Identification of Efflux Substrates Using a Riboswitch-Based Reporter in Pseudomonas aeruginosa

Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention, and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide-sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In the first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate) riboswitch reporter activity in efflux-deficient P. aeruginosa, allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and -proficient strain using high-resolution liquid chromatography-mass spectrometry (LC-MS). This simple yet powerful method, optimized for high-throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multidrug-resistant Gram-negative pathogen. IMPORTANCE Treatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multidrug-resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside or enter the cell and are exported by efflux systems. This approach enables the development of rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.