For many years our research group has been interested in the molecular mechanisms of various antibiotics and the fundamental cellular processes they inhibit. We have primarily focused on drugs that target bacterial cell wall biosynthesis, including the beta-lactams, vancomycin, and moenomycin. We use these molecules to study the protein machines that synthesize and degrade the bacterial cell wall. Mechanistic studies of these classes of enzymes provide insight into the factors that determine bacterial cell shape, growth, and division. Recently, we have also become interested in understanding how the structure of cellular membranes is established and maintained. This is a stereochemical problem since biological membranes are asymmetric and require proper spatial organization of their constituent lipids and proteins in order to function correctly. We use E. coli as our model system and have identified two protein complexes that are involved in assembling lipids and proteins in the outer membrane of Gram-negative bacteria. Our challenge now is to determine how these machines function at a detailed chemical level and to explore their potential as antibacterial targets. A combination of sophisticated organic synthesis, bacterial genetics, biochemistry, and structural biology are required in these efforts.
In light of the public health threat posed by antibiotic resistance, current research in our laboratory is focused on essential processes in bacterial cell envelope biogenesis and on the antibiotics that target them. We seek to increase the fundamental understanding of these pathways and lay the groundwork for new antibacterial strategies. The following projects illustrate some of our current interests:
Bacterial Cell Wall Biosynthesis and Degradation: The cross-linked glycan polymer that surrounds bacterial cells dictates their cell shape and prevents them from lysing due to environmental changes in osmotic pressure. The peptidoglycan glycosyltransferases (PGTs) generate the cell wall, while the amidases, a group of hydrolases, degrade it in a controlled and coordinated manner during cell division. We are studying the mechanisms of these enzymes using chemically synthesized substrates (Lipid II and Lipid IV). We seek to identify the factors that determine bacterial cell shape, growth, and division. Because an intact cell wall is essential to bacterial cell survival, many commonly used antibiotics target enzymes in this pathway. Therefore, we are also studying how these antibiotics, including penicillin, cephalexin, vancomycin, and moenomycin, interact with their targets, alter cell wall synthesis, and induce cell death.
Inhibitory Mechanisms of Moenomycin A: The natural product moenomycin is the only known small molecule inhibitor of the peptidoglycan glycosyltransferases (PGTs). We are using this antibiotic as a tool to study the biochemical function of the PGTs and to determine the structural requirements for their inhibition. Moenomycin has superb in vitro activity but has not been used clinically because it possesses poor physical properties related to its phosphoglycerate lipid. We have developed a synthetic route to moenomycin A using the sulfoxide glycosylation method, which allows ready access to a variety of synthetic analogs. In collaboration with the Walker Lab at Harvard Medical School, we are using these analogs to probe the lipid chain length requirements, the role of the phosphate, the role of the glycerate, and the minimum number of sugar subunits required for transglycosylase inhibition and biological activity. In conjunction with structural studies of PGTs, these experiments will provide information about how PGTs can be inhibited and how moenomycin can be modified to make it more therapeutically useful.
Biogenesis of the Outer Membrane of Gram-negative Bacteria: The outer membrane is an important permeability barrier that consists of an asymmetric bilayer in which the inner leaflet is primarily composed of phospholipids and the outer leaflet is primarily composed of lipopolysaccharides (LPS). Outer membrane proteins (OMPs) of the β-barrel family span the bilayer and serve as channels and transporters. Using a chemical genetic approach, we recently identified two protein complexes in the outer membrane of Escherichia coli that are responsible for the assembly of OMPs and LPS. We are currently pursuing biochemical and structural studies of these complexes in order to understand how β-barrel proteins are folded and inserted into membranes and how the hydrophobic LPS molecule is transported to and assembled in the outer leaflet of the OM. We hope to identify whether there are general principles that guide the assembly of this membrane and to characterize these new protein targets for antibiotic development.
Reconstitution of outer membrane protein assembly from purified components. C.L. Hagan, S. Kim, D. Kahne. Science 2010; 328:890-2.
Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane. S.S. Chng, N. Ruiz, G. Chimalakonda, T.J. Silhavy, D. Kahne. Proc Natl Acad Sci USA 2010; 107:5363-8.
Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine. S. Kim, J.C. Malinverni, P. Sliz, T.J. Silhavy, S.C. Harrison, D. Kahne. Science 2007; 317:961-964.
Identification of a Multi-Component Complex Required for Outer Membrane Biogenesis in Escerichia coli. T. Wu, J. Malinverni, N. Ruiz, S. Kim, T.J. Silhavy, D. Kahne. Cell 2005; 121:235-246.