Identification small molecule inhibitors of the bacterial morphogenetic complex


Principal Investigator: JAMES GOBER
Abstract: A fundamental problem in bacterial cell biology understands the molecular mechanisms orchestrating temporal and spatial control of cell morphogenesis. The bacterial morphogenetic complex is comprised of a highly conserved, widely distributed set of proteins found in both Gram negative and positive organisms. This complex is required for the spatial and temporal synthesis of the peptidoglycan cell wall and functions to position and coordinate cell wall biosynthetic activities on both sides of the cytoplasmic membrane. A critical organizer of this complex, MreB, the bacterial actin homolog, functions by spatially coordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. MreC physically associates with penicillin binding proteins (PBPs) which catalyze the insertion of intracellularly synthesized peptidoglycan precursors into the cell wall. MreC is required for the spatial organization of components of the peptidoglycan synthesizing holoenzyme in the periplasm, and MreB directs the localization of a peptidoglycan precursor synthesis in the cytosol. Thus, bacterial morphogenes, essential for cell growth and division, encode proteins that spatially coordinate cell wall synthetic activities on either side of the cytoplasmic membrane. This proposal focuses on the development of assays to identify small molecule inhibitors of bacterial morphogenetic protein interactions. The specific aims of the proposal are to develop a high-throughput screen to identify small molecule inhibitors of bacterial morphogenetic protein interactions using a robust bacterial two-hybrid interaction assay to screen over 70,000 diverse compounds. Additionally, the effect of small molecule inhibitors of bacterial morphogenetic protein interactions will be characterized in vivo with respect to cell growth, cell shape, subcellular localization of morphogenetic proteins, and cell wall synthesis. A limited genetic screen will also be performed to validate the in vivo target of the inhibitors. Lastly, a validation of the effect of small molecule inhibitors on bacterial morphogenetic protein interactions will be performed using biochemical interaction assays. The small-scale screen developed here can be used as an experimental template to screen larger chemical libraries in the Molecular Libraries Probe Production Centers Network (MLPCN) in the future. Significantly, this project will generate a new set of small molecule tools to be used for probing the assembly and function of the bacterial morphogenetic apparatus and may guide efforts in the development and/or identification of lead compounds for new antimicrobics. PUBLIC HEALTH RELEVANCE: The array of antibiotics that have usefulness in combating bacterial infections is rapidly declining as a consequence emerging resistance. Historically, some of the most effective antimicrobics have targeted the enzymes involved in bacterial cell wall synthesis. Development of small molecule probes targeted at cell wall synthesizing proteins, may stimulate the development of lead compounds for novel antimicrobics that are directed at the organizing centers of bacterial cell wall synthesis, thus providing next-generation targeted antimicrobial therapy.
Funding Period: 2009-09-25 - 2010-08-31
more information: NIH RePORT