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* This work was supported by Grant ORBiMP ANR-14-CE14-0003-01 from the Agence Nationale de la Recherche and used the platforms of the Grenoble Instruct Center (UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI Grant ANR-10-INSB-05-02 and GRAL Grant ANR-10-LABX-49-01 within the Grenoble Partnership for Structural Biology. The authors declare that they have no conflicts of interest with the contents of this article.
Pneumococcus resists β-lactams by expressing variants of its target enzymes, the penicillin-binding proteins (PBPs), with many amino acid substitutions. Up to 10% of the sequence can be modified. These altered PBPs have a much reduced reactivity with the drugs but retain their physiological activity of cross-linking the peptidoglycan, the major constituent of the bacterial cell wall. However, because β-lactams are chemical and structural mimics of the natural substrate, resistance mediated by altered PBPs raises the following paradox: how PBPs that react poorly with the drugs maintain a sufficient level of activity with the physiological substrate. This question is addressed for the first time in this study, which compares the peptidoglycan cross-linking activity of PBP2b from susceptible and resistant strains with their inhibition by different β-lactams. Unexpectedly, the enzymatic activity of the variants did not correlate with their antibiotic reactivity. This finding indicates that some of the numerous amino acid substitutions were selected to restore a viable level of enzymatic activity by a compensatory molecular mechanism.
). β-Lactams hamper formation of the peptidoglycan, which is the main constituent of the bacterial cell wall. Peptidoglycan is a giant polymer encasing the cell and consists of chains of tandemly repeated disaccharides cross-linked by peptide bridges. This cross-linking results from a transpeptidation reaction catalyzed by enzymes that are inhibited by β-lactams, which are mimics of the donor dipeptide of the transpeptidation reaction. The enzymes responsible for the peptidoglycan assembly are called penicillin-binding proteins (PBPs).
). Some PBPs have an additional transglycosylase domain that catalyze the elongation of the glycan strands. The bifunctional transpeptidase and transglycosylase PBPs constitute the class A, whereas the monofunctional transpeptidase PBPs are of class B.
Resistance to antibiotics is recognized as a major threat to human health (
). Resistance to β-lactams, which are the most widely used antimicrobials, is particularly worrisome. A case in point is that of pneumococcus, a major human pathogen that causes otitis, pneumonia, and meningitis and that is estimated to cause over 1.5 million deaths per year (
). The mechanisms responsible for the diminution of the reactivity with the drugs have been investigated biochemically and structurally to various degrees for the three PBPs. For PBPs from a β-lactam-susceptible strain, the common source has been the laboratory strain R6, which has a minimal inhibitory concentration (MIC) below 0.016 μg/ml for penicillin and cefotaxime. The most thoroughly characterized PBPs from resistant pneumococcus are from the highly resistant strain 5204 isolated in France in 1999, with MICs of 6 μg/ml for penicillin and 12 μg/ml for cefotaxime (
The monofunctional transpeptidase PBP2x is the most studied of these PBPs. The role of important amino acid substitutions within the active site of PBP2x has been established by kinetics and structural studies. For example, a mutation two residues downstream of the active site serine found in highly resistant strains was shown to change the orientation of the hydroxyl group of the serine, thereby diminishing its reactivity with β-lactams (
). A comprehensive study of all the substitutions in the transpeptidase domain of PBP2x from the highly resistant strain 5204 determined that only 6 of the 41 substitutions are important for reducing the reactivity with β-lactams (
The other monofunctional class B PBP2b has not been as extensively studied, although the crystal structure has been solved for the variants from the susceptible laboratory strain R6 and the clinical resistant strain 5204 (
). Like in PBP2x, a loop forming one side or “lip” of the active site is flexible in the variant from the resistant strain. Sequence comparison identified the T446A substitution within the active site as critical for resistance, and an early biochemical study demonstrated that this mutation severely reduces the reactivity with β-lactams, although no reaction kinetics could be measured (
). A mutation adjacent to the active site serine was found to modify its orientation, and the loop forming a lip of the active site was destabilized as in PBP2x and PBP2b by a stretch of four substitutions (
). The carbonyl of the β-lactam ring that is attacked by the nucleophilic active site serine is analogous to the carbonyl of the peptide bond that links the two terminal d-alanine residues of the peptidoglycan stem peptide. Because of the similarity between the drugs and physiological substrate, we are facing the following paradox: PBP alterations that affect reaction with β-lactams, as briefly presented above, would be expected to impact negatively on their transpeptidase enzymatic function. To solve this paradox, compensatory mechanisms likely mitigate this problem. Compensation could take place at the cellular level to cope with the effectively reduced enzymatic activity of the PBPs in resistant strains. Alternatively, compensation could occur at the molecular level of the PBPs themselves if the consequences of some substitutions are different on the reactivity with the β-lactams and the transpeptidase activity. The two types of compensatory mechanisms are not mutually exclusive and could operate together.
Investigating cellular mechanisms that could compensate for lower PBP activity is very difficult because the cascade of events that occur in the pneumococcus following β-lactam challenge remains largely mysterious (
). Demonstrating the existence of compensatory mechanism to maintain the enzymatic activity of mosaic PBPs of S. pneumoniae was not possible until recently because there was no assay to evaluate the transpeptidase activity in vitro. This hurdle was removed because the proper peptidoglycan precursor is now available. The nature of the stem pentapeptide of the membrane-linked precursor (lipid II) varies slightly in different bacterial species. In the pneumococcus, the second residue is a γ-d-iso-glutamine. The discovery of the amido transferase enzyme (MurT/GatD) that modifies the second residue γ-d-glutamate into γ-d-iso-glutamine (
To investigate the paradox raised by PBP-based resistance and the structural similarity between β-lactams and the natural substrate of PBPs, we present here a comparison of four variants of PBP2b, (i) from the susceptible laboratory strain R6, (ii) from the clinical resistant strain 5204 (
), (iii) a hybrid form with the N terminus from strain 5204 and the C terminus of strain R6, and (iv) the T446A point mutant (FIGURE 1, FIGURE 2). 5204-PBP2b carries 56 substitutions, of which 43 are within the transpeptidase domain. Hybrid-PBP2b carries 28 substitutions, including 15 in the transpeptidase domain, and requires further presentation. The hybrid form was not designed but results from the transformation of the R6 strain with the pbp2b gene from strain 5204 and selection with piperacillin. S. pneumoniae is naturally competent and readily recombines foreign homologous DNA. An allele conferring a resistance can therefore easily be introduced and selected, provided that flanking regions are provided to allow recombination. With PBP2b, however, the whole gene could not be introduced because recombination repeatedly occurred within the coding region, resulting in a gene with the 5′-region from the resistant strain and the 3′-region retained from the susceptible strain (
). Characterization of the resulting Hybrid-PBP2B might shed light on this incomplete incorporation of the 5204-pbp2b allele in the R6 strain. The T446A substitution, which is found in both 5204- and Hybrid-PBP2b, is the most commonly found in resistant strains and can confer some resistance alone (
For the first time, both the in vitro transpeptidase activity and the reactivity with a panel of β-lactams were evaluated for a set of PBPs. We uncovered a new level of complexity because the impacts of substitutions on the transpeptidase activity and reactivity with β-lactams are not fully correlated, revealing that some substitutions have compensatory effects that restore enzymatic activity. We also discovered that variants of PBP2b can display β-lactamase activity that may contribute to the resistance to some β-lactams.
The properties of the PBP2b variants measured in vitro, both the transpeptidase activity and the reactivity with β-lactams, are consistent with the impossibility to introduce the full PBP2b sequence from a clinical resistant strain (5204) into the susceptible strain R6. Indeed, the Hybrid-PBP2b resulting from the splicing of the N-terminal part of 5204-PBP2b and the C-terminal part of R6-PBP2b has better enzymatic activity and lower drug reactivity than the full 5204-PBP2b. The biochemical properties of Hybrid-PBP2b explain why it is repeatedly selected by β-lactams in laboratory transformation experiments (
). Note that if the c50 is considered, 5204-PBP2b is slightly less susceptible to β-lactam than Hybrid-PBP2b. The level of transpeptidase activity would therefore be the main factor favoring the selection of Hybrid-PBP2b instead of 5204-PBP2b by β-lactams.
Examination of the modeled structure of Hybrid-PBP2b shows that the missing substitutions compared with 5204-PBP2b are distributed in the “upper lip” of the active site and at the “back” of the protein (Fig. 2). The “upper lip” substitutions A619G, D625G, Q628E, and T630N are the most likely to cause the severe diminution of transpeptidase activity of 5204-PBP2b because of their proximity to the active site residues at the entrance of the cleft. The region spanning these substitutions forms a loop connecting strands β3 and β4 that is mobile and not visible in the crystal structure of 5204-PBP2b (
), because Hybrid-PBP2b with a probable “stiff upper lip” is even less reactive with β-lactams than the more “relaxed” 5204-PBP2b. In contrast, substitutions in the lower lip of the active site S412P, N422Y, T426Q, and Q427L likely contribute the most to the decrease in reactivity with the drugs. Interestingly and in agreement with the PBP2b observations, substitutions in the lower lip of PBP2x were found to reduce β-lactam reactivity, whereas substitutions in the β3-β4 upper lip had no effect or even increased the reactivity with the drug (
). The lower lip on the contrary is held tightly against the active site and contacts the bound antibiotic. Unfortunately, no crystal structure of PBP2b with bound antibiotic and no data on the transpeptidase activity of PBP2x variants are available to further compare both proteins.
The deacylation rate (k3), which may also contribute to the resistance, is particularly elevated with 5204-PBP2b. It is possible that the flexibility of the upper lip and the replacement of Ala619 by a glycine very close to the active site Ser386 allow easier access of water to the acyl-enzyme bond.
The T446A substitution is the most important to reduce the acylation by β-lactams (Table 1). Collectively, the 55 other substitutions in 5204-PBP2b cause only a modest additional reduction of the acylation rate (Table 1). Among these, the 27 substitutions at the C terminus must collectively have an opposite effect on the reactivity with β-lactams, because their absence in Hybrid-PBP2b further decreases the reactivity (Table 1). The T446A mutation, however, is very detrimental to the transpeptidase activity, the point mutant being the less active of the four variants (Fig. 3).
Therefore, among the many substitutions found in mosaic PBPs from resistant strains, some substitutions play key roles in reducing the reactivity toward β-lactams, such as the T446A in PBP2b or the six substitutions identified among 41 in the transpeptidase domain of 5204-PBP2x (
). Other substitutions are likely neutral and present solely by virtue of the “hitchhiking” effect resulting from the homologous recombination of long stretches of DNA. What is revealed in the present study is that some substitutions are compensatory and participate to the resistance not by restricting the reaction with the drugs but by restoring an acceptable level of physiological enzymatic activity, which would otherwise be severely impacted by mutations that hamper the reaction with β-lactams.
The findings above raise the question of why is 5204-PBP2b present in a clinical resistant strain, since a sequence with fewer substitutions appears biochemically superior? A clue can be found in a laboratory study of the transfer of β-lactam resistance from Streptococcus mitis to S. pneumoniae (
). The full sequence of PBP2b from a resistant S. mitis strain, including the 16 substitutions at the C terminus following position 590, could be transformed and selected by benzylpenicillin in S. pneumoniae, only if the mosaic variant of the murM gene from the S. mitis was also introduced.
The murMN operon encodes two cytoplasmic enzymes responsible for the “branching” of the stem peptides of the peptidoglycan (
). The precursor of the peptidoglycan is synthesized with a pentapeptide, the third residue of which is a lysine in pneumococcus. The free amine of the lysine side chain of the donor peptide forms the peptide bond with the fourth residue (d-Ala) carboxyl group of the donor peptide to cross-link glycan chains. Alternatively, MurM and MurN can add successively two residues onto the lysine side chain to produce branched stem peptides with additional Ser-Ala or Ala-Ala dipeptides. It is then the N terminus of the dipeptide on the acceptor that forms the cross-linking peptide bond with the donor stem peptide. Strain R6 and its parental strain R36A were found to have ∼45 and 36% branched peptides, respectively (
To explain the special relationship between PBP2b and MurM in β-lactam resistance, two plausible explanations can be considered. More branched peptides may have a compensatory role (i) on the enzymatic activity of PBP2b or (ii) on the cell wall metabolism. (i) Branched stem peptides may be better substrates for altered PBP2bs than the linear form, either as donor or acceptor, thus compensating the decreased transpeptidase activity. It is highly desirable to test this hypothesis in vitro, but suitable substrates are unfortunately not available in sufficient amounts. However, the fact that in the absence of antibiotic challenge, a strain devoid of MurM and branched peptide grows normally with altered PBPs (
). Addition of peptidoglycan material to the cell wall during elongation requires concomitant opening of the existing peptidoglycan to permit insertion. A peptidoglycan lytic transglycosylase has recently been identified that likely works in concert with PBP2b during elongation of the pneumococcus (
). A decreased transpeptidase activity of PBP2b might create an imbalance with the degradative activity of its associated lytic transglycosylase that may trigger full lysis with the participation of other peptidoglycan hydrolases such as the major autolysin LytA. The greater proportion of branched peptides caused by mosaic MurM may protect against lysis. Indeed, elevated level of branched peptides caused by expression of a mosaic hyperactive MurM also protects against cell lysis normally induced by non-β-lactam antibiotics (
). Also, gradual depletion of PBP2b was found to be tolerated to a large extent due to an increased level of branched peptides. In the absence of MurM, and consequently of branched peptides, depletion of PBP2b was much less tolerated (
In conclusion, we propose that the deleterious effect of mutations that greatly diminish the reactivity of PBP2b with β-lactams, such as the T446A, is compensated by a combination of compensatory substitutions within PBP2b that maintain a minimal necessary transpeptidase activity, together with an alteration of the peptidoglycan composition that compensate for the reduced enzymatic activity.
The reactivity with Bocillin FL of the extracellular domain of PBP2b was found to be the same as that of the full-length membrane proteins (Fig. 5). In contrast, the transpeptidase activity was severely impacted by the truncation of the transmembrane segment. Residual activity was detected only with soluble R6-PBP2b (not shown). An influence of the membrane anchor on the active site cannot be ruled out but is unlikely in the light of the reaction with β-lactams. Rather, the glycan chains and the full-length enzymes may be brought into close proximity by their common anchoring into detergent micelles. This efficient concentration effect is lost by truncation of the transmembrane segment. In any case, the in vitro transpeptidase activities reported here and previously are extremely weak and far from realistic physiologic rates. In the future, it will be necessary to study monofunctional PBPs in a membrane environment and/or in the presence of their protein partners (
). This property is not general to cephalosporins, however, because nitrocefin was the most reactive drug of our panel. Examination of the structure of the different cephalosporins that fail to react with PBP2b (
) suggests that the nature of the R2 substituent is important. Bulky and planar substituent in position R2 appear to prevent recognition by PBP2b, whereas smaller and tetrahedral substituent seem compatible. The unfavorable interaction between cefotaxime and PBP2b seems to persist once the β-lactam ring is open and the acyl-enzyme complex is formed, because the thermal stability of the protein is diminished (Table 2).
It is remarkable that substitutions that lead to the thermal stabilization of the apo form of 5204- and Hybrid-PBP2b compared with the apo form of R6-PBP2b have the opposite effect on the acylated forms (Table 2). In the variants from the resistant strains, the covalently bound drugs must make unfavorable interactions with the proteins. Although the “open” forms of the antibiotics that are bound in the active site are different from the original β-lactams, they likely retain interactions that also occur in the preacylation complex or the transition state and may be relevant to the acylation and deacylation kinetics.
Other than nitrocefin, which is not in clinical use, the most important diminution of reactivity measured for 5204-PBP2b compared with R6-PBP2b was with amoxicillin. It is also with amoxicillin that the greatest β-lactamase activity was measured for 5204-PBP2b (Table 1). Amoxicillin has long been a widely prescribed treatment for otitis media and sore throat, even when bacterial infection is not clearly diagnosed (
). The nasopharyngeal flora, including S. pneumoniae has therefore been subjected to a severe selection pressure by amoxicillin, and it is possible that the substitutions found in 5204-PBP2b reflects the clinical practice. Strain 5204 exhibits the highest level of amoxicillin resistance (MIC 6 μg ml−1) in a panel of French clinical isolates, together with isolate 5268, which has a similar PBP2b sequence (
The surprising finding that a laboratory selected PBP2b (Hybrid) performs better in vitro than its clinical parental variant (5204) indicates that β-lactam resistance in pneumococcus is a complex process that is not fully described by biochemical reconstitution experiments. Resistance to β-lactams must be considered as a complete physiological response with compensatory mechanisms at play. Such mechanisms should be studied in the future because they may offer novel therapeutic options.
P. C. and A. Z. designed and conducted most of the experiments and analyzed the results. E. B. synthesized lipid II. D. I. R. purified PBP1a. M. D. performed the thermal shift assays. C. C.-M. modeled the protein structure. A. Z. wrote the manuscript.
We thank Anne-Marie Villard for performing site-directed mutagenesis and Thierry Vernet and Max Maurin for critical discussion of the results.
Penicillin: the medicine with the greatest impact on therapeutic outcomes.