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Antimicrob Agents Chemother. Nov 2005; 49(11): 4622–4627.
PMCID: PMC1280142

Amino Acid Mutations Essential to Production of an Altered PBP 2X Conferring High-Level β-Lactam Resistance in a Clinical Isolate of Streptococcus pneumoniae

Abstract

Altered penicillin-binding protein 2X (PBP 2X) is a primary β-lactam antibiotic resistance determinant and is essential to the development of penicillin and cephalosporin resistance in the pneumococcus. We have studied the importance for resistance of 23 amino acid substitutions located in the transpeptidase domain (TD) of PBP 2X from an isolate with high-level resistance, isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). Strain R62X/2B/1A/mur (for which the MICs are as described for isolate 3191) was constructed by transforming laboratory strain R6 with all the necessary resistance determinants (altered PBPs 2X, 2B, and 1A and altered MurM) from isolate 3191. Site-directed mutagenesis was used to reverse amino acid substitutions in altered PBP 2X, followed by investigation of the impact of these reversions on resistance levels in R62X/2B/1A/mur. Of the 23 substitutions located in the TD of PBP 2X, reversals at six positions decreased the resistance levels in R62X/2B/1A/mur. Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 μg/ml, respectively, and individually displayed the greatest impact on resistance. To a lesser extent, reversal of the Leu364Phe, Ala369Val, Arg384Gly, and Tyr595Phe substitutions individually also decreased the penicillin and cefotaxime MICs. Reversal at all six positions collectively decreased both the penicillin and the cefotaxime MICs of R62X/2B/1A/mur to 0.06 μg/ml. This study confirms the essential role of altered PBP 2X as a resistance determinant. Our data reveal that, for isolate 3191, the six amino acid substitutions described above are collectively essential to the production of an altered PBP 2X required for high-level resistance to penicillin and cefotaxime.

Streptococcus pneumoniae (the pneumococcus) contains a set of six penicillin-binding proteins (PBPs). High-molecular-weight (80- to 90-kDa) PBPs 2X, 2B, 1A, 1B, and 2A have transpeptidase domains (TDs) which play a vital role in cell wall (peptidoglycan cross-linking) synthesis, while low-molecular-weight (45-kDa) PBP 3 is a carboxypeptidase which is thought to regulate the degree of peptidoglycan cross-linking. β-Lactam antibiotics (penicillins and cephalosporins) inhibit the growth of pneumococci by targeting the antibiotic-sensitive transpeptidase activity of high-molecular-weight PBPs, thereby inactivating the protein and inhibiting cell wall synthesis. Pneumococcal resistance to β-lactams is essentially due to a complex production of altered PBPs with decreased affinities for these antibiotics (9, 22, 26, 31). A functional operon for MurMN is required for the expression of PBP-mediated β-lactam resistance in the pneumococcus (6, 30). In conjunction with altered PBPs, alterations in MurM may assist in the development of β-lactam resistance (27). Altered PBPs 2X, 2B, and 1A are the major players in the development of β-lactam resistance in clinical isolates; and this is well documented by extensive data (1, 9, 20, 22, 26, 31). These altered PBPs are encoded by genes of a mosaic organization in which parts of the genes are replaced by allelic variants that differ by up to 25% in DNA sequence (5, 14, 15). On the other hand, only a few reports have described altered PBPs 1B, 2A, and 3 as β-lactam resistance determinants (2, 8, 11, 14, 22, 28). Most recently, Smith and coworkers (28) have shown that an altered PBP 2A was essential to the development of penicillin, cefotaxime, and ceftriaxone resistance in a specific clinical isolate of pneumococcus.

Attempts to isolate a pneumococcal deletion mutant of PBP 2X have failed (10), suggesting that PBP 2X is an essential protein with an indispensable role in peptidoglycan synthesis. Recently, immunofluorescence microscopy of growing and dividing cells has found that PBP 2X displays a specific septal localization and therefore plays a major role in cell division and septation (17). This confirmed previous assumptions that PBP 2X is involved in cell division, as the pbp2X gene is located within a gene cluster devoted to cell division (16). In addition, altered PBP 2X is a primary β-lactam resistance determinant and the first PBP to undergo alteration with respect to the development of penicillin and cephalosporin resistance (12, 22, 23). The amino acid changes in altered PBP 2X that are responsible for the development of β-lactam resistance in clinical isolates of pneumococci are not well defined. A number of studies have used penicillin and cefotaxime to select spontaneous laboratory mutants of low-level resistance and associated resistance with variable amino acid substitutions in PBP 2X, most notably, His394Tyr, Leu403Phe, Arg512Tyr, Thr526Ser, Thr550Ala, Gln552Glu, Gly597Asp, Leu600Trp, and Gly601Glu (12, 13, 23). Among clinical isolates, it has been shown that the Thr550Ala substitution in PBP 2X results in increased cephalosporin resistance accompanied by decreased penicillin resistance (4). Most other studies of clinical isolates have only gone as far as to assert that an association of β-lactam resistance with mutations in altered PBP 2X exists. Those studies have emphasized mostly the mutations within and in close proximity to three conserved amino acid sequence motifs, most notably, a mutation at position 338 within the active-site serine motif. Some studies have taken the process one step further to prove the actual involvement of specific PBP 2X mutations in the development of resistance by using site-directed mutagenesis (SDM) to alter specific residues in PBP 2X, followed by an analysis of the effects of these mutations on the acylation efficiency of PBP 2X by cefotaxime and penicillin. Such studies have proved that Thr338Ala, Met339Phe, Thr550Ala, Gln552Glu, and Ser571Ala are influential mutations that affect acylation efficiencies (3, 18, 19).

The identification of resistance-causing alterations in PBP 2X may assist in the design of improved β-lactam antibiotics to counter bacterial resistance to the currently available antibiotics. It may also assist in the development of genetic tests (multiplex PCRs and microarrays) for the rapid diagnosis of penicillin and cephalosporin resistance in the pneumococcus. No study has yet analyzed mutations in altered PBP 2X from a clinical isolate with respect to their importance in contributing to the development of high-level β-lactam resistance in the presence of other participating resistance determinants and with the interaction of other participating resistance determinants. With this in mind, we have analyzed the altered PBP 2X from highly β-lactam-resistant pneumococcal isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml), a representative of a Hungarian serotype 19A clone isolated during the period from 1997 to 1998. Compared to susceptible strain R6, this isolate has 23 amino acid mutations located in the TD of PBP 2X. We were interested in determining which of these mutations are essential for the development of resistance. Within the genetic background of resistance, we wanted to investigate how resistance would be influenced when SDM was used to reverse specific PBP 2X mutations. Should reversal of a mutation accompany a decrease in resistance of the mutant strain, this would signal the loss of an essential resistance-causing mutation. Using this approach, we report on six amino acid substitutions identified as essential in the development of high-level penicillin and cephalosporin resistance.

MATERIALS AND METHODS

Pneumococcal culture and MIC susceptibility tests.

Pneumococci were routinely cultured at 37°C in 5% CO2 on Mueller-Hinton agar (Oxoid, Basingstoke, England) supplemented with 5% defibrinated horse blood (blood agar). The antibiotic MICs of the isolates were determined by the Etest (AB BIODISK, Solna, Sweden) and the agar dilution method (21).

Genomic DNA isolation, PCR, and DNA sequencing.

Chromosomal DNAs were extracted from bacterial cells, and genes were amplified from the chromosomal DNAs by PCR by methods that have been described previously (25, 27, 29). The PCR products were purified from the agarose gel by using a Geneclean kit (Bio 101, Inc., La Jolla, CA) and sequenced by using the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems model 310 automated DNA sequencer.

Mutagenesis and transformation studies.

Genes from isolate 3191 coding for the full open reading frames for PBP 2X, PBP 2B, PBP 1A, and MurM were separately cloned into plasmid pGEM-3Zf (Promega, Madison, WI) by standard techniques, followed by DNA sequencing to confirm the nucleotide sequence of the entire cloned genes. The above DNA was used for transformation studies; therefore, all transforming DNA was in the form of plasmid DNA. Pneumococci were made competent and transformed, and transformants were selected as described previously (26). Antibiotic-susceptible pneumococcal strain R6 (penicillin and cefotaxime MICs, 0.015 μg/ml) was the initial recipient in transformation experiments. As described previously (26, 27), antibiotic-resistant strain R62X/2B/1A/mur (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml) was created by transforming strain R6 with “resistance” DNA from donor isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). The superscript lettering (2X/2B/1A/mur) of strain R62X/2B/1A/mur indicates that the strain has an altered PBP 2X, an altered PBP 2B, an altered PBP 1A, and an altered MurM. For strain R62X/2B/1A/mur, the TD-encoding region of PBPs and the full murM gene were sequenced to confirm the presence of altered genes. For the sake of our mutagenesis analysis of PBP 2X, we then needed to construct an R62B/1A/mur strain (details of this strain construction are described separately below). The presence of an unaltered PBP 2X in strain R62B/1A/mur resulted in antibiotic susceptibility (penicillin MIC, 0.03 μg/ml; cefotaxime MIC, 0.015 μg/ml). The QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce mutations into the wild-type altered pbp2X gene from donor isolate 3191 and to introduce mutations into an unaltered pbp2X gene. Mutagenized pbp2X DNA was then used to transform strain R62B/1A/mur, followed by plating and selection on blood agar plates containing an increasing penicillin concentrations ranging from 0.06 to 16 μg penicillin per ml of agar. Transformants (mutagenized R62X/2B/1A/mur) were picked from the plates containing the highest penicillin concentration. For each mutagenesis and transformation experiment, four transformants were selected for further analysis. MIC susceptibility tests were performed with the transformants, and their TD-encoding region of PBP 2X was sequenced to confirm the presence of a mutagenized DNA sequence.

Construction of strain R62B/1A/mur.

The construction of strain R62B/1A/mur has been described previously (24). Briefly, it essentially involves the transformation of strain R62X/2B/1A/mur with an unaltered pbp2X gene. Transformants would be converted to a susceptible phenotype, as the presence of an altered PBP 2X is vital for the development of penicillin resistance. To select for this penicillin-susceptible phenotype, we created a transforming DNA fragment which contained an unaltered pbp2X gene and an erythromycin resistance marker. Penicillin-susceptible transformants could therefore be indirectly selected by virtue of their simultaneous transformation to erythromycin resistance. The erythromycin resistance marker was an 860-bp erythromycin resistance gene cassette (PcEm) which replaced an 860-bp internal segment of the yllC gene. This replacement was in frame; therefore, the reading frame of the pneumococcal genome was not affected. On the genome of strain R6, yllC is the second gene upstream of pbp2X, with its start codon located 1,283 nucleotides from the start codon of pbp2X. Although no function has yet been described for the YllC protein, it has been shown to be nonessential for pneumococcal growth; therefore, inactivation of the gene is a nonlethal event. The transforming DNA fragment was 4,938 bp and started 980 nucleotides upstream of yllC and ended 420 nucleotides downstream of pbp2X. Following transformation of R62X/2B/1A/mur, erythromycin-resistant transformants were selected on blood agar plates containing erythromycin at a concentration of 0.5 μg/ml. The TD-encoding region of PBP 2X from the transformants was sequenced to confirm the presence of an unaltered pbp2X gene and to therefore confirm the creation of R62B/1A/mur. MIC susceptibility tests found strain R62B/1A/mur to be antibiotic susceptible (penicillin MIC, 0.03 μg/ml; cefotaxime MIC, 0.015 μg/ml). For control purposes, we also created an erythromycin-resistant transformant in the presence of an altered PBP 2X. This erythromycin-resistant control strain, R62X/2B/1A/mur, was revealed to have full β-lactam resistance (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml), demonstrating that the loss of yllC does not affect β-lactam resistance.

Nucleotide sequence accession numbers.

The PBP 2X sequence data appear in the EMBL, GenBank, and DDJB nucleotide sequence data libraries under accession numbers: AY936480 for isolate 3191 and X16367 for strain R6.

RESULTS AND DISCUSSION

The crystal structure of pneumococcal PBP 2X has revealed a three-domain protein, of which the central domain is a 350-amino-acid residue domain with transpeptidase activity (7). This TD is the β-lactam-sensitive component of PBP 2X. The TD starts at approximately 60 amino acid residues before the active-site serine residue that is acylated by penicillin and ends approximately 60 residues after the conserved Lys547-Ser-Gly motif. The TD-encoding region of PBP 2X from highly β-lactam-resistant pneumococcal isolate 3191 (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml) was sequenced, and this revealed 14% nucleotide sequence divergence compared to the sequence of susceptible strain R6, resulting in 23 amino acid substitutions in the protein (Fig. (Fig.1).1). Only a few of these mutations may be essential for decreasing the affinity of the protein for a β-lactam and so confer β-lactam resistance. Some mutations are probably compensatory effects, which occur to keep the enzymatic function of the protein optimal. Some mutations may not be associated with resistance but, rather, may be associated with the origin of an altered pbp2X gene and its interspecies movements within the streptococcal population.

FIG. 1.
Amino acid sequences of the transpeptidase domain of PBP 2X from susceptible strain R6 (line 1) and resistant isolate 3191 (line 2). Only those positions that differ from the sequence of strain R6 are shown in line 2. The conserved amino acid sequence ...

We present a mutagenesis study which has investigated all amino acid mutations in the TD of altered PBP 2X from isolate 3191 to investigate the importance of every mutation in contributing to the development of high-level β-lactam resistance in the presence of other participating resistance determinants and with the interaction of other participating resistance determinants. Before mutagenesis analysis could commence, we needed to construct a suitable transformation recipient with a suitable genetic background with respect to all participating resistance determinants (PBPs 2X, 2B, and 1A and MurM). Strain R62B/1A/mur was therefore constructed and contained an unaltered PBP 2X, an altered PBP 2B, an altered PBP 1A, and an altered MurM (the superscript lettering indicates the presence of an altered protein). Isolate 3191 was the donor of “resistance” genes coding for all altered PBPs and altered MurM. Strain R62B/1A/mur showed antibiotic susceptibility (penicillin MIC, 0.03 μg/ml; cefotaxime MIC, 0.015 μg/ml). This confirms the vital role that an altered PBP 2X plays in the development of resistance. Even though all other resistance determinants (altered PBP 2B, altered PBP 1A, and altered MurM) are in place, these have no effect on resistance while PBP 2X remains unaltered. When strain R62B/1A/mur is then transformed with an altered pbp2X gene, it results in the selection of R62X/2B/1A/mur transformants with high-level resistance (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). These transformants are selected on blood agar containing a penicillin concentration as high as 12 μg/ml.

For SDM analysis of PBP 2X, amino acid mutations were inactivated (the substitutions were reversed) in altered PBP 2X, followed by investigation of the effect of mutagenized PBP 2X on the level of penicillin and cefotaxime resistance in strain R62X/2B/1A/mur. These mutagenized R62X/2B/1A/mur strains were created by transforming R62B/1A/mur with mutagenized pbp2X genes. Should the inactivated mutation be important for resistance development, then mutagenized R62X/2B/1A/mur transformants would be selected on blood agar containing a penicillin concentration less than 12 μg/ml, i.e., in association with a decreased antibiotic MIC. Table Table11 shows the effect that reversal of amino acid substitutions in altered PBP 2X had on the level of resistance in mutagenized R62X/2B/1A/mur strains compared to that in strain R62X/2B/1A/mur (penicillin MIC, 16 μg/ml; cefotaxime MIC, 4 μg/ml). Of the 23 substitutions, reversal at six positions decreased the levels of penicillin and cefotaxime resistance. Reversal of the Thr338Pro and Ile371Thr substitutions individually decreased the penicillin and cefotaxime MICs to 2 and 1 μg/ml, respectively. These two positions individually displayed the greatest impact on resistance. This was at least expected of the Thr338Pro substitution, as it is located immediately adjacent to the active-site Ser337 residue that is acylated by β-lactams. Reversal of the Tyr595Phe substitution decreased the penicillin and cefotaxime MICs to 4 and 1 μg/ml, respectively. Tyr595Phe is located at the C-terminal end of the TD. This area of the TD has been suggested to be an important area for β-lactam recognition of PBP 2X (7); therefore, it is not surprising that reversal of the Tyr595Phe substitution had an impact on resistance. Furthermore, spontaneous laboratory mutants with low-level β-lactam resistance are also frequently associated with mutations in this area, most notably, Gly597Asp, Leu600Trp, and/or Gly601Glu (12, 13). Lastly, reversal of the Leu364Phe, Ala369Val, and Arg384Gly substitutions individually decreased the penicillin and cefotaxime MICs to 8 and 2 μg/ml, respectively.

TABLE 1.
Reversal of substitutions at amino acid positions in altered PBP 2X and the resulting effect of this mutagenesis on resistance levels in mutagenized R62X/2B/1A/mur strains compared to those for R62X/2B/1A/mura

These results clearly showed that no single amino acid substitution in altered PBP 2X is sufficient to allow the development of high-level resistance. We were interested in creating a mutagenized PBP 2X which had all of the six amino acid substitutions described above reversed in order to investigate their combined effect on altered PBP 2X and its development of resistance in mutagenized R62X/2B/1A/mur. These substitutions could not be reversed in a single step; therefore, we had to start with one reversal and build from there. A logical place to start was at position 338, adjacent to the active-site serine residue, following which more reversals were added, but not necessarily in any specific order. Our aim was not to create any specific combinations of reversals, as there would be too many possible combinations to work with. Our focus was reaching the end result with all six mutations reversed in PBP 2X. However, as combinations of reversals resulted, we do report their effect on resistance in mutagenized R62X/2B/1A/mur (Table (Table1).1). Reversal of the Thr338Pro, Ala369Val, and Ile371Thr substitutions collectively decreased the penicillin and cefotaxime MICs to 0.25 and 0.125 μg/ml, respectively. Reversal of the Thr338Pro, Leu364Phe, Ala369Val, Ile371Thr, and Tyr595Phe substitutions collectively resulted in penicillin and cefotaxime MICs both at 0.125 μg/ml. Finally, reversal at all six positions (Thr338Pro, Leu364Phe, Ala369Val, Ile371Thr, Arg384Gly, and Tyr595Phe) collectively decreased both the penicillin and the cefotaxime MICs to 0.06 μg/ml. These results showed that collectively these six amino acid mutations produce an altered PBP 2X with a low affinity for penicillin and cefotaxime and therefore collectively play an essential role in the development of high-level resistance to these β-lactams. The presence of these mutations in altered PBP 2X switches the penicillin and cefotaxime resistance of a pneumococcal strain from a susceptible phenotype (MIC, 0.06 μg/ml) to a fully resistant phenotype (penicillin MIC, 16 μg/ml; cefotaxime MIC 4, μg/ml).

We then proceeded to investigate whether it was possible to take unaltered PBP 2X, introduce these six amino acid mutations, and so create a functional altered PBP 2X that could mimic that of the wild-type altered PBP 2X from resistant isolate 3191. As before, a couple of steps would be required to introduce these multiple mutations into PBP 2X. The order of introduction of mutations followed the order of the previous approach. As before, our aim was not to create any specific combinations of mutations, as our focus was reaching the end result with all six mutations introduced into PBP 2X. However, as combinations of mutations resulted, we do report their effect on resistance (Table (Table2).2). Altered pbp2X gene constructs were used to transform strain R62B/1A/mur (penicillin MIC, 0.03μg/ml; cefotaxime MIC 0.015, μg/ml), followed by penicillin selection of transformants and an assay for increased resistance (Table (Table2).2). Once again we started at position 338 and so created a PBP 2X with a Thr338Pro substitution. R62X/2B/1A/mur transformants which showed penicillin and cefotaxime MICs increased to 0.125 and 0.06 μg/ml, respectively, were selected. Resistance was further increased, by the combination of Thr338Pro, Ala369Val, and Ile371Thr substitutions, to penicillin and cefotaxime MICs of 0.5 and 0.125 μg/ml, respectively. This increased further by the combination of Thr338Pro, Ala369Val, Ile371Thr, and Tyr595Phe substitutions, to penicillin and cefotaxime MICs of 2 and 0.5 μg/ml, respectively. Even further increases occurred with the combination of the Thr338Pro, Leu364Phe, Ala369Val, Ile371Thr, and Tyr595Phe substitutions, up to penicillin and cefotaxime MICs of 4 and 1μg/ml, respectively (these transformants were selected on blood agar containing a penicillin concentration to a maximum of 2 μg/ml). When an altered pbp2X gene construct containing all six amino acid substitutions was used to transform strain R62B/1A/mur, no transformants with all six substitutions in PBP 2X could be selected. This transformation resulted in transformants which were still limited to growth on blood agar containing a penicillin concentration to a maximum of 2 μg/ml. Twelve transformants were picked and analyzed to discover that they never simultaneously contained Arg384Gly and Tyr595Phe. Transformants either contained a combination of Thr338Pro, Leu364Phe, Ala369Val, Ile371Thr, and Arg384Gly or a combination of Thr338Pro, Leu364Phe, Ala369Val, Ile371Thr, and Tyr595Phe. These transformants all showed penicillin and cefotaxime MICs of 4 and 1 μg/ml, respectively. Our data suggested that a combination of Arg384Gly and Tyr595Phe on PBP 2X was lethal to the bacterium. This was not an unexpected result. There was always a chance that our unnatural laboratory creations of altered PBP 2X would be lethal for the bacterium, because a functional PBP 2X is essential for survival (10). Therefore, in the absence of additional (unknown) compensatory mutations in PBP 2X, which presumably would restructure PBP 2X back to a functional state, we were not able to select for R62X/2B/1A/mur containing a full complement of the six amino acid substitutions in PBP 2X.

TABLE 2.
Addition of amino acid substitutions into unaltered PBP 2X and the resulting effect of this mutagenesis on resistance levels in mutagenized R62X/2B/1A/mur strains compared to those for R62X/2B/1A/mura

The Thr550Ala substitution in PBP 2X has been shown to have a significant impact on the development of β-lactam resistance in the pneumococcus. Coffey and coworkers (4) showed that the presence of this mutation increases the cefotaxime resistance of an isolate by fourfold (from an MIC of 8μg/ml to an MIC of 32 μg/ml) and at the same time decreases the penicillin resistance by 16-fold (from an MIC of 4 μg/ml to an MIC of 0.25 μg/ml). Our resistant isolate 3191 did not show any substitution at position 550. We decided to investigate its possible impact by introducing a Thr550Ala substitution into the altered pbp2X gene from isolate 3191, followed by transformation of strain R62B/1A/mur and cefotaxime selection of the transformants. The presence of Thr550Ala in R62X/2B/1A/mur transformants increased their cefotaxime MICs from 4 to 8μg/ml, while their penicillin MICs were decreased from 16 to 0.5 μg/ml. Although this mutation clearly results in increased cefotaxime resistance, its more pronounced effect is the hypersensitivity to penicillin that it creates.

In conclusion, we have shown the vital role that altered PBP 2X plays in contributing to the development of high-level β-lactam resistance in a clinical isolate of pneumococcus. Mutagenesis analysis of PBP 2X has revealed six amino acid substitutions in altered PBP 2X that play important roles in conferring high-level resistance to penicillin and cefotaxime. These six mutations, however, require a compensatory mutation(s) in PBP 2X to allow a functional protein essential for cell viability. The identity of the compensatory mutation(s) is under investigation. Furthermore, it would be interesting to follow up this study with a crystal structure analysis of mutagenized PBP 2X in order to investigate the structural impacts of these six mutations on the altered protein.

Acknowledgments

We thank Donald Morrison of the University of Illinois at Chicago for his gift of competence-stimulating peptide.

This research was financially supported by the Medical Research Council, National Institute for Communicable Diseases, and University of the Witwatersrand. DNA sequencing was performed with an automated DNA sequencer funded by the Wellcome Trust (grant 061017).

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