Dissecting the Mechanisms Underlying Heterogeneous Antimicrobial Accumulation in Isogenic Bacterial Populations

Antimicrobial resistance increasingly threatens the viability of modern medical interventions. There is a dire need to develop novel therapies, especially to counter resistance mechanisms employed by starved or slow-growing pathogens that are refractory to conventional antimicrobial therapies. Antimicrobial peptides have been advocated as potential therapeutic solutions due to their unique mechanisms of action and low levels of genetic resistance observed in bacteria. This thesis investigates the link between the accumulation and efficacy of the antimicrobial peptide, tachyplesin-1, establishing a clear link between intracellular accumulation and efficacy. Accumulation solely on the bacterial membrane was shown to be insufficient for bacterial eradication. Furthermore, it was shown that certain subpopulations of stationary phase Escherichia coli and Pseudomonas aeruginosa can survive tachyplesin treatment without genetic mutations, relying instead on phenotypic adaptations. Transcriptomic and lipidomic analyses revealed that these phenotypically tachyplesin-tolerant cells differentially regulated processes including efflux, outer membrane vesicle section, proteolytic activity, modifications to their membrane lipids and composition, and metabolism, suggesting potential mechanisms facilitating phenotypic tachyplesin-tolerance. The formation of phenotypically tachyplesin-tolerant cells was prevented in ΔqseB, ΔqseBΔqseC, and ΔacrAΔtolC mutants, as well as when tachyplesin was combined with efflux pump inhibitors, including two clinically approved drugs, sertraline and verapamil, and metabolic stimulants. These findings highlight the role of efflux in phenotypic tachyplesin-tolerance and suggest a potential strategy to combat antimicrobial-refractory stationary phase bacteria. Lastly, virtual high-throughput screening was conducted to identify potential inhibitors of EvgA, the response regulator in the EvgAS two-component regulatory system. This was motivated by the inferred upregulation of EvgA in phenotypically tachyplesin-tolerant cells in the transcriptomics analysis. Although deletion of evgA and evgS did not prevent the emergence of phenotypic tachyplesin-tolerant cells, the role of EvgAS in multidrug and acid resistance may support future efforts to identify adjuvants that could enhance antibiotic efficacy.