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Regine Hakenbeck - One of the best experts on this subject based on the ideXlab platform.

  • beta lactam resistance in streptococcus pneumoniae Penicillin Binding Proteins and non Penicillin Binding Proteins
    Molecular Microbiology, 1999
    Co-Authors: Regine Hakenbeck, Thorsten W Grebe, Dorothea Zahner, Jeffry B Stock
    Abstract:

    The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the Penicillin-Binding Proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of Penicillin-Binding Proteins.

  • mutational analysis of the streptococcus pneumoniae bimodular class a Penicillin Binding Proteins
    Journal of Bacteriology, 1999
    Co-Authors: Johanna Paik, Iza Kern, Rudi Lurz, Regine Hakenbeck
    Abstract:

    One group of Penicillin target enzymes, the class A high-molecular-weight Penicillin-Binding Proteins (PBPs), are bimodular enzymes. In addition to a central Penicillin-Binding–transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.

  • Penicillin Binding Proteins as resistance determinants in clinical isolates of streptococcus pneumoniae
    Microbial Drug Resistance, 1996
    Co-Authors: Peter Reichmann, Andrea Konig, Anna Marton, Regine Hakenbeck
    Abstract:

    ABSTRACT Altered Penicillin-Binding Proteins (PBPs) with reduced affinity for Penicillin are encoded by mosaic genes in Penicillin-resistant clinical isolates of Streptococcus pneumoniae. Generally, members of one bacterial clone contain the same mosaic gene. We report here on a serotype 19A clone of Penicillin- and multiple-resistant S. pneumoniae prevalent in Hungary, members of which are exceptionally diverse in terms of PBP properties. The pbp2x gene of four 19A isolates was sequenced, and a distinct mosaic structure detected in each case. The pbp2x genes also differed from a homologous gene of a high-level Penicillin-resistant S. mitis from Hungary. The contribution of PBPs to resistance development was studied on transformation experiments using the laboratory strain R6 as recipient, and PBP genes from the type 19A isolate Hull. pbp2x and pbp2b function as primary resistance determinants for different β-lactams. Secondary transformation with pbp1a increased the resistance level considerably for peni...

  • Penicillin Binding Proteins 2x and 2b as primary pbp targets in streptococcus pneumoniae
    Microbial Drug Resistance, 1996
    Co-Authors: Jan Kraus, Thorsten W Grebe, Mark Van Der Linden, Regine Hakenbeck
    Abstract:

    ABSTRACT Different Penicillin-Binding Proteins PBPs are affected in cefotaxime-resistant laboratory mutants compared to piperacillin-resistant mutants. PBP2x acts as the primary PBP target in cefotaxime-resistant mutants, whereas PBP2b is the primary target in piperacillin-resistant mutants. Depending on the mutations in PBP2x, it functions as a resistance determinant for cefotaxime only, or for Penicillins as well. Mutations in PBP2x of laboratory mutants are found exclusively in the Penicillin-Binding domain that contains three homology boxes common to all Penicillin-interacting enzymes. Most mutations relevant for resistance occur close to the SXN or the KT/SG box, or at the C-terminal end of the Penicillin-Binding domain, similar to mutations described in PBP2b of laboratory mutants. Amino acid alterations occur at similar sites also in PBP2x of β-lactam-resistant clinical isolates and most of these Proteins also contain changes in the SXXK box with the active site serine, suggesting that these altera...

  • antigenic variation of Penicillin Binding Proteins from Penicillin resistant clinical strains of streptococcus pneumoniae
    The Journal of Infectious Diseases, 1991
    Co-Authors: Regine Hakenbeck, Thomas Briese, Lynda Chalkley, Heinz Ellerbrok, Raili Kalliokoski, Cristina Latorre, Maija Leinonen, C Martin
    Abstract:

    : Penicillin-resistant strains of Streptococcus pneumoniae that are isolated with increasing frequency worldwide contain low-affinity Penicillin-Binding Proteins (PBPs). The relatedness of PBPs from 55 resistant strains isolated on three continents was investigated by testing the reactivity of antibodies specific for PBP 1a or 2b and by comparing the PBP patterns. Seventeen patterns of antibody reactivity could be distinguished, 12 of which were specific to one isolate. Most strains, including all German and South African strains, had a unique PBP profile. A few groups of Spanish and Finnish isolates were identified where the strains within each group shared the same PBP profile, the same antigenic variants of PBPs 1a and 2b, and the same serogroup, suggesting that they represent different clones of S. pneumoniae. The results demonstrated highly variable pathways of resistance development and confirmed that resistant strains have emerged independently in different locations.

Andrea Dessen - One of the best experts on this subject based on the ideXlab platform.

  • Penicillin Binding Proteins pbps and bacterial cell wall elongation complexes
    Sub-cellular biochemistry, 2019
    Co-Authors: Mayara M Miyachiro, Carlos Contrerasmartel, Andrea Dessen
    Abstract:

    The bacterial cell wall is the validated target of mainstream antimicrobials such as Penicillin and vancomycin. Penicillin and other β-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles’ heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.

  • Penicillin Binding Proteins key players in bacterial cell cycle and drug resistance processes
    Fems Microbiology Reviews, 2006
    Co-Authors: Pauline Macheboeuf, Otto Dideberg, C Contrerasmartel, Andrea Dessen
    Abstract:

    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 β-lactam antibiotics. The increasing incidence of β-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.

  • Penicillin Binding Proteins: Key players in bacterial cell cycle and drug resistance processes
    FEMS Microbiology Reviews, 2006
    Co-Authors: Pauline Macheboeuf, Carlos Contreras-martel, Viviana Job, Otto Dideberg, Andrea Dessen
    Abstract:

    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.

Erin E Carlson - One of the best experts on this subject based on the ideXlab platform.

  • harnessing β lactam antibiotics for illumination of the activity of Penicillin Binding Proteins in bacillus subtilis
    ACS Chemical Biology, 2020
    Co-Authors: Shabnam Sharifzadeh, Felix Dempwolff, Daniel B Kearns, Erin E Carlson
    Abstract:

    Selective chemical probes enable individual investigation of Penicillin-Binding Proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution....

  • chemical tools for selective activity profiling of bacterial Penicillin Binding Proteins
    Methods in Enzymology, 2020
    Co-Authors: Shabnam Sharifzadeh, Joshua D Shirley, Malcolm E Winkler, Nathaniel W Brown, Kevin E Bruce, Erin E Carlson
    Abstract:

    Abstract Penicillin-Binding Proteins (PBPs) are membrane-associated Proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These Proteins were discovered and named for their affinity to bind the β-lactam antibiotic Penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4–18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a β-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent β-lactone probes.

  • novel electrophilic scaffold for imaging of essential Penicillin Binding Proteins in streptococcus pneumoniae
    ACS Chemical Biology, 2017
    Co-Authors: Shabnam Sharifzadeh, Michael J Boersma, Ozden Kocaoglu, Alireza Shokri, Clayton L Brown, Joshua D Shirley, Malcolm E Winkler, Erin E Carlson
    Abstract:

    Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-Binding Proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The β-lactam antibiotics, which are among the most clinically effective antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional s...

  • profiling of β lactam selectivity for Penicillin Binding Proteins in escherichia coli strain dc2
    Antimicrobial Agents and Chemotherapy, 2015
    Co-Authors: Ozden Kocaoglu, Erin E Carlson
    Abstract:

    Penicillin-Binding Proteins (PBPs) are integral players in bacterial cell division, and their catalytic activities can be monitored with β-lactam-containing chemical probes. Compounds that target a single PBP could provide important information about the specific role(s) of each enzyme, making identification of such molecules important. We evaluated 22 commercially available β-lactams for inhibition of the PBPs in live Escherichia coli strain DC2. Whole cells were titrated with β-lactam antibiotics and subsequently incubated with a fluorescent Penicillin derivative, Bocillin-FL (Boc-FL), to label uninhibited PBPs. Protein visualization was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and fluorescent scanning. The examined β-lactams exhibited diverse PBP selectivities, with amdinocillin (mecillinam) showing selectivity for PBP2, aztreonam, piperacillin, cefuroxime, cefotaxime, and ceftriaxone for PBP3, and amoxicillin and cephalexin for PBP4. The remaining β-lactams did not block any PBPs in the DC2 strain of E. coli or inhibited more than one PBP at all examined concentrations in this Gram-negative organism.

Jeanmarie Frere - One of the best experts on this subject based on the ideXlab platform.

  • Penicillin Binding Proteins evergreen drug targets
    Current Opinion in Pharmacology, 2014
    Co-Authors: Jeanmarie Frere, Malcolm G P Page
    Abstract:

    The Penicillin-Binding Proteins (PBPs) are well known targets for the β-lactam antibiotics. They continue to be a focus of interest for pharmaceutical design, as exemplified by the number of new agents under clinical investigation as well as novel experimental molecules. Considerable advances have been made in understanding the structure and function of this family of enzymes, through high-resolution structural studies and mechanistic studies in solution. These studies have thrown light on role of the high molecular mass PBPs in mediating β-lactam resistance, although much work remains to be done to enable a full description of the mechanisms by which these Proteins modulate their sensitivity towards β-lactams while retaining their essential activity in cell wall biosynthesis.

  • novel peptide inhibiting both tem 1 β lactamase and Penicillin Binding Proteins
    FEBS Journal, 2010
    Co-Authors: Denis Phichith, Jeanmarie Frere, Moreno Galleni, Severine Padiolleaulefevre, Adeline Guellier, Soun Banh, Daniel Thomas, Alain Friboulet, Berangere Avalle
    Abstract:

    9G4H9, a catalytic antibody displaying β-lactamase-like activity, has been developed by the anti-idiotypic approach using β-lactamase as the first antigen. Thus 9G4H9 represents the ‘internal image‘ of β-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 β-lactamase (Ki = 333 μm) and several Penicillin-Binding Proteins (IC50 values ranging from 6–62 μm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 β-lactamase.

  • A new, highly sensitive method for the detection and quantification of Penicillin-Binding Proteins.
    Biochemical Journal, 1993
    Co-Authors: Moreno Galleni, Bernard Lakaye, Sophie Lepage, Marc Jamin, Iris Thamm, Bernard Joris, Jeanmarie Frere
    Abstract:

    A new method for the identification and quantification of Penicillin-Binding Proteins is described which uses fluorescein-coupled Penicillins. It allows the rapid detection of 0.2 pmol with the naked eye and 2 fmol with the help of an A.L.F. automatic DNA sequencer. Direct labelling can also be performed on whole bacterial cells.

Malcolm G P Page - One of the best experts on this subject based on the ideXlab platform.

  • Penicillin Binding Proteins evergreen drug targets
    Current Opinion in Pharmacology, 2014
    Co-Authors: Jeanmarie Frere, Malcolm G P Page
    Abstract:

    The Penicillin-Binding Proteins (PBPs) are well known targets for the β-lactam antibiotics. They continue to be a focus of interest for pharmaceutical design, as exemplified by the number of new agents under clinical investigation as well as novel experimental molecules. Considerable advances have been made in understanding the structure and function of this family of enzymes, through high-resolution structural studies and mechanistic studies in solution. These studies have thrown light on role of the high molecular mass PBPs in mediating β-lactam resistance, although much work remains to be done to enable a full description of the mechanisms by which these Proteins modulate their sensitivity towards β-lactams while retaining their essential activity in cell wall biosynthesis.

  • Binding of ceftobiprole and comparators to the Penicillin Binding Proteins of escherichia coli pseudomonas aeruginosa staphylococcus aureus and streptococcus pneumoniae
    Antimicrobial Agents and Chemotherapy, 2007
    Co-Authors: Todd A Davies, Malcolm G P Page, Wenchi Shang, Ted Andrew, Malgosia Kania, Karen Bush
    Abstract:

    Ceftobiprole exhibited tight Binding to PBP2a in methicillin-resistant Staphylococcus aureus, PBP2x in Penicillin-resistant Streptococcus pneumoniae, and PBP3 and other essential Penicillin-Binding Proteins in methicillin-susceptible S. aureus, Escherichia coli, and Pseudomonas aeruginosa. Ceftobiprole also bound well to PBP2 in the latter organisms, contributing to the broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria.