Targeting Gram-Negative Bacteria for Antibacterial Strategies

Project Summary/Abstract The cell wall is a structural edifice critical for survival of bacteria. Its biosynthetic machinery and the cell wall itself are targets of antibiotics. Cell wall is a crosslinked polymer that encases the entire bacterium, whose structure has been estimated to be larger than the chromosome. The complex biosynthetic and recycling processes of cell wall and its regulation during homeostasis are largely unknown. The Mobashery laboratory studies these processes in the Gram-negative bacterium Pseudomonas aeruginosa, with emphasis on cell-wall recycling. Two directions are proposed. The first is the study of the cytoplasmic pathway for recycling of muropeptides, which are fragments of the cell wall that have been internalized to the cytoplasm. A sequence of six enzymatic reactions constitutes this pathway, which brings the metabolites to a point of merger with the de novo pathway for the biosynthesis of cell-wall precursors. We proposed to investigate the mechanistic details of these enzymes individually. Meanwhile, all six genes have been cloned and the respective recombinant proteins have been purified to homogeneity. We have reconstituted the pathway in vitro and we have been able to observe the full metabolic flux for it. The reconstituted pathway has been miniaturized as a fluorescence assay for high throughput screening of compounds as inhibitors of these enzymes. An argument is presented that an inhibitor of the de novo biosynthesis (fosfomycin) should synergize well with an inhibitor of any step in the recycling pathway, whereby the action of the two inhibitors should shut down biosynthesis of the peptidoglycan entirely, which will be fatal for the Gram-negative bacteria. In the second direction, the mechanistic nexus between cell-wall recycling and manifestation of resistance to b-lactam antibiotics has been established. Historically, b-lactam antibiotics have been antibiotics of choice for treatment of infections by Gram-negative bacteria, including P. aeruginosa. In an attempt to disrupt this link, we have devised a fluorescent reporter strain of P. aeruginosa that fluoresces when cell-wall recycling takes place. The screening of a focused natural-product library (390 compounds) against the reporter strain for shutting down cell-wall recycling potentiates the activity of the b-lactam antibiotics, which otherwise will be ineffective in treating the resistant bacterium. Several compounds were identified with activity in this screening. We plan to screen two additional compounds libraries, at which time the positive hits will be assessed for selection of one or more for synthesis and evaluation of analogs with enhanced activity against a broader collection of Gram-negative bacteria.