Grant: Operating Grant

The insoluble peptidoglycan (PG) sacculus encloses the cytoplasmic membrane of bacterial cells to maintain their structural integrity. This fact, together with its uniqueness to the bacterial cell, have made the enzymatic machinery responsible for the biosynthesis of PG a popular and prime target for antibacterial therapy. Indeed, the beta-lactams and vancomycin have proven to be very useful classes of antibiotics, but their efficacy now appears to be limited in view of the various forms of resistance that have emerged over the past 50 years. New targets have to be identified which, ideally, should be common to both Gram-positive and Gram-negative pathogens thereby permitting the development of broad spectrum antibiotics. We propose that the lytic transglycosylases (LTs) present an attractive target for the development of new antibacterials. These enzymes would be ideal targets because: i) their PG substrate is unique to, but common amongst, almost all bacteria; ii) the component sugar residues remain consistent in all PGs; iii) the lack of variation in the glycan chain implies less chance for resistance to evolve; iv) they are required for a host of essential functions, including secretion/transport, insertion of appendages, cell growth, and cell division; and v) an inhibitor specific for the active site region for any one of the LTs will likely prove to be reactive against all in the same way that penicillin binds all PG transpeptidases. The major aims of this research proposal are to: i) Continue to characterize the structure and function relationship of LTs; ii)Develop a high throughput screen for the search of inhibitors of LTs; and iii)Continue to investigate the biological role and organization of LTs in PG metabolism. These studies will involve a combination of microbiological, molecular biological, and analytical biochemistry techniques, including insertion and site-directed mutagenesis, enzyme kinetics, and electron microscopy.