Small molecule regulation of an antibiotic resistance efflux pump in pseudomonas aeruginosa

The National Institute of Allergy and Infectious Diseases awarded Emory University $100,228 on October 6, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to conduct research on small molecule regulation of an antibiotic resistance efflux pump in Pseudomonas aeruginosa.

The research examines the MexXY-OprM efflux pump, the only efflux system in P. aeruginosa capable of exporting aminoglycosides, a clinically relevant antibiotic. The project investigates how the PA5471 operon regulates MexXY expression and the role of the SUHB protein in controlling this regulation in the absence of aminoglycosides. The work hypothesizes that aminoglycoside binding to the ribosome, in the presence of the PA5471.1 leader peptide, inhibits SUHB function and triggers ribosome stalling, causing downstream expression of PA5471 through a novel gene regulation mechanism. Aim 1 employs reporter assays to identify critical regions in the PA5471.1 leader peptide and intergenic sequences governing this regulation.

The award supports research at Emory University in Atlanta, Georgia, with performance through April 9, 2029. This project grant is classified as direct assistance funding to a nonprofit educational institution supporting biomedical research addressing antibiotic resistance, a recognized public health priority within the NIAID portfolio.

Abstract

Bacterial resistance to antibiotics is an escalating crisis in healthcare that threatens to fundamentally alter modern medicine. There are many distinct mechanisms of antibiotic resistance in bacteria, but increased production of multidrug efflux pumps is a common response among pathogenic bacteria to remove drugs from the cell. The resistance-nodulation-division (RND) efflux pump superfamily is broadly distributed among gram- negative pathogenic bacteria and contributes extensively to clinical resistance. The important opportunistic pathogen pseudomonas aeruginosa encodes multiple RND efflux systems, at least four of which contribute to its intrinsic multi-drug resistance. A major efflux pump in p. Aeruginosa is mexxy-oprm and IT is the only efflux pump that can export aminoglycosides, an antibiotic that is clinically relevant in the treatment of bacterial infections. Mexxy is regulated by the expression of the PA5471 operon that encodes for a regulatory leader peptide, PA5471.1, and the PA5471 protein. The PA5471.1 leader peptide and aminoglycoside exposure controls the expression of PA5471. The suhb protein, typically known to interact with RNA polymerase as an integral part of the antitermination complex for ribosomal RNA biosynthesis in e. Coli, is also involved in this regulation in the absence of aminoglycosides by preventing both ribosome stalling and protein production of PA5471. However, the molecular and structural bases of this novel gene regulation are unknown. My hypothesis is that aminoglycoside binding to the ribosome in the presence of the PA5471.1 leader peptide inhibits suhb function and causes ribosome stalling to confer downstream gene expression of PA5471, suggesting a novel mode of gene regulation. In aim 1, i will use reporter assays to determine the regions in the PA5471.1 leader peptide and the downstream intergenic region between PA5471.1 and PA5471 that are important for PA5471 production and thus mexxy production. I will also identify PA5471.1 leader peptide amino acids important for ribosome stalling using biochemical assays. Lastly, i will determine the structural basis of aminoglycoside-mediated ribosomal stalling in the context of actively translating PA5471.1 by cryogenic-electron microscopy. In aim 2, i will determine the important suhb residues for the regulation of PA5471 using reporter and biochemical assays to test whether suhb functions as a transcription or translation factor. Finally, i will determine the structural basis of suhb interaction with the ribosome by cryogenic-electron microscopy. Collectively, my results will reveal important aspects of p. Aeruginosa antibiotic-mediated gene regulation and intrinsic antibiotic resistance. These studies will provide the foundation to identify new targets for novel antibiotic drug development to combat the increasing antibiotic resistance in p. Aeruginosa.