Using Synthetic-Lethal Interactions to Discover Antibacterial Drug Targets

Abstract

Natural products, small molecules conferring a fitness advantage to their producers, havebeen an integral source of medicines for much of written history. Discovery efforts intensi- fied in the past century, leading to the development of, among others, antimicrobials which changed the practice of clinical medicine: fatal diseases are now managed simply by oral medicines. Frustratingly, as discovery has declined, resistance has spread rapidly, in part due to the indiscriminate use of broad-spectrum agents. This rise in resistance is compounded by our limited knowledge of antibacterial targets: among hundreds of antbiotics, most act upon a few conserved targets, so resistance to one agent renders its entire class unusable. Focusing on limited targets also limits our ability to selectively inhibit pathogens without affecting our commensal microbiome. In addition, while we know the canonical targets, we do not under- stand the downstream pathways which lead to bacterial death. Previous development efforts focused on the effects of single compounds on survival, but this does not often reflect the modern approach to antibiotic therapy, which relies on combination therapies with multi- ple drugs with different mechanisms of action leading to synergistic or antagonistic effects. We have started to address these issues using the Gram-negative model bacterium for the tropical disease melioidosis Burkholderia thailandensis. Our lab has previously shown that low doses of antibiotics such as the anti-folate trimethoprim can have a stimulatory effect on B. thailandensis’s metabolism, including upregulation of the alternate folate biosynthetic enzyme FolE2, likely the stress-induced activation of a compensatory response. In this work, we show that treating B. thailandensis with a combination of two otherwise noninhibitory agents, low-dose trimethoprim and an inhibitor of FolE2, results in cell death, a scenario we refer to as chemical synthetic lethality. We demonstrate that this combination also works to inhibit the pathogenic caustive agent of melioidosis, B. pseudomallei, but has minimal impact on microbiome-associated commensal strains. We then construct a transposon mu- tant library to search the entirety of the Burkholderia genome for additional targets of syn- theticlethality, discoveringadiversityoftargetsincludingtranscriptionalregulation, primary metabolism, and motility. For example, we find that loss of either component of a two- component system or its regulon leads to cell death with otherwise subinhibitory doses of trimethoprim, suggesting the system’s importance in antibiotic-stress response. We also find in vitro inhibitors of another hit, methionine synthase, and demonstrate that they can reca- pitulate the synthetic lethal phenotype in vivo. Finally, we extend our transposon method to the human pathogen Pseudomonas aeruginosa which drives much morbitidy and mortality in the United States.