Explore the vast range of titles available.

The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS 256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp 2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp 4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.

Elongation of rod-shaped bacteria is mediated by a dynamic peptidoglycan-synthetizing machinery called the Rod complex. Here we report that, in Bacillus subtilis , this complex is functional in the absence of all known peptidoglycan polymerases. Cells lacking these enzymes survive by inducing an envelope stress response that increases the expression of RodA, a widely conserved core component of the Rod complex. RodA is a member of the SEDS (shape, elongation, division and sporulation) family of proteins, which have essential but ill-defined roles in cell wall biogenesis during growth, division and sporulation. Our genetic and biochemical analyses indicate that SEDS proteins constitute a family of peptidoglycan polymerases. Thus, B. subtilis and probably most bacteria use two distinct classes of polymerase to synthesize their exoskeleton. Our findings indicate that SEDS family proteins are core cell wall synthases of the cell elongation and division machinery, and represent attractive targets for antibiotic development. SEDS proteins are core peptidoglycan polymerases involved in bacterial cell wall elongation and division. SEDS proteins key to bacterial cell wall integrity It has been generally accepted that the cell wall peptidoglycans of the bacterial exoskeleton are synthesized by penicillin binding proteins (PBPs) known as class A PBPs. Now, using genetic manipulation, phylogenetic analysis and functional experiments in Bacillus subtilis , David Rudner and colleagues have identified SEDS family proteins as the main peptidoglycan polymerases more broadly conserved than class A PBPs. Specifically in B. subtilis , they show that the SEDS protein RodA, a widely conserved component of the Rod complex involved in elongation of rod-shaped bacteria, acts with class B PBPs as the core cell wall synthase of the cell elongation and division machinery. The authors conclude that B. subtilis and probably most bacteria use two distinct classes of polymerases to synthesize their exoskeleton. This work also suggests that SEDS family proteins should be attractive targets for antibiotic development.

Streptococcus pneumoniae ( S. pneumoniae ) has developed resistance to β-lactam antibiotics, largely due to mutations in penicillin-binding protein 2x (PBP2x), particularly within conserved motifs such as STMK and KSG. PBP2x mutations are frequently reported in multidrug-resistant pneumococcal strains associated with pneumonia, meningitis, and septicaemia. especially in serotypes 19A, 19F, and 23F, showing reduced susceptibility to β-lactam antibiotics. These mutations in the PBP2x disrupt antibiotic binding and enzymatic functions, highlighting the need for alternative therapeutic strategies. This study focused on five clinically relevant PBP2x mutations (T338A/G/P and K547G/T) within its active site. A library of phytocompounds was screened using a machine learning model trained to identify antibacterial compounds. Top candidates were filtered based on ADMET properties, and their electronic characteristics were assessed using HOMO–LUMO analysis and electrostatic potential mapping, through density functional theory (DFT). Glucozaluzanin C, a phytochemical derived from Elephantopus scaber , emerged as a potential candidate. Molecular docking and dynamics simulations revealed strong binding affinity and structural integrity with all PBP2x mutants, over a 100-ns timescale. RMSD, RMSF, and hydrogen bonding analysis confirmed stable interactions, suggesting Glucozaluzanin C may effectively interact with PBP2x mutants. Overall, the study highlights an effective strategy for identifying plant-derived inhibitors against β-lactam-resistant S. pneumoniae.

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species ( Bacillus subtilis , Listeria monocytogenes and Streptococcus pneumoniae ) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae . We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner. GpsB is a cytosolic protein that modulates bacterial cell wall synthesis by interacting with cytoplasmic domains of peptidoglycan synthases. Here, Cleverley et al. describe structural features that are important for these interactions, and identify new interacting partners of GpsB in three bacterial species.

The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria 1 . For decades, it was thought that only class A penicillin-binding proteins (PBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA—a member of the unrelated SEDS protein family—also acts as a peptidoglycan polymerase 2 – 4 . Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly 5 , 6 . FtsW was previously proposed to translocate the peptidoglycan precursor lipid II across the cytoplasmic membrane 7 , 8 . Here, we report that purified FtsW polymerizes lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP. We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate class B PBP to produce septal peptidoglycan during cell division. Classically, peptidoglycan (PG) synthesis was thought to be mediated solely by class A penicillin-binding proteins (PBPs) and related enzymes, a view changed by the identification of RodA as a PG polymerase. Now FtsW is also shown to polymerize PG, in a process that requires complex formation with a partner class B PBP.

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd -transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd -transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

A number of ways and means have evolved to provide resistance to eubacteria challenged by β-lactams. This review is focused on pathogens that resist by expressing low-affinity targets for these antibiotics, the penicillin-binding proteins (PBPs). Even within this narrow focus, a great variety of strategies have been uncovered such as the acquisition of an additional low-affinity PBP, the overexpression of an endogenous low-affinity PBP, the alteration of endogenous PBPs by point mutations or homologous recombination or a combination of the above.

Infections caused by multidrug-resistant Streptococcus pneumoniae remain the leading cause of pneumonia-related deaths in children < 5 years globally, and mutations in penicillin-binding protein (PBP) 2 × have been identified as the major cause of resistance in the organism to beta-lactams. Thus, the development of new modulators with enhanced binding of PBP2x is highly encouraged. In this study, phenolics, due to their reported antibacterial activities, were screened against the active site of PBP2x using structure-based pharmacophore and molecular docking techniques, and the ability of the top-hit phenolics to inhibit the active and allosteric sites of PBP2x was refined through 120 ns molecular dynamic simulation. Except for gallocatechin gallate and lysidicichin, respectively, at the active and allosteric sites of PBP2x, the top-hit phenolics had higher negative binding free energy (ΔG bind ) than amoxicillin [active site (− 19.23 kcal/mol), allosteric site (− 33.75 kcal/mol)]. Although silicristin had the best broad-spectrum effects at the active (− 38.41 kcal/mol) and allosteric (− 50.54 kcal/mol) sites of PBP2x, the high thermodynamic entropy (4.90 Å) of the resulting complex might suggest the need for its possible structural refinement for enhanced potency. Interestingly, silicristin had a predicted synthetic feasibility score of < 5 and quantum calculations using the DFT B3LYP/6-31G+ (dp) revealed that silicristin is less stable and more reactive than amoxicillin. These findings point to the possible benefits of the top-hit phenolics, and most especially silicristin, in the direct and synergistic treatment of infections caused by S. pneumoniae . Accordingly, silicristin is currently the subject of further confirmatory in vitro research.

While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens . The development of fluorescent d- amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens , enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld -transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella . This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales . Our findings suggest that penicillin-binding protein 1a, along with a subset of ld -transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales .

Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.