This era may come to be remembered as one in which infectious diseases made a dramatic worldwide resurgence, owing to the rise of antibiotic resistance and the limited number of antibiotics in the drug development pipeline. New antibiotics are needed and the emergence of resistance to almost any antibiotic underscores the need to understand the molecular details of antibiotic modes of action to help guide the development of new antibiotics. Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Yet, as a heterogeneous insoluble polymeric matrix, the cell wall poses a challenge to non-perturbative analyses of composition and architecture. Solid-state NMR has emerged as a powerful tool to measure parameters of composition and architecture in the context of intact cell walls and whole cells and was employed to define the mode of action of vancomycin analogs including oritvancin (OrbactivTM), focusing on specifically labeled samples and two key peptidoglycan sites (crosslinks and bridgelinks). Analyses of unlabeled or uniformly-labeled samples report more broadly on overall carbon and nitrogen composition in cell walls and whole cells, can be readily extended to other organisms, do not require considerations of label scrambling and dilution, and can identify alterations due to different classes of antibiotics. In this project, we will introduce new protocols, integrate these with existing approaches, and build and describe an effective solid-state NMR toolkit to define and quantify bacterial cell-wall composition, including peptidoglycan and teichoic acid components, in intact preparations of cell walls and whole cells. These approaches will integrate different labeling strategies coupled with appropriate NMR pulse sequences. The results from these approaches complement extensive biochemical data usually already available for old and new antibiotics and will be uniquely enabling in their ability to identify and quantitatively compare cell-wall components in intact samples without degradative hydrolyses and perturbations that limit quantitative comparisons in solution-based assays, particularly important for Gram-positive organisms with cell walls that resist complete dissolution. The methods will be tested with a panel of established antibiotics and will be used to investigate different bacterial strains and the modes of action of four new antibiotics.

Public Health Relevance

This era may come to be remembered as one in which infectious diseases made a dramatic worldwide resurgence, owing to the rise of antibiotic resistance and the limited number of antibiotics in the drug development pipeline. Staphylococcus aureus infections are often resistant to multiple drugs and new methods are needed to identify the modes of action of candidate antibiotics to develop next generation inhibitors, particularly those that target cell-wall synthesis in Gram-positive bacteria. We have designed new approaches to define and quantify bacterial cell-wall composition and will deliver needed discoveries regarding the modes of action of new and emerging antibiotics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Fabian, Miles
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Stanford University
Schools of Arts and Sciences
United States
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Romaniuk, Joseph A H; Cegelski, Lynette (2018) Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance. Biochemistry 57:3966-3975
Yang, Hao; Staveness, Daryl; Ryckbosch, Steven M et al. (2018) REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment. ACS Cent Sci 4:89-96
Antonoplis, Alexandra; Zang, Xiaoyu; Huttner, Melanie A et al. (2018) A Dual-Function Antibiotic-Transporter Conjugate Exhibits Superior Activity in Sterilizing MRSA Biofilms and Killing Persister Cells. J Am Chem Soc 140:16140-16151
Nygaard, Rie; Romaniuk, Joseph A H; Rice, David M et al. (2017) Whole Ribosome NMR: Dipolar Couplings and Contributions to Whole Cells. J Phys Chem B 121:9331-9335
Cegelski, Lynette (2017) Disentangling Nanonets: Human ?-Defensin 6 Targets Candida albicans Virulence. Biochemistry 56:1027-1028