Penicillin-Binding Proteins in Escherichia coli

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1982, p /82/ $02.00/0 Vol. 21, No. 3 Penem Derivatives: 3-Lactamase Stability and Affinity for Penicillin-Binding Proteins in Escherichia coli SATOSHI OHYA,l* YUKIO UTSUI,1 SHINICHI SUGAWARA, AND MITSUO YAMAZAKI' Biological Research Laboratories' and Research Institute,2 Sankyo Company, Ltd., Shinagawa-k, Tokyo 140, Japan Received 8 October 1981/Accepted 15 December 1981 Penem derivatives, a new group of,b-lactam antibiotics with potent activities against a wide range of bacteria, including Pseudomonas aeruginosa, were tested for their stability against hydrolysis by 3-lactamases purified from clinical isolates of Morganella morganii, Proteus vulgaris, and Escheric hia coli and by a penicillinase from Bacillus cerelus. Penems having 6oa substituents, such as hydroxyethyl, hydroxymethyl, and ethyl groups, were very stable against hydrolysis by each of the enzymes. Penems having no 6(x substituents were easily hydrolyzed by P. vulgaris and E. coli enzymes, whereas they were rather stable against hydrolysis by M. morganii and B. cereus enzymes, a typical cephalosporinase and penicillinase, respectively. Affinity of the penems for E. coli penicillinbinding proteins (PBPs) was also tested.,b-lactamase-stable penems having a 60- hydroxyethyl group showed high affinity for PBP-4, -5, and -6 as well as for PBP- 1A, -lbs, and -2. However, the penems having no 6oc substituents showed a far lower affinity for PBP-4, -5, and -6 than that shown by the corresponding 6zxhydroxyethyl penems. Among the penems tested, affinity for PBP-4, -5, and -6 was closely related to their 3-lactamase stability, as was the case among cephamycins and cephalosporins. Effects of the penems on the morphology of a strain of E. coli are also described. Penem derivatives are a new group ot 3- lactam antibiotics having a fused thiazoline ring with a 3-lactam ring. Penems are structurally similar to the new,b-lactam antibiotic known as thienamycin, which is a 1-carbadethiapenem (carbapenem). Thienamycin has been reported to have extremely potent antibacterial activity against both gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa (17, 20). This antibiotic is active against even,blactamase-producing strains that are resistant to penicillins and cephalosporins (H. Kropp, J. S. Kahan, F. M. Kahan, J. Sundelof, G. Darland, and J. Birnbaum, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 16th, Chicago, Ill., abstr. no. 228, 1976). The mode of action of thienamycin seems to be different from penicillins and cephalosporins, and its affinity for target proteins, i.e., penicillin-binding proteins (PBPs), has been reported by Spratt et al. (16). Cephamycins have been shown to be stable against,b-lactamase hydrolysis (1, 2), and the 7cx-methoxy group of cephamycin is considered to be responsible for the stability. In a previous paper (13), we showed that the 7a-methoxy group was also responsible for the binding of the antibiotic to Escherichia coli PBP-4, -5, and -6. In the present study, we examined penem derivatives for their stability against hydrolysis by several types of 3-lactamases, affinity for E. coli PBPs, and morphological effect on E. coli. The 6cx substituent of penem was found to be responsible for both the resistant mechanism against 3- lactamase hydrolysis and binding to PBP-4, -5, and -6. The analogous role of the 60 substituent in penem derivatives to the 7cx-methoxy group in cephamycins is discussed. MATERIALS AND METHODS Chemicals. Cephaloridine (Torni Pharmaceutical Co.), cephalothin, cephalexin (both from Shionogi Pharmaceutical Co.), cefazolin (Fujisawa Chemical Industries), and cefmetazole (Sankyo Co.) were commercially available. All other chemical compounds used in this study were synthesized in the Chemical Research Laboratories of our company. Synthesis of some penem derivatives and their antibacterial activities was reported by Oida et al. (14).,-Lactamases. Three 3-lactamases were purified from clinical isolates of Morganella morganii, Proteus vulgaris (M. Tajima, Y. Takenouchi, S. Ohya, and S. Sugawara, Microbiol. Immunol., in press), and E. coli. Each enzyme preparation gave a single protein band by polyacrylamide gel electrophoresis. The purification and properties of M. morganii enzyme have been reported previously (5). A purified penicillinase from Bacillus cereus was purchased from Calbiochem Co. Determination of hydrolysis rate. Hydrolysis of penems and other,b-lactam compounds, except penicillins, by 3-lactamases was determined spectrophoto- 492

2 VOL. 21, 1982 PENEM 3-LACTAMASE STABILITY AND PBP BINDING 493 metrically by measuring their decrease in UV absorbance as reported previously (5). Wavelengths used for the assay were (in nanometers): cephaloridine and cefuroxime, 260; cephalothin, 262; cefazolin, 263; cefotaxime, 264; cephalexin, 267; cefmetazole, 275; cephalosporin analog of cefmetazole (7a-H-cefmetazole), 273; compounds 1, 2, 3, 4, 5, 6, and 7, 320. Hydrolysis of penicillins was determined by the microiodometric method devised by Novick (11). The hydrolysis rates are given as values relative to that of cephaloridine or penicillin G taken as 100. Assay of PBPs. Affinities of P-lactam compounds for E. coli (K-12 strain JE 1011) PBPs were determined by the competition method with [14C]benzylpenicillin as reported previously (13). The level of [14C]benzylpenicillin remaining bound to each PBP was quantified by microdensitometry of its images on the X-ray films with an Ozumor model OZ-802 densitometer. Morphology of E. coli. The effect of penems, cefmetazole, and 7a-H-cefmetazole on morphological changes of a clinical isolate of E. coli (strain 29) was observed with a phase-contrast microscope as described by Nishino and Nakazawa (10). RESULTS,-Lactamase stability of penems. Substrate specificity profiles of four,-lactamases used in this study are summarized in Table 1. M. morganii P-lactamase is a typical cephalosporinase. The enzyme shows high activity against cephalosporins and low activity against penicillins. P. vulgaris and E. coli 1-lactamases show high activity against both penicillins and cephalosporins. The P. vulgaris enzyme shows an activity against even cefuroxime and cefotaxime, which are known as 3-lactamase-stable oxyiminocephalosporins (4, 9). B. cereus,-lactamase shows activity only against penicillins. None of the four,-lactamases shows ability to hydrolyze cephamycin or cefmetazole, whereas the cephalosporin analog of cefmetazole (7a-H-cefmetazole) is easily hydrolyzed by all but B. cereus enzymes. Figure 1 shows the chemical structures of the Compound 2 3 RI R2 LI, N R2 C02 S-R3 H CH<OH CH3 (R) H CH< OH CH3 (5) H CH20H 4 CH20H H 5 H H 6 H CH<OH CH3 (R) 7 H H NCCH2SCH2CONHNy ) N-N CHCSJ%NON C02- CH3 Cefmetazole R = OCH3 R3 CH2CH2NH2 CH2CH2CH2NH2 7a-H Cefmetazole: R = H FIG. 1. Chemical structures of the compounds used in this study. penems used in this study, cefmetazole and 7a- H-cefmetazole. Relative hydrolysis rates (relative Vmax'S) of penems having either an aminoethylthio or aminopropylthio group at their C-2 positions for the four enzymes are shown in Table 2 with their minimal inhibitory concentrations (MICs) against two E. coli strains: one,blactamase nonproducing and one,-lactamase producing (NIHJ JC-2 and 609, respectively). All of the penems were stable against hydrolysis by both M. morganii cephalosporinase and B. cereus penicillinase. However, they showed different stabilities against hydrolysis by P. vulgar- TABLE 1. Substrate specificity profiles of four,-lactamases Relative Vm,ax Substrate M. morganii P. vulgaris E. coli B. cereus Cephaloridine Cephalothin <0.1 Cefazolin <0.1 Cephalexin <0.1 Cefuroxime <0.1 Cefotaxime < <0.1 Cefmetazole <0.1 <0.1 <0.1 <0.1 7a-H-cefmetazole <0.1 Penicillin G Ampicillin a The hydrolysis of cephaloridine (M. morganii, P. vulgaris, and E. coli) or penicillin G (B. cereus) was given a value of 100.

3 494 OHYA ET AL. TABLE 2. Relative hydrolysis rates of penems by,b-lactamases of M. morganii, P. iulgaris, E. coli, and B. cereus and their MICs against E. coli strains Relative Vmax' MIC (,ug/ml) Compound M. morganii P. vulgaris E. coli E. coli E. coli B. cer-euis NIHJ609I <0.1 <0.1 < <0.1 <0.1 < <0.1 <0.1 <0.1 < < < <0.1 <0.1 <0.1 < < a The hydrolysis of cephaloridine (M. morganii, P. vulgaris, and E. coli) or penicillin G (B. cereus) was given a value of 100. b A,B-lactamase producer. is and E. coli enzymes. The four penems having 6a substituents (compounds 1, 2, 3, and 6) showed high resistance against hydrolysis by both of the enzymes and gave relative Vmax values lower than 0.1. In contrast, the three penems having no 6ot substituents (compounds 4, 5, and 7) were easily hydrolyzed by the two enzymes. By comparing the hydrolysis rates of two stereo isomers, i.e., 6a.-hydroxymethyl penem (compound 3) and 6,B-hydroxymethyl penem (compound 4), we found that the 6at substituent plays an important role in the 1- lactamase stability. Stability of the 6Q-hydroxymethyl penem against hydrolysis by P. vulgaris and E. coli,b-lactamases was 40 or more times greater than that of its 61 counterpart. Substituents at the C-2 position of the penem nucleus were less effective for the f3-lactamase stability. Two hydroxyethyl groups (R and S configurations) at the 6a position of the penems (compounds 1 and 2, respectively) were nearly equally effective for showing the high stability against 1-lactamase hydrolysis, whereas their contributions to antibacterial activity were very different. The results of MICs of the penems against two E. coli strains were considered to reflect the,3-lactamase stability of the penems (Table 2). Each MIC of penems having a 6a substituent against strain 609, which produced a 1-lactamase and showed a high level of resistance against penicillins and cephalosporins, was equal to or only twice greater than its MIC against the NIHJ strain. However, MICs of penems having no 6ot substituents against strain 609 were far greater than those against the NIHJ strain. Affinity of penems for E. coli PBPs. The affinity of compounds 1, 5, 6 and 7 for E. coli PBPs was examined by using the competition method. Figure 2 shows the competition of increasing concentrations of compounds 6 and 7 for the binding of [i4c]benzylpenicillin to the E. coli PBPs. Table 3 shows the concentrations of compounds 1, 5, 6, and 7 required to give 50% competition for [14C]benzylpenicillin binding to the PBPs compared with those of cefmetazole and 7(x-H-cefmetazole reported previously (12, 13). 1-Lactamase-stable penems having 6x-hydroxyethyl groups, compounds 1 and 6, showed high affinities for PBP-4, -5, and -6 as well as for PBP-1A, -lbs, and -2. On the contrary, the lactamase-labile penems having no 6o substituents, compounds 5 and 7, showed low affinity foi 7BP-4 and almost no affinities for PBP-5 and -6, although they showed affinities for PBP-1A, -lbs, and -2 nearly equal to those of compounds 1 and 6. Similar results have been obtained with cefmetazole and 7cx-H-cefmetazole (13). Cefmetazole, which has a 7ot-methoxy group, showed high affinity for PBP-4, -5, and -6 as well as PBP-1A, -lbs, and -3. In contrast, 7oa-Hcefmetazole showed a far lower affinity for PBP- 4, -5, and -6 than that shown by cefmetazole. Pop 1A_: A ANTIMICROB. AGENTS CHEMOTHER. 2- ; 3- i~~~~~~~~~~~~~~~~~~~~~~ 4 -_fwo FIG. 2. Fluorography showing competition of compounds 6 (A) and 7 (B) for ["4C]benzylpenicillin binding to E. coli PBPs. Concentrations of antibiotics are expressed as molar ratios to ['4C]benyzlpenicillin. The same membrane preparations and protein concentrations were used for both compounds. B

4 VOL. 21, 1982 PENEM J3-LACTAMASE STABILITY AND PBP BINDING 495 TABLE 3. Competition of cefmetazole, 7a-H-cefmetazole, and compounds 1, 5, 6, and 7 for binding of [14C]benzylpenicillin to E. coli PBPs Competitor ID50' for [14Clbenzylpenicillin binding to following PBP: compound 1A lbs < < < >125 > < < >125 >125 Cefmetazole > a-H-cefmetazole >125 >125 a ID50, 50% infective dose (molar ratio to ['4C]benzylpenicillin). The P-lactamase stability of cefmetazole was also higher than that of 7a-H-cefmetazole (Table 1). All of the penems tested showed the highest affinity for PBP-2 but very reduced affinity for PBP-3. Cefmetazole and 7a-H-cefmetazole showed high affinity for PBP-3 but low affinity for PBP-2. Effect of penems on morphology of E. coli. Figure 3 shows the effects of compounds 1 and 5 on the morphology of an E. coli strain compared with those of cefmetazole and 7a-H-cefmetazole. Concentrations of compound 1 up to 0.2,ug/ml (Fig. 3a) had little influence on the morphology of the E. coli. Above 0.20 pg/ml, compound 1 caused production of large osmotically stable round cells (Fig. 3b) which were similar to those produced by thienamycin (16; D. Hashizume, F. Ishino, J. Nakagawa, S. Tamaki, and M. Matsuhashi, submitted for publication). Then, lysis of the cells occurred with the release of the cell contents as spheroplasts (Fig. 3c). Compound 5 showed a similar effect on the morphology of the E. coli to that obtained with compound 1 at the same concentration (Fig. 3d). This indicates that the high affinity of compound 1 for PBP-4, -5, and -6 had little effect on the morphology of the E. coli. On the other hand, cefmetazole, which showed a high affinity for PBP-3 and a low affinity for PBP-2, resulted in the production of filamentous cells at concentrations of 0.05,g/ml or higher (Fig. 3e). 7ot-Hcefmetazole also showed a similar effect on the morphology at the same concentration (Fig. 3f), also indicating that high affinity for PBP-4, -5, and -6 had little effect on the morphology. DISCUSSION Penem derivatives tested showed different stability against hydrolysis by P. vulgaris and E. coli,-lactamases, although they were all stable against hydrolysis by M. morganii cephalosporinase and B. cereus penicillinase. Penems having 6ot substituents were stable against hydrolysis by P. vulgaris and E. coli,b-lactamases. In contrast, penems having no 6cx substituents were easily hydrolyzed by both enzymes. This indicates that the 6cc substituents increase the 3- lactamase stability of the penems. A similar result has been obtained with cephamycins and their corresponding cephalosporin analogs (1). Cephamycins are far more stable against I- lactamase hydrolysis than their corresponding cephalosporin analogs. The 7ot-methoxy group of cephamycins may prevent the enzymic attack from the a side of the P-lactam ring. The results obtained in this study strongly suggest that the 6a substituent of penems may play a similar role against,-lactamase hydrolysis. Thienamycin and PS-5, members of the new P-lactam antibiotics known as carbapenems, also have 6a substituents (hydroxyethyl and ethyl groups, respectively). The two carbapenems have also been reported to be stable against 1-lactamase hydrolysis (15, 17). Therefore, we concluded that the 6a substituents of penems and carbapenems also prevent the attack of,-lactamase from the ca side of the,b lactam ring as well as does the 7ocmethoxy group of cephamycins. The results of PBP-binding of penems were also comparable with those of cefmetazole and 7a-H-cefmetazole (Table 3). Penems having the 6a-hydroxyethyl group showed a far higher affinity for PBP-4, -5, and -6 than their corresponding penems having no 6ac-substituents (6cx- H-penems). Cefmetazole, which has the 7amethoxy group, also showed a far higher affinity for PBP-4, -5, and -6 than its cephalosporin analog. It may be reasonable to consider that the 6ac position of penems corresponds to the 7cx position of cephamycins with respect to their sterically relative position to the P-lactam ring. Therefore, the 6ot-hydroxyethyl group in penems may play a role similar to that of the 7ccmethoxy group of cephamycins with respect to both the resistant mechanism against P-lactamase hydrolysis and the binding to PBP-4, -5, and -6. We cannot explain precisely at present why,blactamase stability is related to affinity for PBP- 4, -5, and 6 in both penems and cephems (cepha-

5 496 OHYA ET AL. ANTIMICROB. AGENTS CHEMOTHER. Downloaded from FIG. 3. Phase-contrast micrographs of E. coli 29: a, normal E. (oli showing typical rod shape; b and c, E. coli exposed to 0.78,ug of compound 1 per ml for 90 min (b) and 180 min (c); d, exposed to 0.78 p.g of compound 5 per ml for 90 min; e, exposed to 0.78,ug of cefmetazole per ml for 90 min; f, exposed to 0.78 V.g of 7ox-H-cefmetazole per ml for 90 min. Bar, 1 p.m. on September 20, 2018 by guest mycins and cephalosporins). However, one reason might be that PBPs and 1-lactamases have something in common with respect to their molecular structures. Tipper and Strominger (19) hypothesized that 1-lactamases have evolved from enzymes inhibited by P-lactam antibiotics. In fact, Tamura et al. (18) reported that in E. coli, penicillin-sensitive D-alanine carboxypeptidase 1A, which was identified as PBP- 5 and -6, showed,b-lactamase activity and yielded benzylpenicilloic acid from benzylpenicillin. Moreover, 1-lactamase activity has also been demonstrated for D-alanine carboxypeptidases of Bacillus stearothermophilus (7), Actinomadura sp. (6), Streptomyces sp. (3, 6) and Staphylococcus aureus (8). Therefore, penems and cephems that are easily hydrolyzed by P-lactamases may also be hydrolyzed by PBP-4, -5, and -6. Hence, they may show practically no affinity for the PBPs. Cephamycins and 6ox-hydroxyeth-

6 VOL. 21, 1982 yl penems may be stable against hydrolysis by PBP-4, -5, and -6. We have reported kinetic release of cefmetazole from PBP-5 and -6 of E. coli (13). Cefmetazole was not released from PBP-5 and -6. This may also indicate that I- lactamase-stable 1-lactam is not hydrolyzed by PBP-4, -5, and -6. Among both penems and cephems, we could not find any correlation between their affinity for PBP-4, -5, and -6 and their effect on the morphology of E. coli. However, their affinity for PBP-2 and -3 was closely related to their effect on the morphology. Penems, which showed higher affinity for PBP-2 than for PBP-3, produced ovoid cells. Cephems, which showed higher affinity for PBP-3 than for PBP-2, produced filamentous cells. ACKNOWLEDGMENTS We thank M. Matsuhashi, the Institute of Applied Microbiology, University of Tokyo, for generous advice and helpful discussions. We also thank S. Oida and co-workers at the Chemical Research Laboratories of our company for supplying the penem derivatives. LITERATURE CITED 1. Birnbaum, J., E. 0. Stapley, A. K. Miller, E. Celozzi, H. Wallick, B. A. Pelak, S. B. Zimmerman, D. Hendlin, and H. B. Woodruff Development of the semisynthetic cephamycin, cefoxitin, as a clinical candidate. Infection 7(Suppl. 1): Daoust, D. R., H. R. Onishi, H. Wallick, D. Hendlin, and E. 0. Stapley Cephamycins, a new family of 13- lactam antibiotics: antibacterial activity and resistance to,b-lactamase degradation. Antimicrob. Agents Chemother. 3: Frere, J. M., J. M. Ghuysen, H. Vanderhaeghe, P. Adriaens, J. Degelaen, and J. de Graeve Fate of thiazolidine ring during fragmentation of penicillin by exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61. Nature (London) 260: Fu, K. P., and H. C. Neu Beta-lactamase stability of HR756, a novel cephalosporin, compared with that of cefuroxime and cefoxitin. Antimicrob. Agents Chemother. 14: Fujii-Kuriyama, Y., M. Yamamoto, and S. Sugawara Purification and properties of beta-lactamase from Proteus morganii. J. Bacteriol. 131: Ghuysen, J. M Penicillin-sensitive enzymes of pep- PENEM t-lactamase STABILITY AND PBP BINDING 497 tidoglycan metabolism, p In D. Schiessinger (ed.), Microbiology American Society for Microbiology, Washington, D.C. 7. Hammerstrom, S., and J. L. Strominger Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus. Proc. Natl. Acad. Sci. U.S.A. 72: Kozarich, J. W Penicillinase, carboxypeptidase, and transpeptidase activities from Staphylococcus aureus H., p In D. Schlessinger (ed.), Microbiology American Society for Microbiology, Washington, D.C. 9. Neu, H. C., and K. P. Fu Cefuroxime, a betalactamase-resistant cephalosporin with a broad spectrum of gram-positive and -negative activity. Antimicrob. Agents Chemother. 13: Nishino, T., and S. Nakazawa Morphological changes in Staphylococcus aureus and Escherichia coli exposed to cephalexin. Jpn. J. Microbiol. 16: Novick, R. P Micro-iodometric assay for penicillinase. Biochem. J. 83: Ohya, S., M. Yamazaki, S. Sugawara, and M. Matsuhashi Penicillin-binding proteins in Proteus species. J. Bacteriol. 137: Ohya, S., M. Yamazaki, S. Sugawara, S. Tamaki, and M. Matsuhashi New cephamycin antibiotic, CS-1170: binding affinity to penicillin-binding proteins and inhibition of peptidoglycan cross-linking reactions in Escherichia coli. Antimicrob. Agents Chemother. 14: Oida, S., A. Yoshida, T. Hayashi, N. Takeda, T. Nishimura, and E. Ohki Synthesis of penems and their antibacterial activities. J. Antibiot. 33: Sakamoto, M., H. Iguchi, K. Okamura, S. Hori, Y. Fukagawa, and T. Ishikura PS-5, A new,b-lactam antibiotic. II. Antimicrobial activity. J. Antibiot. 32: Spratt, B. G., V. Jobanputra, and W. Zimmermann Binding of thienamycin and clavulanic acid to the penicillin-binding proteins of Escherichia coli K-12. Antimicrob. Agents Chemother. 12: Tally, F. P., N. V. Jacobus, and S. L. Gorbach In vitro activity of thienamycin. Antimicrob. Agents Chemother. 14: Tamura, R., Y. Imae, and J. L. Strominger Purification to homogeneity and properties of two D-alanine carboxypeptidases I from Escherichia coli. J. Biol. Chem. 251: Tipper, D. J., and J. L. Strominger Mechanism of action of penicillins: a proposal based on their structural similarity to acyl-d-alanyl-d-alanine. Proc. Natl. Acad. Sci. U.S.A. 255: Weaver, S. S., G. P. Bodey, and B. M. LeBlanc Thienamycin: new beta-lactam antibiotic with potent broad-spectrum activity. Antimicrob. Agents Chemother. 15: