Vancomycin kills Gram-positive enterococci and staphylococci by interfering with cell-wall biosynthesis. We have shown that in wild-type Staphylococcus aureus, vancomycin binds to the stem termini of cell-wall precursors and inhibits transglycosylation (glycan chain extension). Recently, vancomycin-resistant enterococci and staphylococci have emerged. These bacteria have altered peptidoglycan structures and reduced vancomycin binding. We have used solid-state NMR to characterize the cell-wall complexes of five fluorinated glycopeptides with improved potency against vancomycin-resistant enterococci. We obtained two of the drugs from Eli Lilly Company, two from the Cause Institute of New Antibiotics (Russia), and one we synthesized ourselves. We have correlated structure and activity for these drugs for the first time and have formulated their mode of action. In the next grant period, we plan to prove or disprove the general hypothesis, that these drugs kill vancomycin-resistant enterococci and staphylococci by interfering with template recognition during peptidoglycan biosynthesis. We believe that new peptidoglycan strands must be pre-ordered to fit into a tightly cross-linked three-dimensional network, and that this is the reason that an existing nearest-neighbor strand is used as a template for the synthesis of a new strand. We will test these notions using solid-state NMR detection of drug-complex formation and biosynthesis in whole cells of a variety of vancomycin-susceptible and vancomycin-resistant enterococci and staphylococci, organisms which we have acquired from our colleague- collaborators, P. Courvalin (France) and H. Labischinski (Germany). We will use highly selective stable-isotope labeling protocols with detection by new, specially designed solid-state NMR experiments. None of the glycopeptides with improved potency against vancomycin-resistant enterococci and staphylococci that we have examined are approved for clinical use in the United States because of deleterious side effects. We are hopeful that the insights into the mode(s) of action of these glycopeptides that will result from the work proposed in this application will stimulate the search for new and potent antibiotics with tolerable side effects.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Mclaughlin, Alan Charles
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Washington University
Schools of Arts and Sciences
Saint Louis
United States
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Yang, Hao; Singh, Manmilan; Kim, Sung Joon et al. (2017) Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis. Biochim Biophys Acta Biomembr 1859:2171-2180
Singh, Manmilan; Kim, Sung Joon; Sharif, Shasad et al. (2015) REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant. Biochim Biophys Acta 1848:363-8
Kim, Sung Joon; Chang, James; Singh, Manmilan (2015) Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR. Biochim Biophys Acta 1848:350-62
Chen, Ying-Jr; Huang, Xiaojing; Mahieu, Nathaniel G et al. (2014) Differential incorporation of glucose into biomass during Warburg metabolism. Biochemistry 53:4755-7
Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad et al. (2014) Cross-link formation and peptidoglycan lattice assembly in the FemA mutant of Staphylococcus aureus. Biochemistry 53:1420-7
Sharif, Shasad; Kim, Sung Joon; Labischinski, Harald et al. (2013) Uniformity of glycyl bridge lengths in the mature cell walls of fem mutants of methicillin-resistant Staphylococcus aureus. J Bacteriol 195:1421-7
Kim, Sung Joon; Tanaka, Kelly S E; Dietrich, Evelyne et al. (2013) Locations of the hydrophobic side chains of lipoglycopeptides bound to the peptidoglycan of Staphylococcus aureus. Biochemistry 52:3405-14
Kim, Sung Joon; Singh, Manmilan; Preobrazhenskaya, Maria et al. (2013) Staphylococcus aureus peptidoglycan stem packing by rotational-echo double resonance NMR spectroscopy. Biochemistry 52:3651-9
Kim, Sung Joon; Singh, Manmilan; Wohlrab, Aaron et al. (2013) The isotridecanyl side chain of plusbacin-A3 is essential for the transglycosylase inhibition of peptidoglycan biosynthesis. Biochemistry 52:1973-9
Sharif, Shasad; Kim, Sung Joon; Labischinski, Harald et al. (2009) Characterization of peptidoglycan in fem-deletion mutants of methicillin-resistant Staphylococcus aureus by solid-state NMR. Biochemistry 48:3100-8

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