(228cl) Characterizing Complex Gene-Drug Interactions: Gene Perturbation and Antibiotic Treatment As Combination Therapy

Bacteria possess the ability to survive in and gain resistance to diverse antibiotic treatments, necessitating new approaches by which to combat bacterial infection. One useful strategy has been to utilize antibiotics in combination. Combination therapies can lead to increased antibiotic effect especially in the case of synergistic interactions, or could potentially be used to select against resistance in the case of antagonistic interactions. As new multi-drug resistant strains continue to emerge, the need to locate and pursue alternative antibiotic targets increases in priority. In the face of new technologies allowing for precise interference with the expression of specific genes, next-generation therapeutics do not need to be compounds that specifically bind to an enzyme or cellular components; any region of the genome has the potential to be utilized as a therapeutic target. We hypothesized that combining antibiotic treatment with gene perturbation would reveal complex interactions, advantageous in the design of new therapeutics. We characterized the impact of single gene knockout as well as single gene perturbation with CRISPR-dCas9 in combination with antibiotic agents targeting various pathways, including cell wall synthesis, translation (impacting 50S, 30S, and RNA), and nucleic acid synthesis. The thirty target genes included many global regulators as well as other genes involved with the general stress response, the SOS response, the mar-regulon, efflux, motility, and several of unknown function that have been previously connected to adaptive resistance. Using a growth assay, we demonstrate that the gene-drug combinations result in synergistic, antagonistic, and additive relationships. We describe the landscape of select interactions by titrating the degree of repression in conjunction with varying concentrations of antibiotic. Finally, network analysis reveals how specific functional classes of genes generally interact with various drug classes. This work provides evidence for the applicability of drug-gene combination therapies in promoting complex synergistic and antagonistic behaviors, an area which has great impact on the design of future treatments.