Most bacteria polymerize peptidoglycan (PG) into a mesh-like sacculus that surrounds the cytoplasmic membrane and protects it against osmotic lysis. The PG sacculus also provides cell shape and serves as a scaffold to which virulence factors are anchored. Biogenesis of the PG sacculus is essential for viability, and our long-term goal is to understand it at the molecula level. This proposal focuses on the transport of PG intermediates across the cytoplasmic membrane, a poorly understood step in PG biogenesis. Bacteria build their PG sacculus in the extracytoplasmic space by polymerizing a disaccharide pentapeptide into glycan strands that are later crosslinked. The disaccharide pentapeptide is made in the cytoplasm as a lipid intermediate known as lipid II. Therefore, lipid II must be flipped across the membrane by a transporter (a flippase) through an unknown mechanism. The membrane protein MurJ has been proposed to be the lipid II flippase in Escherichia coli since it is essential for PG synthess and it belongs to the MOP (multidrug/oligosaccharidyl- lipid/polysaccharide) superfamily of exporters. This family is conserved in diverse organisms and it includes flippases of lipid-linked oligosaccharides that are similar to lipid II. FtsW and RodA have also been proposed to be lipid II flippases in E. coli, but there is no in vivo evidence supporting this hypothesis.
Aim 1 of thi proposal is to determine whether MurJ, FtsW, and RodA are involved in lipid II translocation in vivo. Two analytical assays based on differential labeling and mass spectrometry will be developed to assess how depletion of these factors affects the levels and membrane topology of lipid II. The data obtained will be crucial for understanding the roles of MurJ, FtsW, and RodA in PG biogenesis.
Aim 2 of this proposal is to determine how MurJ functions. Structural information will be obtained by determining the membrane topology of MurJ. Functional partners of MurJ will be identified biochemically and genetically. Mutation and suppression analyses will be conducted to identify domains of MurJ required for stability, intra- and inter- molecular interactions, and activity. Together, the data obtained will uncover the essential function of MurJ in PG biogenesis. Many of the most effective antibiotics target PG biogenesis. MurJ and other proteins required for PG biogenesis are potential targets for antibiotics since PG is essential in most bacteria and is absent in humans. The proposed work will aid in the development of MurJ inhibitors, which may prove to be much-needed novel antibiotics. In addition, understanding MurJ function will advance knowledge of the MOP superfamily of exporters, which includes members that are involved in bacterial pathogenesis and certain types of human congenital disorders of glycosylation.
The increase in antibiotic resistance is a world-wide problem that can be combated by developing new antibiotics. This project seeks to understand the function of an unexplored antibiotic target, the essential bacterial protein MurJ. Our research will (1) facilitate the futur development and characterization of novel MurJ inhibitors, and (2) advance our knowledge of a family of proteins related to MurJ that includes factors involved in bacterial pathogenesis and certain forms of the human congenital metabolic diseases collectively known as CDG.
|May, Janine M; Owens, Tristan W; Mandler, Michael D et al. (2017) The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport. J Am Chem Soc 139:17221-17224|
|Elhenawy, Wael; Davis, Rebecca M; Fero, Jutta et al. (2017) Correction: The O-Antigen Flippase Wzk Can Substitute for MurJ in Peptidoglycan Synthesis in Helicobacter pylori and Escherichia coli. PLoS One 12:e0170518|
|Qiao, Yuan; Srisuknimit, Veerasak; Rubino, Frederick et al. (2017) Lipid II overproduction allows direct assay of transpeptidase inhibition by ?-lactams. Nat Chem Biol 13:793-798|
|Chamakura, Karthik R; Sham, Lok-To; Davis, Rebecca M et al. (2017) A viral protein antibiotic inhibits lipid II flippase activity. Nat Microbiol 2:1480-1484|
|Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J et al. (2016) Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function. MBio 7:|
|Okuda, Suguru; Sherman, David J; Silhavy, Thomas J et al. (2016) Lipopolysaccharide transport and assembly at the outer membrane: the PEZ model. Nat Rev Microbiol 14:337-45|
|Lee, James; Xue, Mingyu; Wzorek, Joseph S et al. (2016) Characterization of a stalled complex on the ?-barrel assembly machine. Proc Natl Acad Sci U S A 113:8717-22|
|Ruiz, Natividad (2016) Filling holes in peptidoglycan biogenesis of Escherichia coli. Curr Opin Microbiol 34:1-6|
|Elhenawy, Wael; Davis, Rebecca M; Fero, Jutta et al. (2016) The O-Antigen Flippase Wzk Can Substitute for MurJ in Peptidoglycan Synthesis in Helicobacter pylori and Escherichia coli. PLoS One 11:e0161587|
|May, Janine M; Sherman, David J; Simpson, Brent W et al. (2015) Lipopolysaccharide transport to the cell surface: periplasmic transport and assembly into the outer membrane. Philos Trans R Soc Lond B Biol Sci 370:|
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