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.
The discovery of natural product antimicrobials in the past century has drastically changed the practice of clinical medicine: diseases which were surely fatal are managed simply by once daily oral medicines; however, frustratingly, as the pace of discovery has declined, resistance has grown rapidly. Previous antibiotic development efforts have focused on the effects of isolated compounds on bacterial survival, but this does not often reflect the modern approach to antibiotic therapy, which relies on combination therapies with multiple drugs with different mechanisms of action leading to synergistic or antagonistic effects. We hypothesize that focusing discovery efforts on synthetic lethality by finding compounds which can work with subinhibitory concentrations of known antibiotics can lead to the discovery of innovative precision antibiotics with new mechanisms of action.