An in situ high-throughput screen identifies inhibitors of intracellularBurkholderia pseudomalleiwith therapeutic efficacy

Abstract

Burkholderia pseudomallei(Bp) andBurkholderia 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 ofBurkholderiapathogenesis, 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 byBurkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread byBpand/orBm. Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellularBurkholderia, 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 intracellularBurkholderia. 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.