Wednesday, April 2, 2025
4/2/2025
Investigating ploidy modulation as a strategy to improve antibiotic activity - Project Summary Persisters are antibiotic-tolerant cells that are genetically identical to the overall population that succumbs to treatment, but they occupy a favorable phenotypic niche that enables survival. Persisters are an important health concern because they are thought to contribute to chronic and recurrent infections, and recently, studies have demonstrated that persisters can foster the development of antibiotic resistance. Fluoroquinolone (FQ) persisters are particularly worrisome because FQs are one of the few antibiotic classes that can kill growth- limited bacteria, and it has been shown that FQ persisters from stationary-phase populations experience de novo mutation following treatment that not only accelerates resistance development for FQs but for independent antibiotics as well (e.g., rifampicin, carbenicillin, D-cycloserine, fosfomycin). Recent work has found that the chromosome copy number in individual bacteria (ploidy) is an important determinant of FQ persistence, due to the inability of monoploids to conduct highly efficient homologous recombination to repair FQ-induced DNA damage. Interestingly, polyploidy has been reported to increase the tolerances of bacteria to diverse stresses. Here, we hypothesize that ploidy modulation could be a strategy to improve antibiotic killing of growth-limited bacteria and reduce relapse infections. To test this hypothesis, we will identify DNA repair requirements of monoploid and diploid persisters to FQs (levofloxacin, ciprofloxacin, moxifloxacin: most commonly prescribed FQs); examine whether ploidy impacts killing by other antibiotics (UTI treatments: nitrofurantoin, trimethoprim-sulfamethoxazole; digestive tract treatment: rifaximin); and screen for compounds (e.g., FDA-approved compound repurposing library) that enhance monoploidy (least tolerant ploidy state). To accomplish these tasks, we will use Escherichia coli MG1655 (laboratory model), CFT073 (urosepsis isolate), and UTI89 (uropathogenic), and measure ploidy with Hoechst 33342 (live cell nucleic acid stain), PicoGreen (dsDNA stain for fixed cells), and a fluorescent protein-based origin reporter where the number of fluorescent foci indicate the number of chromosomes. We will use fluorescence activated cell sorting (FACS) to segregate populations based on ploidy and then conduct persistence and antibiotic tolerance assays on those subpopulations for wild-type and DNA repair mutants. In addition, we will screen a focused (FDA-approved compound repurposing library) and diverse small molecule library for the ability to increase monoploidy in growth-inhibited bacteria using Hoechst 33342 and high-throughput flow cytometry. Collectively, these experiments will investigate how a phenotypic variable (ploidy) that often goes ignored impacts persistence to FQs and other antibiotics. Further, this work will begin to examine that feature as a druggable target, with an eye toward ploidy modulators (foster monoploidy) as new antibiotic adjuvants with anti-persister activity.
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