Cell-Wall Recycling and Antibiotic Resistance

Summary

Principal Investigator: Shahriar Mobashery
Abstract: DESCRIPTION (provided by applicant): Many members of Enterobacteriaceae and Pseudomonas aeruginosa (subject of this grant application) have the ability to sense damage inflicted to their cell wall by [unreadable]-lactam antibiotics. A primary mechanism for this sensing involves the events of cell-wall recycling, and results in the induction of resistance mechanisms. These events have led to the obsolescence of many of the [unreadable]-lactam antibiotics against these Gram-negative bacteria. These multiple complex steps are poorly elucidated, and are the subject of this grant proposal. Three Specific Aims are proposed. In Specific Aim 1 I propose to elucidate the reactions of all lytic transglycosylases, enzymes that initiate the cell-wall recycling events. This undertaking utilizes methodologies developed in my lab for highly sensitive high-resolution identification of reaction products of these enzymes by proteomics approaches. Furthermore, a link between the function of penicillin-binding protein 4 (PBP4) of Pseudomonas aeruginosa and the sensing of the presence of the [unreadable]-lactam antibiotic has been made. I disclose my views on how this process could take place and I propose the means to the identification of the signal molecule, which triggers the antibiotic resistance processes. Specific Aim 2 proposes to study and identify a key enzyme in the resistance induction process, the AmpD protease. Three homologous enzymes have been annotated in P. aeruginosa for this activity. We have outlined studies that will delineate which of the three, or a subset thereof, are bona fide enzymes involved in cell-wall recycling and antibiotic resistance induction. Specific Aim 3 will delineate the transcriptional events that lead to [unreadable]-lactam antibiotic resistance in P. aeruginosa. This includes identification of the key transcriptional activator molecule and the kinetics of the processes. I anticipate that the successful completion of this proposed science will not only lead to the elucidation of these events, but also will identify opportunities for their interruption as means to circumvent the elaborate mechanisms of resistance.
Funding Period: 2000-07-01 - 2017-06-30
more information: NIH RePORT

Top Publications

  1. pmc Three-dimensional structure of the bacterial cell wall peptidoglycan
    Samy O Meroueh
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 5670, USA
    Proc Natl Acad Sci U S A 103:4404-9. 2006
  2. pmc Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae
    Beatriz Maestro
    Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Elche, Spain
    FEBS Lett 585:357-63. 2011
  3. pmc High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli
    Cecilia Artola-Recolons
    Department of Crystallography and Structural Biology, Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
    Biochemistry 50:2384-6. 2011
  4. pmc Messenger functions of the bacterial cell wall-derived muropeptides
    Marc A Boudreau
    Department of Chemistry and Biochemistry, Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana 46556, USA
    Biochemistry 51:2974-90. 2012
  5. pmc Synthesis and NMR characterization of (Z,Z,Z,Z,E,E,ω)-heptaprenol
    Dusan Hesek
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 134:13881-8. 2012
  6. pmc Bacterial cell-wall recycling
    Jarrod W Johnson
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    Ann N Y Acad Sci 1277:54-75. 2013
  7. pmc Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall
    Mijoon Lee
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
    J Am Chem Soc 135:3311-4. 2013
  8. pmc Reactions of the three AmpD enzymes of Pseudomonas aeruginosa
    Weilie Zhang
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
    J Am Chem Soc 135:4950-3. 2013
  9. pmc Reaction products and the X-ray structure of AmpDh2, a virulence determinant of Pseudomonas aeruginosa
    Siseth Martínez-Caballero
    Department of Crystallography and Structural Biology, Institute of Physical Chemistry Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
    J Am Chem Soc 135:10318-21. 2013
  10. pmc Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa
    Mijoon Lee
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 135:12604-7. 2013

Detail Information

Publications24

  1. pmc Three-dimensional structure of the bacterial cell wall peptidoglycan
    Samy O Meroueh
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 5670, USA
    Proc Natl Acad Sci U S A 103:4404-9. 2006
    ..The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed...
  2. pmc Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae
    Beatriz Maestro
    Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, Elche, Spain
    FEBS Lett 585:357-63. 2011
    ....
  3. pmc High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli
    Cecilia Artola-Recolons
    Department of Crystallography and Structural Biology, Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
    Biochemistry 50:2384-6. 2011
    ..It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane...
  4. pmc Messenger functions of the bacterial cell wall-derived muropeptides
    Marc A Boudreau
    Department of Chemistry and Biochemistry, Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana 46556, USA
    Biochemistry 51:2974-90. 2012
    ....
  5. pmc Synthesis and NMR characterization of (Z,Z,Z,Z,E,E,ω)-heptaprenol
    Dusan Hesek
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 134:13881-8. 2012
    ..The availability by this synthesis of (Z(4),E(2),ω)-heptaprenol 4 in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope...
  6. pmc Bacterial cell-wall recycling
    Jarrod W Johnson
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    Ann N Y Acad Sci 1277:54-75. 2013
    ..We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall-recycling inhibitors...
  7. pmc Reactions of all Escherichia coli lytic transglycosylases with bacterial cell wall
    Mijoon Lee
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
    J Am Chem Soc 135:3311-4. 2013
    ..These discoveries underscore the importance of the functions of these enzymes for recycling of the cell wall...
  8. pmc Reactions of the three AmpD enzymes of Pseudomonas aeruginosa
    Weilie Zhang
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
    J Am Chem Soc 135:4950-3. 2013
    ..These findings define for the first time the events mediated by these three enzymes that lead to turnover of a key cell-wall recycling metabolite as well as the cell wall itself in its maturation. ..
  9. pmc Reaction products and the X-ray structure of AmpDh2, a virulence determinant of Pseudomonas aeruginosa
    Siseth Martínez-Caballero
    Department of Crystallography and Structural Biology, Institute of Physical Chemistry Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain
    J Am Chem Soc 135:10318-21. 2013
    ..This binding surface can accommodate the 3D solution structure of the cross-linked cell wall...
  10. pmc Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa
    Mijoon Lee
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 135:12604-7. 2013
    ....
  11. pmc Sulfonylation-induced N- to O-acetyl migration in 2-acetamidoethanol derivatives
    Takao Yamaguchi
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Org Chem 75:3515-7. 2010
    ..Furthermore, the results reveal that 2-acetamidoethanol derivatives with a sterically encumbered hydroxyl group result in the migration products in high yields. The mechanism of the migration reaction is discussed...
  12. pmc Thermodynamics of interactions of vancomycin and synthetic surrogates of bacterial cell wall
    Mikhail Rekharsky
    Entropy Control Project, ICORP, JST, 4 6 3 Kamishinden, Toyonaka 560 0085, Japan
    J Am Chem Soc 128:7736-7. 2006
    ..These data were reconciled with the recently determined NMR solution structure for the peptidoglycan fragment 4 and its implications for the larger cell wall...
  13. pmc Structural insights into the bactericidal mechanism of human peptidoglycan recognition proteins
    Sangwoo Cho
    Center for Advanced Research in Biotechnology, W M Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA
    Proc Natl Acad Sci U S A 104:8761-6. 2007
    ....
  14. pmc Restoration of susceptibility of methicillin-resistant Staphylococcus aureus to beta-lactam antibiotics by acidic pH: role of penicillin-binding protein PBP 2a
    Sandrine Lemaire
    Unité de Pharmacologie Cellulaire et Moléculaire, Universite Catholique de Louvain, B 1200 Brussels, Belgium
    J Biol Chem 283:12769-76. 2008
    ....
  15. pmc Facile preparation of a highly functionalized tetrahydropyran by catalytic hydrogenation of an oxazoline
    Dusan Hesek
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Org Chem 73:7349-52. 2008
    ..A simple catalytic hydrogenation at C-1 of an oxazoline constructed from the corresponding 2-aminopyranose results in the desired product...
  16. pmc Lytic transglycosylase MltB of Escherichia coli and its role in recycling of peptidoglycan strands of bacterial cell wall
    Maxim Suvorov
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 130:11878-9. 2008
    ..Furthermore, turnover of the E. coli sacculus (whole cell wall) by MltB was characterized. It is documented that each MltB molecule is able to process the cell wall 14000 times in the course of a single doubling time for E. coli...
  17. pmc Complications from dual roles of sodium hydride as a base and as a reducing agent
    Dusan Hesek
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Org Chem 74:2567-70. 2009
    ..The structural nature of these byproducts is revealed in this report...
  18. pmc Total synthesis of N-acetylglucosamine-1,6-anhydro-N-acetylmuramylpentapeptide and evaluation of its turnover by AmpD from Escherichia coli
    Dusan Hesek
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 131:5187-93. 2009
    ..Compound 1 is shown to be a substrate of the AmpD enzyme of the gram-negative bacterium Escherichia coli, an enzyme that removes the peptide from the disaccharide scaffold in the early cytoplasmic phase of cell wall turnover...
  19. pmc Bacterial AmpD at the crossroads of peptidoglycan recycling and manifestation of antibiotic resistance
    Mijoon Lee
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 131:8742-3. 2009
    ..The implications of the function of this catalyst for the entry into the cell wall recycling events and the reversal of induction of the production of beta-lactamase, an antibiotic resistance determinant, are discussed...
  20. pmc Crystal structures of penicillin-binding protein 6 from Escherichia coli
    Yu Chen
    Department of Pharmaceutical Chemistry, University of California San Francisco, Byers Hall, Room 508D, 1700 Fourth Street, San Francisco, California 94158 2550, USA
    J Am Chem Soc 131:14345-54. 2009
    ....
  21. pmc Elucidation of the structure of the membrane anchor of penicillin-binding protein 5 of Escherichia coli
    Peter I O'Daniel
    Department of Chemistry and Biochemistry, 423 Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana 46556, USA
    J Am Chem Soc 132:4110-8. 2010
    ....
  22. pmc Use of silver carbonate in the Wittig reaction
    Lukas Jedinak
    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
    J Org Chem 78:12224-8. 2013
    ..These reactions proceed overnight at room temperature, under weakly basic conditions, and as such extend the applicability of the Wittig reaction to base-sensitive reactants. ..

Research Grants34

  1. Challenges in beta-Lactamase Mediated Resistance
    Robert A Bonomo; Fiscal Year: 2013
    ..We chose these [unreadable]-lactamases as they are among the most important class A and class C enzymes in Gram-negative bacteria. ..
  2. Identifying and validating new antibiotic targets in cell wall synthesis pathways
    Suzanne Walker; Fiscal Year: 2013
    ..coli will be broadly relevant to our understanding of cell wall polymer biogenesis in other microorganisms and should significantly impact and inform efforts to generate therapies against MRSA and Gram-negative ESKAPE pathogens. ..
  3. Inducible Antibiotic Resistance in Methicillin-Resistant Staphylococcus Aureus
    Shahriar Mobashery; Fiscal Year: 2013
    ..Furthermore, we propose to study antibiotic resistance mechanisms that arise by alterations in the allosteric site. ..
  4. Studies on the biological mechanisms of antibiotics
    DANIEL E KAHNE; Fiscal Year: 2013
    ..Since these compounds target a fundamental metabolic process in bacteria, a better understanding of this process could lead to new antibiotic targets or strategies as well. ..
  5. Novel Compounds Targeting Bacterial PBPs
    LUIGI XERRI; Fiscal Year: 2013
    ..Such new products could serve as a critical advance in the search for new treatments for serious multi-resistant bacterial infections in the nosocomial and community settings. ..
  6. Mechanistic Studies of Carbapenem Synthase (CarC)
    COURTNEY JAMES HASTINGS; Fiscal Year: 2013
    ..These studies are particularly important since structural studies on the CarC enzyme leave many questions unanswered about the precise nature of interactions between the substrate and active site. . ..
  7. Gene silencing therapeutics for chronic infections in cystic fibrosis
    DAVID ELIHU GREENBERG; Fiscal Year: 2013
    ..This work has implications not only for these infections in CF, but for other medically important Gram-negative pathogens. ..
  8. Transplant Tolerance in Non-Human Primates
    STUART JOHNSTON KNECHTLE; Fiscal Year: 2013
    ..This goal will be accomplished via four interrelated projects and two supporting cores. ..
  9. Preserving beta-lactam utility vs pathogens producing any class of beta-lactamase
    LUIGI XERRI; Fiscal Year: 2013
    ..The Phase II application endeavors to select a pre-development candidate from these two "Finalists" and drive this compound through IND-enabling studies to an Investigational New Drug filing. ..
  10. Harvard-wide Program on Antibiotic Resistance
    Suzanne Walker; Fiscal Year: 2013
    ..One compound already has been validated, and the goal of this program is to deliver 10 more along with advancing the attendant science ..
  11. Analysis of metallo-beta-lactamase sequence constraints at high resolution
    Timothy Palzkill; Fiscal Year: 2013
    ..The detailed knowledge of how active site residue positions contribute to [unreadable]-lactam hydrolysis will facilitate the design of inhibitors that interact with the critically importnt MBL residues. ..
  12. Cell wall synthesis enzymes and beta-lactam resistance in Enterococcus faecium
    Louis B Rice; Fiscal Year: 2013
    ..They will also enhance our understanding of cell wall synthesis mechanisms in Gram-positive cocci and reveal promising new targets for antibacterial therapy. ..
  13. Discovery of Polymyxin-based Antibacterial Agents Active Against Multi-Drug Resis
    Lawrence H Lash; Fiscal Year: 2013
    ..This will lead to in vivo proof-of-principle for drug action and pharmacokinetic studies for the selection of compounds for future pre-clinical evaluation. ..
  14. Protein-protein interaction essential for bacterial growth and virulence
    Mark A Saper; Fiscal Year: 2013
    ..Moreover, the mode of ac- tion of such an inhibitor would be complementary with, but different from, ?-lactams. ..
  15. Peptidoglycan Biogenesis in Escherichia Coli
    Thomas G Bernhardt; Fiscal Year: 2013
    ....
  16. Transport and Insertion of Outer Membrane Proteins
    Marcelo C Sousa; Fiscal Year: 2013
    ..We will (i) determine the structure of the BAM complex;(ii) test mechanistic hypotheses derived from the structures and (iii) develop an integrated model of OMP transport folding and insertion in the outer membrane. ..
  17. Class A Carbapenemases
    Sergei Vakulenko; Fiscal Year: 2013
    ..Elucidation of the catalytic mechanisms and structures of class A carbapenemases has the potential to result in development of the next generations of ?-lactam antibiotics and inhibitors of these enzymes. ..
  18. Elucidating beta-lactamase functional mechanisms via evolutionary conservation
    Dennis R Livesay; Fiscal Year: 2013
    ..As such, we propose a series of additional studies along these lines to expand our understanding of BL structure and function, potentially paving the way to new therapeutic opportunities. ..
  19. Molecular Targets in Peptidoglycan Synthesis
    Christopher Davies; Fiscal Year: 2013
    ..To understand the functional role of AmiC in peptidoglycan breakdown, but also to pave the way for drug discovery against its two active sites, we will obtain essential structural and biochemical information for AmiC. ..
  20. Pacific NorthWest Regional Center of Excellence (PNWRCE)
    Jay A Nelson; Fiscal Year: 2013
    ..pseudomallei host pathogen response during both the septicemic as well as the intracellular phases of the disease. ..
  21. New England Regional Center of Excellence in Biodefense and Emerging Infectious D
    Dennis L Kasper; Fiscal Year: 2013
    ..NERCE will also continue its Developmental Projects program and Career Development in Biodefense program in an effort to initiate new research efforts and to attract new investigators to this field. ..
  22. BIOSYNTHESIS OF BETA-LACTAM ANTIBIOTICS
    CRAIG ARTHUR TOWNSEND; Fiscal Year: 2013
    ..abstract_text> ..
  23. Molecular Analyses and Interventions for Biodefense and Emerging Pathogens
    Olaf Schneewind; Fiscal Year: 2013
    ..Research and training at the GLRCE is governed by a mechanism involving ongoing review of scientific excellence and translational goals, inter-institutional advisory boards and external scientific advisory bodies. ..
  24. The use of mass spectrometry for rapid detection of carbapenemase-producing bacte
    DAVID RYAN PEAPER; Fiscal Year: 2013
    ..Future phases of development will apply the technology to primary specimens, which will be followed by multicenter clinical trials in pursuit o FDA 510(k) clearance of the final product. ..
  25. Novel Mechanisms of Beta-lactam Resistance in Staph Aureus
    Henry F Chambers; Fiscal Year: 2013
    ..Recombinant GdpP also will be purified and analyzed by x-ray crystallography to identify its critical structural properties. Achieving these aims will increase knowledge of ?-lactam antibiotic effects and mechanisms of resistance. ..
  26. Aminoglycoside Resistance in Bacteria
    Sergei Vakulenko; Fiscal Year: 2013
    ..Design of such novel antibacterial agents will be a breakthrough for treatment of life-threatening infectious diseases. ..
  27. Pacific Southwest RCE for Biodefense &Emerging Infectious Diseases Research
    Alan G Barbour; Fiscal Year: 2013
    ..abstract_text> ..
  28. BIOLOGICALLY ACTIVE CYCLIC PEPTIDES
    Dale L Boger; Fiscal Year: 2013
    ....