherichia coli K-12 and Bacillus VOL. 18, 1980 ,8-LACTAMS AND S. PNEUMONIAE PBPs 637

megaterium KM. Antimicrob. Agents Chemother. 14: binding proteins. Proc. Natl. Acad. Sci. U.S.A. 75:664- 625-627. 668. 19. Reynolds, P. E., S. T. Shepherd, and H. A. Chase. 26. Tamaki, S., S. Nakajima, and M. Matsuhashi. 1977. 1978. Identification of the binding protein which may Thermosensitive mutation in Escherichia coli simul- be the target of penicillin action in Bacillus megate- taneously causing defects in penicillin-binding proteins rium. Nature (London) 271:568-570. lB's and in enzyme activity for peptidoglycan synthesis 20. Rodriguez, W. J., and A. K. Saz. 1978. Differential in vitro. Proc. Natl. Acad. Sci. U.S.A. 74:5472-5476. binding of penicillin by membrane fractions from peni- 27. Tomasz, A., and S. Waks. 1975. Mechanism of action of and -resistant Antimicrob. cillin-susceptible gonococci. penicillin: triggering of the pneumococcal autolytic en- Agents Chemother. 13:589-597. zyme by inhibitors of cell wall synthesis. Proc. Natl. 21. Shepherd, S. T., H. A. Chase, and P. E. Reynolds. Acad. Sci. U.S.A. 72:4162-4166. 1977. The separation and properties of two penicillin- binding proteins from Salmonella typhimurium. Eur. J. 28. Williamson, R., R. Hakenbeck, and A. Tomasz. 1980. Biochem. 78:521-532. The penicillin-binding proteins of Streptococcus pneu- 22. Spratt, B. G. 1975. Distinct penicillin-binding proteins moniae grown under lysis-permissive and lysis-protec- involved in the division, elongation, and shape of Esch- tive (tolerant) conditions. FEMS Microbiol. Lett. 7: erichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:2999-3003. 127-131. 23. Spratt, B. G. 1977. Properties of the penicillin-binding 29. Zighelboim, S., and A. Tomasz. 1979. Stepwise acqui- proteins of Escherichia coli. Eur. J. Biochem. 72:341- sition of resistance to oxacillin in Streptococcus pneu- 352. moniae, p. 290-292. In D. Schlessinger (ed.), Microbi- 24. Spratt, B. G., and A. Pardee. 1975. Penicillin-binding ology-1979. American Society for Microbiology, Wash- proteins and cell shape in Escherichia coli. Nature ington, D.C. (London) 254:516-517. 30. Zighelboim, S., and A. Tomasz. 1980. Penicillin-binding 25. Suzuki, H., Y. Nishimura, and Y. Hirota. 1978. On the proteins of multiply antibiotic-resistant South African process of cell division in Escherichia coli: a series of strains of Streptococcus pneumoniae. Antimicrob. mutants of Escherichia coli altered in the penicillin- Agents Chemother. 17:434-442.

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