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An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy

P. Bulterys,Isabelle J. Toesca,Michael H. Norris,J. Maloy,S. Fitz-Gibbon,Bryan France,Babak Toffig,M. Morselli,Nawarat Somprasong,M. Pellegrini,H. Schweizer,A. Tuanyok,R. Damoiseaux,C. French,Jeff F. Miller

Published 2019 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance Burkholderia pseudomallei, the etiologic agent of melioidosis, is an environmental organism that inhabits tropical soils and kills an estimated 90,000 people each year. Caused by an intracellular and often drug-resistant pathogen, melioidosis is notoriously difficult to treat, with mortality rates approaching 50% in some settings despite appropriate diagnosis and clinical management. Using a high-throughput, cell-based phenotypic screen we have discovered 2 antibiotic candidates with improved in vivo efficacy compared to the current standard of care: a fluoroquinolone analog, burkfloxacin, and an FDA-approved antifungal drug, flucytosine. As a widely used antifungal with a well-known safety profile, the potential to repurpose flucytosine for treating melioidosis may represent a rapid route to clinical translation. Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm. Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell–cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia. In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

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