My laboratory focuses on the investigation of host-pathogen interactions and metabolic changes associated during the infection to provide insights into the molecular mechanisms of microbial pathogenesis, thereby facilitating the development of novel therapeutic agents for the treatment and the prevention of infectious disease.  The structural characterization of host-pathogen interactions in vivo involving cell surfaces is difficult due to the complexity and heterogeneity, making it incompatible with the conventional structural methods such as x-ray diffraction and solution-state NMR.  Our approach is to use solid-state NMR to provide structural insights with atomic resolution and the changes in chemical and metabolic compositions during the infection. 


NEWS: Congratulations to James Chang for The Glasscock Energy Research Endowed Scholarship award.


Abstracts from Selected Publications




Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy

Highlighted in the current Biochemistry journal's home page (May 28, 2013 Volume 52, Issue 21).

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Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-13C]alanine and l-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C–15N average distance of 4.1–4.4 Å between d- and l-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.


Locations of the Hydrophobic Side Chains of Lipoglycopeptides Bound to the Peptidoglycan of Staphylococcus aureus (Biochemistry. 2013 May 8)

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Glycopeptides whose aminosugars have been modified by attachment of hydrophobic side chains are frequently active against vancomycin-resistant microorganisms. We have compared the conformations of six such fluorinated glycopeptides (with side chains of varying length) complexed to cell walls labeled with d-[1-13C]alanine, [1-13C]glycine, and l-[ε-15N]lysine in whole cells of Staphylococcus aureus. The internuclear distances from 19F of the bound drug to the 13C and 15N labels of the peptidoglycan, and to the natural abundance 31P of lipid membranes and teichoic acids, were determined by rotational-echo double resonance NMR. The drugs did not dimerize, and their side chains did not form membrane anchors but instead became essential parts of secondary binding to pentaglycyl bridge segments of the cell-wall peptidoglycan.


Uniformity of Glycyl Bridge Lengths in the Mature Cell Walls of Fem Mutants of Methicillin-Resistant Staphylococcus aureus (J Bacteriol. 2013 Apr;195(7):1421-7)

Fig 1

Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.


The Isotridecanyl Side Chain of Plusbacin-A3 Is Essential for the Transglycosylase Inhibition of Peptidoglycan Biosynthesis (Biochemistry. 2013 Mar 19;52(11):1973-9)

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Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm, and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer, thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [2H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [2H]pb-A3 has an isotopically 2H-labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [2H]pb-A3 bound to [1-13C]glycine-labeled whole cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [2H]pb-A3 during the growth of S. aureus resulted in the accumulation of Park’s nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.


Oritavancin binds to isolated protoplast membranes but not intact protoplasts of Staphylococcus aureus
(J. Mol. Biol
. 2009 Aug 14;391(2):414-25)

Full-size image (52 K)

Solid-state NMR has been used to examine the binding of N'-4-[(4-fluorophenyl)benzyl)]chloroeremomycin, a fluorinated analogue of oritavancin, to isolated protoplast membranes and whole-cell sucrose-stabilized protoplasts of Staphylococcus aureus, grown in media containing [1(13)C]glycine and L-[epsilon-(15)N]lysine. Rotational-echo double-resonance NMR was used to characterize the binding by estimating internuclear distances from (19)F of oritavancin to (13)C and (15)N labels of the membrane-associated peptidoglycan and to the (31)P of the phospholipid bilayer of the membrane. In isolated protoplast membranes, both with and without 1 M sucrose added to the buffer, the nascent peptidoglycan was extended away from the membrane surface and the oritavancin hydrophobic side chain was buried deep in the exposed lipid bilayer. However, there was no N'-4-[(4-fluorophenyl)benzyl)]chloroeremomycin binding to intact sucrose-stabilized protoplasts, even though the drug bound normally to the cell walls of whole cells of S. aureus in the presence of 1 M sucrose. As shown by the proximity of peptidoglycan-bridge (13)C labels to phosphate (31)P, the nascent peptidoglycan of the intact protoplasts was confined to the membrane surface.


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