Using new modalities of synthetic-lethal interactions to find alternative antibacterial drug targets

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7. Project Summary/Abstract The proposed project seeks to discover new narrow spectrum molecular targets for Gram-negative antimicrobials by exploiting synthetic-lethal interactions. Natural products discovery efforts in the past century have produced over 70% of the current collection of clinical antibiotics; however, frustratingly, as the pace of discovery has declined, the rate of resistance is rapidly growing. This is also the case for the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis, which is endemic or hyperendemic in parts of the world. We propose to use Burkholderia thailandensis , a nonpathogenic Gram-negative bacterium and a model strain for B. pseudomallei , to investigate the synthetic-lethal interactions for the discovery of antibiotics with new mechanisms of action. Synthetic lethality refers to a lethal inactivation of two genes which are not individually lethal. We are using a similar terminology to refer to the interaction of two otherwise non-toxic small molecules (at doses used) to create a synthetic-lethal combination. To this end, we have already demonstrated that low doses of antibiotics can induce changes in bacterial secondary metabolism, including upregulation of the folate biosynthetic protein FolE2 by trimethoprim (TMP). In addition, the deletion of folE2 is not growth-defective, but is lethal in the presence of otherwise nonlethal doses of TMP, suggesting that loss of FolE2 and partial inhibition of dihydrofolate reductase by TMP create a synthetic-lethal scenario. Therefore, we hypothesize that a small molecule inhibitor of FolE2 would represent a valuable tool in our antimicrobial arsenal, allowing us to use lower doses of antibiotics, thereby increasing their therapeutic window (Aim 1). Creating a library of transposon mutants would allow us to study the effects of TMP or other low-dose antibiotics in combination with loss of function mutations across the entire genome, further probing interactions that would enable us to discover potential new targets (Aim 2). Our characterization of bacterial synthetic lethality would lay the groundwork for development of targeted therapies for not just Burkholderia, but a broad spectrum of human pathogens using an entirely new approach.