JOURNAL OF BACTERIOLOGY, Sept. 1994, p. 5574-5577 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Vol. 176, No. 17
Penicillin-Binding Protein 2b of Streptococcus pneumoniae Piperacillin-Resistant Laboratory Mutants
in
REGINE HAKENBECK,l* CHRISTIANE MARTIN,1 CHRIS DOWSON,2 AND THORSTEN GREBE' Max-Planck Institut fir Molekulare Genetik D-14195 Berlin, Germany, 1 and Microbial Genetics Group, School of
Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom2 Received 5 May 1994/Accepted 27 June 1994
In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of P-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective I-lactams.
(10), it was not surprising that PBP 2b appeared not to be affected in any of the cefotaxime-resistant mutants nor to be involved in cefotaxime resistance of clinical isolates (20). In contrast to the C mutants, a low-affinity PBP 2b was common to all three P mutant families (13). Another P mutant-specific phenotype, an apparent reduced amount of PBP la, was noted in two cases. Similar to the C mutants, two P mutants also contained a PBP 2x with slightly reduced affinity. We now investigated mutations in PBP genes of these strains to identify the basis for the phenotypic PBP alterations. The P mutants. P504, P506, and P408 are the most highly resistant members isolated after four or five selection steps (indicated by the first digit of the mutant number) of the three P mutant families 004, 006, and 008 (indicated by the last digit of the mutant number). Properties of their PBPs have been reported elsewhere (13). Common to all three mutants is the appearance of a low-affinity PBP 2b. In addition, reduced affinity was noted in PBP 2x of P506 and P408, and PBP la was apparently produced at reduced amounts in P504 and P506 as judged after labeling with PBP la-specific antibodies. In Fig. 1, the PBP profiles of the three mutants are shown after labeling with 10 ,ug of benzylpenicillin per ml, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of cell lysates, and then Western blotting (immunoblotting) and immunostaining with the help of anti-penicilloyl antibodies (8). All PBPs can be detected in this way. This is in contrast to cefotaxime-resistant mutants of three families in which PBP 2a could not be labeled at penicillin concentrations of as high as 100 ,ug/ml and another C mutant which contained a PBP 2x that could not be labeled (13). In one P mutant, cross-resistance to cefotaxime has been reported (13). We now compared the MICs for piperacillin and cefotaxime in the three most highly resistant mutants, P504, P506, and P408. The MICs for piperacillin were 0.1 to 0.2 ,ug/ml in all three mutants compared with 0.016 ,ug/ml in the parental R6 strain, whereas cefotaxime resistance was noted only in P506 and P408 (0.04 to 0.06 and 0.08 to 0.1 ,ug/ml, respectively, compared with 0.015 to 0.02 ,ug/ml for R6). Mutations in PBP 2b genes. The number of mutations in the PBP 2b genes (penA) was determined after direct sequencing
Penicillin resistance in Streptococcus pneumoniae involves alterations in high-molecular-weight penicillin-binding proteins (PBPs), target enzymes of penicillin action, that result in reduced penicillin affinity. PBPs are members of the superfamily of penicilloyl serine transferases that interact with 1-lactam antibiotics via the formation of a (covalent) serine ester-linked penicilloyl complex (6). In low-penicillin-affinity PBP variants, higher concentrations are required for PBP inhibition and hence for biological activity of 1-lactams. S. pneumoniae contains six PBPs: PBP la, lb, 2x, 2a, and 2b and the lowmolecular-weight DD-carboxypeptidase PBP 3 (9). Changes in penicillin affinity have been observed in as many as four high-molecular-weight PBPs, i.e., la, 2x, 2a, and 2b, of highly resistant clinical isolates (16). To identify point mutations responsible for reduced penicillin affinity and thus to map PBP regions relevant for the interaction with the 1-lactam inhibitor, a series of spontaneous mutants was isolated from the parental S. pneumoniae R6 strain on increasing concentrations of either cefotaxime (C mutants) or piperacillin (P mutants) (13). In all of the more highly resistant C mutants, PBP 2x showed reduced penicillin affinity, and the point mutations were mapped in five independently obtained mutants (14). Two protein regions of PBP 2x appeared to be especially important for 1-lactam interaction since they were affected in several of the mutant proteins: PBP 2x of four mutants contained one or two mutations at the C-terminal end of the penicillin-binding domain between amino acids 597 and 601, and in three mutant proteins, a Thr-550-to-Ala. change had occurred directly adjacent to the Lys-547SerGly triad. The LysSerGly box is one of the three conserved ami.,io acid motifs common to all penicilloyl serine transferases (6); no mutations were found in the vicinity of the other two motit*:, the SerXxxXxxLys tetrad with the active-site serine or the SerXxxAsn triad. Since cefotaxime belongs to a group of 1-lactam antibiotics that do not interact with PBP 2b * Corresponding author. Mailing address: Max-Planck Institut fiir Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany. Phone: 49 30 8413 1340. Fax: 49 30 8413 1385. Electronic mail address:
[email protected]. 5574
NOTES
VOL. 176, 1994
R6 P504 P506 P408 R6
PBP
PBP 2b (615)
A (P506)
la lb 2x -2a 2b
-
5575
D
(P504, P408)
XT2TAESYVAD ...... GQQATNTNAVAYAPSDNPQIAVAVVFPHNTNLTNQVGPSIARDIINLYQKYHPMN-
III
11 I
11
s
KSGTAQIADEKNGGYLVGLTDYIFSAVSMSPAENPDFILYVTVQQPEHYSQIQLGEFANPILERASAMKDSL... D (P506)
PBP 2x (547)
FIG. 2. Point mutations in PBP 2b and PBP 2x of piperacillinresistant mutants. The C-terminal end of the penicillin-binding domain aligned at the KSG and KTG triads is shown. The amino acids affected in the P mutants are underlined; altered amino acids are indicated above or below the peptide sequence. Arrows mark the positions mutated in PBP 2x of the C mutants (14). -3
FIG. 1. PBP profiles of piperacillin-resistant mutants. Cells were labeled with benzylpenicillin (10 ,ug/ml) and lysed with Triton X-100 prior to separation of the proteins in SDS-polyacrylamide gel electrophoresis as described previously (13). PBPs were detected after transfer to a nitrocellulose membrane and labeling with the help of anti-benzylpenicilloyl antibodies (8). PBPs are indicated on the right. PBP la in P504 and P506 appears to be less labeled than it does in P408 and the parental R6 strain because of the apparently reduced amounts of protein (13).
of PCR-amplified chromosomal DNA isolated as described previously (16) by using the oligonucleotide primers, with terminal EcoRI restriction sites, of pGCGAATTCT205T'TA AGTI'AGAAATGAGAC and pGCGAATTCT2283TCCIT TCTA-GTTCATTGG (4, 5). In some cases, overlapping fragments were amplified by using one of the primers indicated above plus an oligonucleotide that primed in the sequence. The conditions used for the PCR have been described elsewhere (19). For direct sequencing, DNA fragments were isolated after separation from agarose gels by using the Biotrap electroseparation system (Schleicher und Schiill, Dassel, Germany) and further purified over miniprep spun columns (Pharmacia, Uppsala, Sweden) as described by the manufacturer. Sequencing was performed by the dideoxynucleotide chain termination method with the Sequenase Version 2.0 sequencing kit (U.S. Biochemical Corp., Cleveland, Ohio) (21). The PBP 2b genes of all three mutants contained one single mutation. In P408 and P504, the same mutation, GG2211T to GAT, resulting in the amino acid substitution Gly-660 to Asp, was observed. In the P506 PBP 2b gene, the GGT-to-GCT mutation changed the Gly-617 to Ala, which is part of the LysSerGly triad. The different PBP 2b mutations are reflected by the different binding properties to piperacillin, the selective antibiotic: labeling of the P504 and P408 PBP 2b required 3- to 6-fold-higher, and that of P506 required approximately 100fold-higher, antibiotic concentrations than the parental R6 strain did (13). It is striking that the PBP 2b mutations map at sites homologous to those observed in PBP 2x of cefotaximeresistant mutants, i.e., after selection with different classes of P-lactams (Fig. 2). It strongly suggests that these sites are crucial for ,-lactam interaction, although PBP 2x and PBP 2b differ in important aspects of their modular design (7). The effect on enzymatic activity of the mutants can be studied with soluble PBP 2b derivatives and substrates as described for the parental protein (1), but the impact on protein structure will ultimately require crystallization of the protein. Homologous sites are also affected in PBP 3 of cephalexin-resistant Escherichia coli mutants obtained after mutagenesis (11, 12). PBP 2
of Neisseria gonorrhoeae and PBP 2b of S. pneumoniae of resistant clinical isolates also carry mutations at the C-terminal domain of the penicillin-binding domain, but amino acid changes close to the SXN triad appear to be primarily responsible for reduced penicillin affinity in both cases (2, 3). Since only one mutation was found in each of the PBP 2b genes, the step at which this was selected should correspond to the appearance of a low-affinity PBP 2b in the respective mutant family. In the two families where the same PBP 2b mutation was observed (004 and 008), the low-affinity PBP 2b appeared at different selection steps, i.e., at step five in P504 and at step two in P208. In the P006 family, the step two mutant contained the low-affinity PBP 2b (13). Thus, no correlation between selection step and mutation was apparent, similar to the situation with PBP 2x in the cefotaxime-resistant mutants (15). Mutations in PBP 2x. P506 and P408, but not P504, contained a PBP 2x with a slightly reduced affinity. The first two mutants were clearly cross-resistant to cefotaxime, but P504 was only marginally so. pbpX genes encoding a low-affinity PBP 2x can be readily selected with cefotaxime in transformation assays using the R6 strain as the recipient (15, 16). To determine whether the cefotaxime resistance was indeed due to an altered PBP 2x, pbpX DNA of the three mutants was amplified by PCR using the oligonucleotide primers Pn2xUP and Pn2xDOWN (16) and was used to transform the parental strain R6 to increased cefotaxime resistance. With both P506 and P408 DNA, transformants could be selected with 0.05 ,ug of cefotaxime per ml. However, transformation with pbpX of P504 did not result in obtaining cefotaxime-resistant mutants. These observations are consistent with the suggestion mentioned above. PCR-amplified pbpX DNA of each of the mutants of the P006 and P008 families was used in transformation assays to determine at which step the PBP 2x mutation had been selected. In both cases, pbpX of the step three mutant (P306 and P308) but not of the step two mutant conferred cefotaxime resistance, demonstrating that, in both mutant families, PBP 2b had been altered prior to PBP 2x. This is different from the situation in penicillin-resistant clinical isolates in which alterations in PBP 2b appear to depend on an altered PBP 2x (4). The P506 pbpX was further investigated. Direct sequencing of PCR-amplified pbpX DNA revealed one mutation, GG2042T to GAT, corresponding to a Gly-597-to-Asp mutation (Fig. 2). The Gly-to-Asp mutation has also been found in pbpX genes of two independent C mutants (18). This site has been suggested to be involved in the interaction of ,-lactam antibiotics in general rather than being specific for cefotaxime. In agreement with this suggestion is the observation that the same mutation has now been identified in the piperacillinresistant mutant. It is also possible that the bulky side chains of
NOTES
5576
J. BACTERIOL.
account for the number of selection steps with which the respective mutants were isolated. This strongly suggests that, at least under laboratory conditions, non-PBP genes are also involved in resistance development.
Piperacillin Resistant Mutants
This work was supported by the Deutsche Forschungsgemeinschaft and the Lister Institute. C.G.D. is a Lister Institute Centenary Research Fellow. REFERENCES
step
zD
2
P208
.$/3 4
5
1. Adam, M., C. Damblon, M. Jamin, W. Zorzi, V. Dusart, M. Galleni, A. El Kharroubi, G. Piras, B. G. Spratt, W. Keck, J. Coyette, J.-M. Ghuysen, M. Nguyen-Disteche, and J.-M. Frere. 1991. Acetyltransferase activities of the high molecular mass essential penicillin-binding proteins. Biochem. J. 279:601-604. 2. Brannigan, J. A., I. A. Tirodimos, Q.-Y. Zhang, C. G. Dowson, and B. G. Spratt. 1990. Insertion of an extra amino acid is the main cause of the low affinity of penicillin binding protein 2 in penicillinresistant strains of Neisseria gonorrhoeae. Mol. Microbiol. 4:913-
919.
family FIG. 3. Alterations in PBP genes of piperacillin-resistant mutants. The mutants isolated at successive steps on increasing concentrations of piperacillin are depicted as circles. The PBP alterations in the mutants are indicated at the step where they appear: (la), reduced amount of PBP la; 2ba, PBP 2b with a G660D mutation; 2bb, PBP 2b with a G617A mutation; 2x, altered PBP 2x.
P-lactams
the two play a role. Whereas the change of Gly-597 to Asp represented the first (and only) mutation in PBP 2x of the P mutant, it was the second mutation introduced in PBP 2x in the C mutant C206 (14). This underlines the notion that the mutational pathway for one PBP is not strictly predetermined. Mutations in PBP la. In P504, where PBP la appeared to be produced in smaller amounts than in the parental R6 strain, the DNA sequence, including the structural gene and 670 bp of the 5' flanking region containing the putative regulatory region (18), was determined from overlapping DNA fragments obtained after PCR amplification. It was completely identical to the R6 sequence (18) and the P408 sequence (17), which was determined for comparison, strongly suggesting that another gene is responsible for the altered PBP la phenotype of this mutant (and presumably also of P506). Identification of this gene is currently under investigation. Interestingly, in both mutant families where the PBP la phenotype was apparent, the step where it occurred was always prior to the selection of a low-affinity PBP 2b. Figure 3 summarizes the results obtained. In all three P mutant families, PBP 2b represents the first PBP gene altered during selection for 1-lactam antibiotic resistance, and this was followed by mutations in PBP 2x in two mutant families. This is in contrast to penicillin-resistant clinical isolates where PBP 2x appears to be the primary target (14), and selection of a low-affinity PBP 2b required the presence of an altered PBP 2x (4). The PBP 2b alteration of the P mutants never occurred at the first selection step, unlike the PBP 2x mutations in C mutants (18). In two P mutants, phenotypic changes in the amount of PBP la which could not be correlated to a mutation in the ponA gene preceded the PBP 2b mutation. Again, C mutants differed in this respect: in addition to PBP 2x, only PBP 2a was affected in three C mutants (13). This suggests that different regulatory systems are responsible for resistance development in the P and C mutant families. The number of mutations in the PBP genes in any of the P mutants did not
3. Dowson, C. G., T. J. Coffrey, C. Kell, and R. A. Whiley. 1993. Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae. Mol. Microbiol. 9:635-643. 4. Dowson, C. G., A. Hutchison, and B. G. Spratt. 1989. Extensive remodelling of the transpeptidase domain of penicillin-binding protein 2B of penicillin resistant South African isolate of Streptococcus pneumoniae. Mol. Microbiol. 3:95-102. 5. Dowson, C. G., A. Hutchison, and B. G. Spratt. 1989. Nucleotide sequence of the penicillin-binding protein 2B gene of Streptococcus pneumoniae. Nucleic Acids Res. 17:7518. 6. Ghuysen, J.-M. 1991. Serine ,B-lactamases and penicillin-binding proteins. Annu. Rev. Microbiol. 45:37-67. 7. Ghuysen, J.-M., and G. Dive. 1994. Biochemistry of the penicilloyl serine transferases, p. 103-129. In J.-M. Ghuysen and R. Hakenbeck (ed.), Bacterial cell wall. Elsevier Sciences BV, Amsterdam. 8. Hakenbeck, R., T. Briese, and H. Ellerbrok. 1986. Antibodies against the benzylpenicilloyl moiety as a probe for penicillinbinding proteins. Eur. J. Biochem. 157:101-106. 9. Hakenbeck, R., T. Briese, H. Ellerbrok, G. Laible, C. Martin, C. Metelmann, H.-M. Schier, and S. Tornette. 1988. Targets of P-lactams in Streptococcus pneumoniae, p. 390-399. In P. Actor, L. Daneo-Moore, M. L. Higgins, M. R. J. Salton, and G. D. Shockman (ed.), Antibiotic inhibition of bacterial cell surface assembly and function. American Society for Microbiology, Washington, D.C. 10. Hakenbeck, R., S. Tornette, and N. F. Adkinson. 1987. Interaction of non-lytic ,B-lactams with penicillin-binding proteins in Streptococcus pneumoniae. J. Gen. Microbiol. 133:755-760. 11. Hedge, P. J., and B. G. Spratt. 1985. Resistance to ,-lactam antibiotics by remodelling the active site of an E. coli penicillinbinding protein. Nature (London) 318:478-480. 12. Hedge, P. J., and B. G. Spratt. 1985. Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin. Eur. J. Biochem. 151:111-121. 13. Laible, G., and R. Hakenbeck. 1987. Penicillin-binding proteins in P-lactam resistant laboratory mutants of Streptococcus pneumoniae. Mol. Microbiol. 1:355-363. 14. Laible, G., and R. Hakenbeck. 1991. Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae. J. Bacteriol. 173:19861990. 15. Laible, G., R. Hakenbeck, M. A. Sicard, B. Joris, and J.-M. Ghuysen. 1989. Nucleotide sequences of the pbpX genes encoding the penicillin binding protein 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506. Mol. Microbiol. 3:1337-1348. 16. Laible, G., B. G. Spratt, and R Hakenbeclk 1991. Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Mol. Microbiol. 5:1993-2002. 17. Martin, C. 1992. Molekulargenetische Untersuchung des Penicillin-bindenden Proteins (PBP la) von Streptococcus pneumoniae:
VOL. 176, 1994
Verwandtschaft von PBP la Mosaikgenen in Penicillin resistenten klinischen Stammen. Ph.D. dissertation. Freie Universitit, Berlin. 18. Martin, C., T. Briese, and R. Hakenbeck. 1991. Nucleotide sequences of genes encoding penicillin-binding proteins from Streptococcus pneumoniae and Streptococcus oralis with high homology to Escherichia coli penicillin-binding proteins 1A and 2B. J. Bacteriol. 174:4517-4523. 19. Martin, C., C. Sibold, and R. Hakenbeck. 1992. Relatedness of penicillin-binding protein la genes from different clones of peni-
NOTES
5577
cillin-resistant Streptococcus pneumoniae isolates in South Africa and Spain. EMBO J. 11:3831-3836. 20. Mufnoz, R., C. G. Dowson, M. Daniels, T. J. Coffey, C. Martin, R. Hakenbeck, and B. G. Spratt. 1992. Genetics of resistance to third generation cephalosporins in clinical isolates of Streptococcus pneumoniae. Mol. Microbiol. 6:2461-2465. 21. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.
