A pathway-directed screen for inhibitors of the bacterial cell elongation machinery [Mechanisms of Action]

New antibiotics are needed to combat the growing problem of resistant bacterial infections. An attractive avenue towards the discovery of such next generation therapies is to identify novel inhibitors of clinically validated targets like cell wall biogenesis. We have therefore developed a pathway-directed, whole-cell screen for small molecules that block the activity of the Rod system of Escherichia coli. This conserved multi-protein complex is required for cell elongation and the morphogenesis of rod-shaped bacteria. It is composed of cell wall synthases and membrane proteins of unknown function that are organized by filaments of the actin-like MreB protein. Our screen takes advantage of the conditional essentiality of the Rod system and the ability of the beta-lactam mecillinam to cause a toxic malfunctioning of the machinery. Rod system inhibitors can therefore be identified as molecules that promote growth in the presence of mecillinam under conditions permissive for the growth of Rod^– cells. A screen of ~690,000 identified 1,300 compounds active against E. coli. Pathway-directed screening of a majority of this subset of compounds for Rod inhibitors successfully identified eight analogs of the MreB-antagonist A22. Further characterization of the A22 analogs identified showed that their antibiotic activity under conditions where the Rod system is essential was strongly correlated with their ability to suppress mecillinam toxicity. This result combined with additional biological studies reinforce the notion that A22-like molecules are relatively specific for MreB and suggest that the lipoprotein transport factor LolA is unlikely to be a physiologically relevant target as previously proposed.

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