In Bacterial Membranes Lipid II Changes the Stability of Pores Formed by the Antimicrobial Peptide Nisin.

Resistance to available antibiotics poses a growing challenge to modern medicine, as this often disallows infections to be controlled. This problem can only be alleviated by the development of new drugs. Nisin, a natural lantibiotic with broad antimicrobial activity, has shown promise as a potential candidate for combating antibiotic-resistant bacteria. However, nisin is poorly soluble and barely stable at physiological pH, which despite attempts to address these issues through mutant design has restricted its use as an antibacterial drug. Therefore, gaining a deeper understanding of the antimicrobial effectiveness, which relies in part on its ability to form pores, is crucial for finding innovative ways to manage infections caused by resistant bacteria. Using large-scale molecular dynamics simulations, we find that the bacterial membrane-specific lipid II increases the stability of pores formed by nisin and that the interplay of nisin and lipid II reduces the overall integrity of bacterial membranes by changing the local thickness and viscosity.

Elucidating Daptomycin's Antibacterial Efficacy: Insights into the Tripartite Complex with Lipid II and Phospholipids in Bacterial Septum Membrane.

This study elucidated the mechanism of formation of a tripartite complex containing daptomycin (Dap), lipid II, and phospholipid phosphatidylglycerol in the bacterial septum membrane, which was previously reported as the cause of the antibacterial action of Dap against gram-positive bacteria via molecular dynamics and enhanced sampling methods. Others have suggested that this transient complex ushers in the inhibition of cell wall synthesis by obstructing the downstream polymerization and cross-linking processes involving lipid II, which is absent in the presence of cardiolipin lipid in the membrane. In this work, we observed that the complex was stabilized by Ca2+-mediated electrostatic interactions between Dap and lipid head groups, hydrophobic interaction, hydrogen bonds, and salt bridges between the lipopeptide and lipids and was associated with Dap concentration-dependent membrane depolarization, thinning of the bilayer, and increased lipid tail disorder. Residues Orn6 and Kyn13, along with the DXDG motif, made simultaneous contact with constituent lipids, hence playing a crucial role in the formation of the complex. Incorporating cardiolipin into the membrane model led to its competitively displacing lipid II away from the Dap, reducing the lifetime of the complex and the nonexistence of lipid tail disorder and membrane depolarization. No evidence of water permeation inside the membrane hydrophobic interior was noted in all of the systems studied. Additionally, it was shown that using hydrophobic contacts between Dap and lipids as collective variables for enhanced sampling gave rise to a free energy barrier for the translocation of the lipopeptide. A better understanding of Dap's antibacterial mechanism, as studied through this work, will help develop lipopeptide-based antibiotics for rising Dap-resistant bacteria.

The Bacterial Cell Wall: From Lipid II Flipping to Polymerization.

The peptidoglycan (PG) cell wall is an extra-cytoplasmic glycopeptide polymeric structure that protects bacteria from osmotic lysis and determines cellular shape. Since the cell wall surrounds the cytoplasmic membrane, bacteria must add new material to the PG matrix during cell elongation and division. The lipid-linked precursor for PG biogenesis, Lipid II, is synthesized in the inner leaflet of the cytoplasmic membrane and is subsequently translocated across the bilayer so that the PG building block can be polymerized and cross-linked by complex multiprotein machines. This review focuses on major discoveries that have significantly changed our understanding of PG biogenesis in the past decade. In particular, we highlight progress made toward understanding the translocation of Lipid II across the cytoplasmic membrane by the MurJ flippase, as well as the recent discovery of a novel class of PG polymerases, the SEDS (shape, elongation, division, and sporulation) glycosyltransferases RodA and FtsW. Since PG biogenesis is an effective target of antibiotics, these recent developments may lead to the discovery of much-needed new classes of antibiotics to fight bacterial resistance.

Identification of the potential active site of the septal peptidoglycan polymerase FtsW.

SEDS (Shape, Elongation, Division and Sporulation) proteins are widely conserved peptidoglycan (PG) glycosyltransferases that form complexes with class B penicillin-binding proteins (bPBPs, with transpeptidase activity) to synthesize PG during bacterial cell growth and division. Because of their crucial roles in bacterial morphogenesis, SEDS proteins are one of the most promising targets for the development of new antibiotics. However, how SEDS proteins recognize their substrate lipid II, the building block of the PG layer, and polymerize it into glycan strands is still not clear. In this study, we isolated and characterized dominant-negative alleles of FtsW, a SEDS protein critical for septal PG synthesis during bacterial cytokinesis. Interestingly, most of the dominant-negative FtsW mutations reside in extracellular loops that are highly conserved in the SEDS family. Moreover, these mutations are scattered around a central cavity in a modeled FtsW structure, which has been proposed to be the active site of SEDS proteins. Consistent with this, we found that these mutations blocked septal PG synthesis but did not affect FtsW localization to the division site, interaction with its partners nor its substrate lipid II. Taken together, these results suggest that the residues corresponding to the dominant-negative mutations likely constitute the active site of FtsW, which may aid in the design of FtsW inhibitors.

Computational evaluation of modified peptides from human neutrophil peptide 1 (HNP-1).

The development of bacterial resistance toward antibiotics has been led to pay attention to the antimicrobial peptides (AMPs). The common mechanism of AMPs is disrupting the integrity of the bacterial membrane. One of the most accessible targets for α-defensins human neutrophil peptide-1 (HNP-1) is lipid II. In the present study, we performed homology modeling and geometrical validation of human neutrophil defensin 1. Then, the conformational and physicochemical properties of HNP-1 derived peptides 2Abz14S29, 2Abz23S29, and HNP1ΔC18A, as well as their interaction with lipid II were studied computationally. The overall quality of the predicted model of full protein was -5.14, where over 90% of residues were in the most favored and allowed regions in the Ramachandran plot. Although HNP-1 and HNP1ΔC18A were classified as unstable peptides, 2Abz14S29 and 2Abz23S29 were stable, based on the instability index values. Molecular docking showed similar interaction pattern of peptides and HNP-1 to lipid II. Molecular dynamic simulations revealed the overall stability of conformations, though the fluctuations of amino acids in the modified peptides were relatively higher than HNP-1. Further, the binding affinity constant (Kd) of HNP-1 and 2Abz23S29 in complex with lipid II was 10 times stronger than 2Abz14S29 and HNP1ΔC18A. Overall, computational studies of conformational and interaction patterns have signified how derived peptides could have displayed relatively similar antimicrobial results compared to HNP-1 in the reported experimental studies. Chemical modifications not only have improved the physicochemical properties of derived peptides compared to HNP-1, but also they have retained the similar pattern and binding affinity of peptides. Communicated by Ramaswamy H. Sarma.

Lipid II Binding and Transmembrane Properties of Various Antimicrobial Lanthipeptides.

There has been an alarming rise in antibacterial resistant infections in recent years due to the widespread use of antibiotics, and there is a dire need for the development of new antibiotics utilizing novel modes of action. Lantibiotics are promising candidates to engage in the fight against resistant strains of bacteria due to their unique modes of action, including interference with cell wall synthesis by binding to lipid II and creating pores in bacterial membranes. In this study, we use atomic-scale molecular dynamics computational studies to compare both the lipid II binding ability and the membrane interactions of five lanthipeptides that are commonly used in antimicrobial research: nisin, Mutacin 1140 (MU1140), gallidermin, NVB302, and NAI107. Among the five peptides investigated, nisin is found to be the most efficient at forming water channels through a membrane, whereas gallidermin and MU1140 are found to be better at binding the lipid II molecules. Nisin's effectiveness in facilitating water transport across the membrane is due to the creation of several different water trajectories along with no significant water delay points along the paths. The shorter peptide deoxyactagardine B (NVB302) was found to not form a water channel. These detailed observations provide insights into the dual mechanisms of the action of lantibiotic peptides and can facilitate the design and development of novel lanthipeptides by strategic placement of different residues.

THCz: Small molecules with antimicrobial activity that block cell wall lipid intermediates.

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop in vitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials.

Class A PBPs: It is time to rethink traditional paradigms.

Until recently, class A penicillin-binding proteins (aPBPs) were the only enzymes known to catalyze glycan chain polymerization from lipid II in bacteria. Hence, the discovery of two novel lipid II polymerases, FtsW and RodA, raises new questions and has consequently received a lot of attention from the research community. FtsW and RodA are essential and highly conserved members of the divisome and elongasome, respectively, and work in conjunction with their cognate class B PBPs (bPBPs) to synthesize the division septum and insert new peptidoglycan into the lateral cell wall. The identification of FtsW and RodA as peptidoglycan glycosyltransferases has raised questions regarding the role of aPBPs in peptidoglycan synthesis and fundamentally changed our understanding of the process. Despite their dethronement, aPBPs are essential in most bacteria. So, what is their function? In this review, we discuss recent progress in answering this question and present our own views on the topic.

Structure-based modeling and dynamics of MurM, a Streptococcus pneumoniae penicillin resistance determinant present at the cytoplasmic membrane.

Branched Lipid II, required for the formation of indirectly crosslinked peptidoglycan, is generated by MurM, a protein essential for high-level penicillin resistance in the human pathogen Streptococcus pneumoniae. We have solved the X-ray crystal structure of Staphylococcus aureus FemX, an isofunctional homolog, and have used this as a template to generate a MurM homology model. Using this model, we perform molecular docking and molecular dynamics to examine the interaction of MurM with the phospholipid bilayer and the membrane-embedded Lipid II substrate. Our model suggests that MurM is associated with the major membrane phospholipid cardiolipin, and experimental evidence confirms that the activity of MurM is enhanced by this phospholipid and inhibited by its direct precursor phosphatidylglycerol. The spatial association of pneumococcal membrane phospholipids and their impact on MurM activity may therefore be critical to the final architecture of peptidoglycan and the expression of clinically relevant penicillin resistance in this pathogen.

Outer-membrane-acting peptides and lipid II-targeting antibiotics cooperatively kill Gram-negative pathogens.

The development and dissemination of antibiotic-resistant bacterial pathogens is a growing global threat to public health. Novel compounds and/or therapeutic strategies are required to face the challenge posed, in particular, by Gram-negative bacteria. Here we assess the combined effect of potent cell-wall synthesis inhibitors with either natural or synthetic peptides that can act on the outer-membrane. Thus, several linear peptides, either alone or combined with vancomycin or nisin, were tested against selected Gram-negative pathogens, and the best one was improved by further engineering. Finally, peptide D-11 and vancomycin displayed a potent antimicrobial activity at low µM concentrations against a panel of relevant Gram-negative pathogens. This combination was highly active in biological fluids like blood, but was non-hemolytic and non-toxic against cell lines. We conclude that vancomycin and D-11 are safe at >50-fold their MICs. Based on the results obtained, and as a proof of concept for the newly observed synergy, a Pseudomonas aeruginosa mouse infection model experiment was also performed, showing a 4 log10 reduction of the pathogen after treatment with the combination. This approach offers a potent alternative strategy to fight (drug-resistant) Gram-negative pathogens in humans and mammals.

Genes encoding lipid II flippase MurJ and peptidoglycan hydrolases are required for chloroplast division in the moss Physcomitrella patens.

KEY MESSAGE: Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and DD-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and LD-carboxypeptidase ldcA in addition to a putative DD-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.

Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization.

Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2-4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.

A Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of the Bacterial Response to Vancomycin Treatment.

A pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which the bacterial cell wall is the front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical d-amino acid (d-X) that was edited in a small proportion into the terminal acyl-d-Ala-d-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in the bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional high-performance liquid chromatography-based methods. Newly synthesized azide-d-Leu and ketone-d-Met were used together with alkynyl-d-Ala for their metabolic assembly and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu, and azide-DOTA-Sm tags. This approach allows direct quantification of the d-X in situ in the cell wall using 158Gd, 153Eu, and 154Sm species-unspecific isotope dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex "cell-broken" pretreatment procedures that might induce racemization of the d-X. The obtained site-specific and accurate in situ information about the d-X enables quantitative monitoring of the bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of azide-d-Leu and ketone-d-Met assembled are more important after determining the structure- and composition-dependent bacterial antibiotic resistance mechanisms. In addition, we found that the combined use of vancomycin and d-Ala restores the efficacy of vancomycin and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

CbrA Mediates Colicin M Resistance in Escherichia coli through Modification of Undecaprenyl-Phosphate-Linked Peptidoglycan Precursors.

Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway.IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

Mode of action of teixobactins in cellular membranes.

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.

Environmental and dynamic effects explain how nisin captures membrane-bound lipid II.

Antibiotics (AB) resistance is a major threat to global health, thus the development of novel AB classes is urgently needed. Lantibiotics (i.e. nisin) are natural compounds that effectively control bacterial populations, yet their clinical potential is very limited. Nisin targets membrane-embedded cell wall precursor - lipid II - via capturing its pyrophosphate group (PPi), which is unlikely to evolve, and thus represents a promising pharmaceutical target. Understanding of exact molecular mechanism of initial stages of membrane-bound lipid II recognition by water-soluble nisin is indispensable. Here, using molecular simulations, we demonstrate that the structure of lipid II is determined to a large extent by the surrounding water-lipid milieu. In contrast to the bulk solvent, in the bilayer only two conformational states remain capable of nisin binding. In these states PPi manifests a unique arrangement of hydrogen bond acceptors on the bilayer surface. Such a "pyrophosphate pharmacophore" cannot be formed by phospholipids, which explains high selectivity of nisin/lipid II recognition. Similarly, the "recognition module" of nisin, being rather flexible in water, adopts the only stable conformation in the presence of PPi analogue (which mimics the lipid II molecule). We establish the "energy of the pyrophosphate pharmacophore" approach, which effectively distinguishes nisin conformations that can form a complex with PPi. Finally, we propose a molecular model of nisin recognition module/lipid II complex in the bacterial membrane. These results will be employed for further study of lipid II targeting by antimicrobial (poly)cyclic peptides and for design of novel AB prototypes.

Fluorescence anisotropy assays for high throughput screening of compounds binding to lipid II, PBP1b, FtsW and MurJ.

Lipid II precursor and its processing by a flippase and peptidoglycan polymerases are considered key hot spot targets for antibiotics. We have developed a fluorescent anisotropy (FA) assay using a unique and versatile probe (fluorescent lipid II) and monitored direct binding between lipid II and interacting proteins (PBP1b, FtsW and MurJ), as well as between lipid II and interacting antibiotics (vancomycin, nisin, ramoplanin and a small molecule). Competition experiments performed using unlabelled lipid II, four lipid II-binding antibiotics and moenomycin demonstrate that the assay can detect compounds interacting with lipid II or the proteins. These results provide a proof-of-concept for the use of this assay in a high-throughput screening of compounds against all these targets. In addition, the assay constitutes a powerful tool in the study of the mode of action of compounds that interfere with these processes. Interestingly, FA assay with lipid II probe has the advantage over moenomycin based probe to potentially identify compounds that interfere with both donor and acceptor sites of the aPBPs GTase as well as compounds that bind to lipid II. In addition, this assay would allow the screening of compounds against SEDS proteins and MurJ which do not interact with moenomycin.

An Engineered Double Lipid II Binding Motifs-Containing Lantibiotic Displays Potent and Selective Antimicrobial Activity against Enterococcus faecium.

Lipid II is an essential precursor for bacterial cell wall biosynthesis and thereby an important target for various antibiotics. Several lanthionine-containing peptide antibiotics target lipid II with lanthionine-stabilized lipid II binding motifs. Here, we used the biosynthesis system of the lantibiotic nisin to synthesize a two-lipid II binding motifs-containing lantibiotic, termed TL19, which contains the N-terminal lipid II binding motif of nisin and the distinct C-terminal lipid II binding motif of one peptide of the two-component haloduracin (i.e., HalA1). Further characterization demonstrated that (i) TL19 exerts 64-fold stronger antimicrobial activity against Enterococcus faecium than nisin(1-22), which has only one lipid II binding site, and (ii) both the N- and C-terminal domains are essential for the potent antimicrobial activity of TL19, as evidenced by mutagenesis of each single and the double domains. These results show the feasibility of a new approach to synthesize potent lantibiotics with two different lipid II binding motifs to treat specific antibiotic-resistant pathogens.

Detection of Transport Intermediates in the Peptidoglycan Flippase MurJ Identifies Residues Essential for Conformational Cycling.

Bacterial cell wall synthesis is an essential process in bacteria and one of the best targets for antibiotics. A critical step on this pathway is the export of the lipid-linked cell wall monomer, Lipid II, by its transporter MurJ. The mechanism by which MurJ mediates the transbilayer movement of Lipid II is not understood because intermediate states of this process have not been observed. Here we demonstrate a method to capture and detect interactions between MurJ and its substrate Lipid II by photo-cross-linking and subsequent biotin-tagging. We show that this method can be used to covalently capture intermediate transport states of Lipid II on MurJ in living cells. Using this strategy we probed several lethal arginine mutants and found that they retain appreciable substrate-binding ability despite being defective in Lipid II transport. We propose that Lipid II binding to these residues during transport induces a conformational change in MurJ required to proceed through the Lipid II transport cycle. The methods described to detect intermediate transport states of MurJ will be useful for characterizing mechanisms of inhibitors.

Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

In oval-shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan syntheses are performed by independent functional complexes: the divisome and the elongasome. Penicillin-binding proteins (PBPs) were long considered the key peptidoglycan-synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with transmembrane glycosyltransferases (FtsW and RodA) from the shape, elongation, division, and sporulation (SEDS) family to make up the core peptidoglycan-synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD that targets the septum of S. pneumoniae cells to show that class A PBPs have an autonomous role during pneumococcal cell wall synthesis. Using assays to specifically inhibit the function of PBP2x and FtsW, we demonstrate that CbpD attacks nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP-mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes recently formed peptidoglycan synthesized by FtsW/PBP2×. Our results support a model in which mature pneumococcal peptidoglycan is synthesized by three functional entities, the divisome, the elongasome, and bifunctional PBPs. The latter modify existing peptidoglycan but are probably not involved in primary peptidoglycan synthesis.