Penicillin binding proteins: key players in bacterial cell cycle and drug resistance processes

Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful beta-lactam antibiotics. The increasing incidence of beta-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.     

Interaction of beta-lactam antibiotics with penicillin-binding proteins from Bacillus megaterium

The binding properties of 25 beta-lactam antibiotics to Bacillus megaterium membranes have been studied. The affinities of the antibiotics for the penicillin-binding proteins (PBPs) are also reported. We found that PBP 4 has the highest affinity for nearly all the antibiotics studied whereas PBP 5 has the lowest affinity. Both PBP 4 and PBP 5 appear to be dispensable for the maintenance of bacterial growth and survival and appear to be DD-carboxypeptidases. Only the beta-lactam cefmetazol bound preferentially to PBP 5 and has been used to study the inhibition of DD-carboxypeptidase. Comparative studies with beta-lactam that simultaneously result in (a) binding to PBPs 1 and 3, (b) inhibition of cell growth and (c) lysis, stressed the importance of PBPs 1 and 3 for cell growth and survival.     

Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases

Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in?vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in?vivo. We further show that LpoB promotes PG synthesis by its partner PBP in?vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.     

Expression and characterization of the isolated glycosyltransferase module of Escherichia coli PBP1b

The enzymes involved in the biosynthesis of peptidoglycan are targets for the development of new antibiotics. The bifunctional high molecular weight (HMW) penicillin-binding proteins (PBPs), which contain both glycosyltransferase (GTase) and transpeptidase (TPase) activities, are particularly attractive targets because of their extracellular location. However, there is limited mechanistic or structural information about the GTase modules of these enzymes. In this paper, we describe the overexpression and characterization of the GTase module of Escherichia coli PBP1b, a paradigm of the HMW PBPs. We define the C-terminal boundary of the GTase module and show that the isolated module can be overexpressed at significantly higher levels than the full-length protein. The catalytic efficiency and other characteristics of the isolated module are comparable in most respects to the full-length enzyme. This work lays the groundwork for mechanistic and structural analysis of GTase modules.     

Different Penicillin-Binding Protein Profiles in Amoxicillin-Resistant Helicobacter pylori

Background. The β-lactam group of antibiotics kills bacteria by inhibiting the terminal stages of peptidoglycan metabolism. We have recently identified amoxicillin-resistant Helicobacter pylori , none of which expressed β-lactamase. Penicillin-binding proteins (PBPs) represent a group of target enzymes for the β-lactam antibiotic family, and alterations in PBPs have been described in other penicillin-resistant bacteria. The amoxicillin-resistant phenotype characteristically was lost after freezing but could be restored by consecutive transfers into gradient plates.
Materials and Methods. To determine whether amoxicillin resistance in H. pylori was related to alterations in any of the H. pylori PBPs, five H. pylori strains resistant to amoxicillin and three amoxicillin-sensitive strains were tested. PBPs were extracted from bacteria grown to logarithmic phase, labeled in vivo with ³H-benzylpenicillin, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Four main PBPs were separated from all amoxicillin-sensitive H. pylori strains.
Results. Only three of the four main PBPs were found in the amoxicillin-resistant H. pylori strains. The differentially detectable PBP (PBP D) had an apparent molecular weight of 30 to 32 kD.
Conclusion. These results suggest that PBP D might play a role in the amoxicillin-resistant phenotype of H. pylori strains lacking β-lactamase activity.     

Labelling of penicillin-binding proteins with a photoreactive peptidoglycan-peptide analogue

Transpeptidases, DD-carboxypeptidases and endopeptidases from bacteria are usually detected by labelling with radioactive beta-lactam antibiotics, due to a selective stabilization of the enzyme-antibiotic complex, and are therefore generally known as penicillin-binding proteins (PBPs). However, as a general rule, PBPs cannot be detected by labelling with real peptidoglycan substrate analogues other than beta-lactams, partly due to the fact that the acyl intermediates formed do not usually accumulate. We here report the chemical synthesis of a radioactive photoreactive derivative of the peptidoglycan substrate L-lysyl-D-alanyl-D-alanine which is able, due to the shortness of its activated state, to label a number of PBPs of Escherichia coli by quenching the reaction at the intermediate step. Furthermore, by using this derivative we have been able to label other PBPs of higher molecular mass (190, 170, 146, 125 and 87 kDa) that were previously detected only by using either photoreactive derivatives of beta-lactam or bis-beta-lactam antibiotics.     

Molecular mechanisms of β-lactam resistance in Streptococcus pneumoniae

Alterations in the target enzymes for β-lactam antibiotics, the penicillin-binding proteins (PBPs), have been recognized as a major resistance mechanism in Streptococcus pneumoniae. Mutations in PBPs that confer a reduced affinity to β-lactams have been identified in laboratory mutants and clinical isolates, and document an astounding variability of sites involved in this phenotype. Whereas point mutations are selected in the laboratory, clinical isolates display a mosaic structure of the affected PBP genes, the result of interspecies gene transfer and recombination events. Depending on the selective β-lactam, different combinations of PBP genes and mutations within are involved in conferring resistance, and astoundingly in non-PBP genes as well.     

Physiological functions of D-alanine carboxypeptidases in Escherichia coli

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of beta-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell morphology.     

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.     

Conformational analysis of bacterial cell wall peptides indicates how particular conformations have influenced the evolution of penicillin-binding proteins,beta-lactam antibiotics and antibiotic resistance mechanisms

Our aim was to use a conformational analysis technique developed for peptides to identify structural relationships between bacterial cell wall peptides and beta-lactam antibiotics that might help to explain their different actions as substrates and inhibitors of penicillin binding proteins (PBPs). The conformational forms of the model cell wall peptide Ac-L-Lys(Ac)-D-Ala-D-Ala are described by just a few backbone torsion combinations: three C-terminal carboxylate regions, with Tor8 (psi(i+1)) ranges of D3 region (50 degrees to 70 degrees ), D6 region (140 degrees to 170 degrees ) and D9 region (-50 degrees to -70 degrees ) are combined with either of two Tor6 (phi(i))-Tor4 (psi(i)) combinations, C4 region (-50 degrees to -80 degrees ) with B8 region (-40 degrees to -70 degrees ) or C11 region (30 degrees to 50 degrees ) with B2 region (30 degrees to 70 degrees ). From these results, and comparisons with conformational analyses of various beta-lactams and Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that molecular recognition of cell wall peptide substrates by PBPs requires conformers with backbone torsion angles of D3C4B8. beta-Lactam antibiotics are constrained compounds with fewer conformational forms; these match well the backbone torsions of cell wall peptides at D3C4, allowing their recognition and acylation by PBPs, whereas their unique Tor4 produces differently orientated CO and N atoms that appear to prevent subsequent deacylation, leading to their action as suicide substrates. The results are also related to the selective pressures involved in evolution of beta-lactamases from PBPs. From analysis of conformers of Ac-L-Lys(Ac)-D-Ala-D-Ala and the vancomycin-resistant analogue Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that vancomycin may recognise D6C11B2 conformers, giving it complementary substrate specificity to PBPs. This approach could have applications in the rational design of antibiotics targeted against PBPs and their substrates.     

The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance

Penicillin-binding proteins (PBPs), the primary targets for beta-lactam antibiotics, are periplasmic membrane-attached proteins responsible for the construction and maintenance of the bacterial cell wall. Bacteria have developed several mechanisms of resistance, one of which is the mutation of the target enzymes to reduce their affinity for beta-lactam antibiotics. Here, we describe the structure of PBP2x from Streptococcus pneumoniae determined to 2.4 A. In addition, we also describe the PBP2x structure in complex with cefuroxime, a therapeutically relevant antibiotic, at 2.8 A. Surprisingly, two antibiotic molecules are observed: one as a covalent complex with the active-site serine residue, and a second one between the C-terminal and the transpeptidase domains. The structure of PBP2x reveals an active site similar to those of the class A beta-lactamases, albeit with an absence of unambiguous deacylation machinery. The structure highlights a few amino acid residues, namely Thr338, Thr550 and Gln552, which are directly related to the resistance phenomenon.     

Rational antibiotic design: in silico structural comparison of the functional cavities of penicillin-binding proteins and ß-lactamases

The class of ß-lactam antibiotics has proven highly efficient in targeting bacterial penicillin-binding proteins (PBP) leading to the blocking of the bacterial cell wall synthesis. However, the benefit of these drugs is limited because of bacterial resistance mechanisms; the most widespread resistance involves ß-lactamase enzymes (ßLACT) that inactivate ß-lactam-based molecules. We focused on PBPs and ßLACTs from enterobacteria, and performed a detailed in silico study of PBPs whose inactivation is lethal for the bacteria and of ßLACTs that have a PBP-type catalytic mechanism. The comparison of the sequences and structures of PBPs and ßLACTs shows an almost perfect conservation of the catalytic site, and a high spatial resemblance of the whole functional cavity despite a very low overall sequence identity. Some notable differences in the functional cavity were observed in the vicinity of the catalytic site: four tyrosines are well conserved in the PBPs, whereas the residues occurring at equivalent positions in the ßLACT families present other physicochemical properties. These tyrosines are thus good candidates to be targeted in designing new antibiotic molecules with increased affinity and specificity for PBPs, with the goal of overcoming drug resistance. Our analysis also identified residues that have similar characteristics in most ßLACT families and different properties in PBPs; these are interesting targets for new ligands that specifically inhibit ßLACT proteins. The in silico approach presented here can be extended to other protein systems in view of guiding and improving rational drug design.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.     

Penicillin binding proteins of Vibrio cholerae

Eleven penicillin binding proteins (PBPs) of Vibrio cholerae have been identified using [125I] labelled p-hydroxybenzyl penicillin (PenX). These proteins are localised in the inner membrane and have molecular weights ranging from 97,000 to 22,000. Neutral hydroxylamine released the labelled PenX from the PBPs and pretreatment with cold benzyl penicillin inhibited labelling completely. The PBP 4 is the most sensitive target for cephaloridine and aztreonam. Cephaloridine also binds to three other high molecular weight PBPs, 1, 2 and 3. Aztreonam, in addition to PBP 4, has affinity for another low molecular weight PBP, PBP 7. Mecillinam has affinity for PBPs 1, 4 and 11.     

Multivariate geometrical analysis of catalytic residues in the penicillin-binding proteins

Penicillin-binding proteins (PBPs) are bacterial enzymes involved in the final stages of cell wall biosynthesis, and are targets of the β-lactam antibiotics. They can be subdivided into essential high-molecular-mass (HMM) and non-essential low-molecular-mass (LMM) PBPs, and further divided into subclasses based on sequence homologies. PBPs can catalyze transpeptidase or hydrolase (carboxypeptidase and endopeptidase) reactions. The PBPs are of interest for their role in bacterial cell wall biosynthesis, and as mechanistically interesting enzymes which can catalyze alternative reaction pathways using the same catalytic machinery. A global catalytic residue comparison seemed likely to provide insight into structure-function correlations within the PBPs. More than 90 PBP structures were aligned, and a number (40) of active site geometrical parameters extracted. This dataset was analyzed using both univariate and multivariate statistical methods. Several interesting relationships were observed. (1) Distribution of the dihedral angle for the SXXK-motif Lys side chain (DA_1) was bimodal, and strongly correlated with HMM/transpeptidase vs LMM/hydrolase classification/activity (P<0.001). This structural feature may therefore be associated with the main functional difference between the HMM and LMM PBPs. (2) The distance between the SXXK-motif Lys-NZ atom and the Lys/His-nitrogen atom of the (K/H)T(S)G-motif was highly conserved, suggesting importance for PBP function, and a possibly conserved role in the catalytic mechanism of the PBPs. (3) Principal components-based cluster analysis revealed several distinct clusters, with the HMM Class A and B, LMM Class C, and LMM Class A K15 PBPs forming one "Main" cluster, and demonstrating a globally similar arrangement of catalytic residues within this group.     

Approaching the physiological functions of penicillin-binding proteins in Escherichia coli

A rigid shell of peptidoglycan encases and shapes bacteria and is constructed and maintained by a diverse set of enzymes, among which are the penicillin-binding proteins (PBPs). Although a great deal has been learned about how these proteins synthesize and modify peptidoglycan, the physiological functions of the multitude of bacterial PBPs remain enigmatic. We approached this problem by combining PBP mutations in a comprehensive manner and screening for effects on biochemical processes involving the passage of proteins or nucleic acids across the cell wall. The results indicate that the PBPs or their peptidoglycan product do have significant biological functions, including roles in determination of cell shape, in phage resistance, in induction of capsule synthesis, and in regulation of autolysis.     

Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.