Discovery of repurposed drugs that disrupt intracellular replication of Burkholderia pseudomallei and Burkholderia mallei and protect against lethal aerosol infection
5 affiliations
- 1Department of Biomedical Sciences, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America
- 2Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, United States of America
- 3Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America
- 4Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America
- 5Division of Scripps Research, Calibr-Skaggs Institute for Innovative Medicines, La Jolla, California, United States of America
Abstract
Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are the etiological agents of the fatal diseases melioidosis and glanders, respectively. No licensed vaccine is available to protect against these facultative intracellular bacteria and treatment of infection is difficult, requiring intensive and prolonged antimicrobial therapy with low success rates due to intrinsic resistance of Bp and Bm to most antibiotics. This, combined with concerns regarding their adversarial use as biological warfare agents, emphasize an immediate need to identify novel and effective countermeasures for the organisms. Herein, we developed an in vitro high-content imaging, high-throughput screening platform and tested a Library of Pharmacologically Active Compounds (LOPAC) and the Repurposing, Focused Rescue, and Accelerated MEdchem (ReFRAME) library of small molecules for bioactive compounds protecting against bacterial intracellular replication, which is a key pathogenicity trait of Bp and Bm. We first analyzed confocal microscopy images of Burkholderia-infected monolayers of Vero E6 cells and established a reproducible endpoint readout that quantifies cytopathic effects and the formation of plaques during infection. Following this, we screened ~14,800 small molecules and identified 170 unique structures providing ≥ 75% inhibition of intracellular replication of Bp and Bm with EC50 values ranging from 1 pM to 9.99 µM. Of these hits, 23 were found to disrupt the intracellular replication of both organisms including 8 compounds targeting dihydrofolate reductase, 3 tetracycline-class antibiotics, an inhibitor of bacterial leucyl-tRNA synthetase (epetraborole), and 2 host-directed compounds targeting receptors on the surface of mammalian cells. Selected compounds were evaluated in vivo using a BALB/c mouse model of aerosol infection and we discovered that daily treatment with epetraborole (20 mg/kg) and the fluoroquinolone levofloxacin (5 mg/kg) completely protected mice against exposure to lethal doses of Bm and Bp, reducing bacterial burdens in the lungs and spleen to undetectable levels in all animals by day 14 post-challenge. Author summary: Pathogens that can survive inside host cells such as Burkholderia pseudomallei and Burkholderia mallei are difficult to treat because they damage host cells from within and are resistant to many conventional antimicrobial therapies. We developed an in vitro high-throughput, high‑content imaging platform that allows us to monitor this process and measure how well different compounds protect infected cells. Using this system, we screened nearly 14,800 pharmacologically active molecules. Many caused a measurable reduction in cell damage, revealing both known antibiotic classes and host‑directed compounds with activity against intracellular Burkholderia. We selected a small subset of candidates for in vivo testing and found that epetraborole and levofloxacin provided full protection against both Burkholderia species in a mouse aerosol infection model, eliminating detectable bacteria from target organs. Our study shows how image‑based phenotypic screening can rapidly identify new therapeutic opportunities, including drugs already available for other uses. This approach may help accelerate treatment discovery for pathogens that depend on intracellular survival and cause severe disease.
Related
- Genomic and morphological analysis of human clinical Burkholderia pseudomallei isolates from New Caledonia (2021−2022)
- Comparative evaluation of sample preparation and enrichment methods for culture and molecular detection of Burkholderia pseudomallei in naturally contaminated soils from Thailand over one year.
- The Pseudomonas protegens PBL3 secretome exerts antimicrobial activity against Burkholderia glumae by disrupting cell envelope homeostasis and impairing bacterial replication
- Development and evaluation of a multiplex PCR for simultaneous detection of Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Burkholderia pseudomallei in clinical specimens
- A genetic variant of Burkholderia mallei detected in Kuwait: Consequences for the PCR diagnosis of glanders
BibTeX
@article{orrburks2026discovery,
title = {{Discovery of repurposed drugs that disrupt intracellular replication of Burkholderia pseudomallei and Burkholderia mallei and protect against lethal aerosol infection}},
author = {Nichole Orr-Burks and Sarah R. Hosking and Steven P. Maher and Dennis E. Kyle and Malina A. Bakowski and Robert J. Hogan and Eric R. Lafontaine},
journal = {PLOS Pathogens},
year = {2026},
month = sep,
doi = {10.1371/journal.ppat.1014596},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014596},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Discovery of repurposed drugs that disrupt intracellular replication of Burkholderia pseudomallei and Burkholderia mallei and protect against lethal aerosol infection
AU - Nichole Orr-Burks
AU - Sarah R. Hosking
AU - Steven P. Maher
AU - Dennis E. Kyle
AU - Malina A. Bakowski
AU - Robert J. Hogan
AU - Eric R. Lafontaine
PY - 2026
DA - 2026/09/16
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014596
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014596
AB - Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are the etiological agents of the fatal diseases melioidosis and glanders, respectively. No licensed vaccine is available to protect against these facultative intracellular bacteria and treatment of infection is difficult, requiring intensive and prolonged antimicrobial therapy with low success rates due to intrinsic resistance of Bp and Bm to most antibiotics. This, combined with concerns regarding their adversarial use as biological warfare agents, emphasize an immediate need to identify novel and effective countermeasures for the organisms. Herein, we developed an in vitro high-content imaging, high-throughput screening platform and tested a Library of Pharmacologically Active Compounds (LOPAC) and the Repurposing, Focused Rescue, and Accelerated MEdchem (ReFRAME) library of small molecules for bioactive compounds protecting against bacterial intracellular replication, which is a key pathogenicity trait of Bp and Bm. We first analyzed confocal microscopy images of Burkholderia-infected monolayers of Vero E6 cells and established a reproducible endpoint readout that quantifies cytopathic effects and the formation of plaques during infection. Following this, we screened ~14,800 small molecules and identified 170 unique structures providing ≥ 75% inhibition of intracellular replication of Bp and Bm with EC50 values ranging from 1 pM to 9.99 µM. Of these hits, 23 were found to disrupt the intracellular replication of both organisms including 8 compounds targeting dihydrofolate reductase, 3 tetracycline-class antibiotics, an inhibitor of bacterial leucyl-tRNA synthetase (epetraborole), and 2 host-directed compounds targeting receptors on the surface of mammalian cells. Selected compounds were evaluated in vivo using a BALB/c mouse model of aerosol infection and we discovered that daily treatment with epetraborole (20 mg/kg) and the fluoroquinolone levofloxacin (5 mg/kg) completely protected mice against exposure to lethal doses of Bm and Bp, reducing bacterial burdens in the lungs and spleen to undetectable levels in all animals by day 14 post-challenge. Author summary: Pathogens that can survive inside host cells such as Burkholderia pseudomallei and Burkholderia mallei are difficult to treat because they damage host cells from within and are resistant to many conventional antimicrobial therapies. We developed an in vitro high-throughput, high‑content imaging platform that allows us to monitor this process and measure how well different compounds protect infected cells. Using this system, we screened nearly 14,800 pharmacologically active molecules. Many caused a measurable reduction in cell damage, revealing both known antibiotic classes and host‑directed compounds with activity against intracellular Burkholderia. We selected a small subset of candidates for in vivo testing and found that epetraborole and levofloxacin provided full protection against both Burkholderia species in a mouse aerosol infection model, eliminating detectable bacteria from target organs. Our study shows how image‑based phenotypic screening can rapidly identify new therapeutic opportunities, including drugs already available for other uses. This approach may help accelerate treatment discovery for pathogens that depend on intracellular survival and cause severe disease.
SN - 1553-7374
ER -