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

Inhibiting Type VI Secretion System Activity with a Biomimetic Peptide Designed To Target the Baseplate Wedge Complex

“…Although the T6SS is structurally well characterized and meets all the criteria as a promising virulence factor candidate, a very limited number of studies have attempted to develop anti-T6SS molecules or peptides. Certain studies have used phenotypic screening ( 58 ), and others have employed a targeted approach, such as blocking the interaction between the two sheath proteins (TssB and TssC) or the dissociation of a toxin immunity complex ( 28 , 59 ). Another study employed a nanobody intracellularly expressed to block the assembly of the membrane complex (MC) ( 60 ), which is limited in terms of a possible therapeutic application, since nanobodies cannot cross the bacterial outer membrane.…”

Genomic loss in environmental and isogenic morphotype isolates of Burkholderia pseudomallei is associated with intracellular survival and plaque-forming efficiency

“…Plaque formation is the final scenario of infection in vitro that represents the virulence of intracellular B . pseudomallei [ 40 , 42 ]. This study demonstrated that B .…”

“…Plaque formation is a phenotypic characterization technique that is used as a surrogate for virulence of intracellular microbes such as Rickettsia rickettsii, Shigella flexneri, Listeria monocytogenes as well as B. pseudomallei and B. thailandensis [34][35][36][37][38][39][40][41]. Recently, plaque formation has been used as a high-throughput screen for inhibitors of the intracellular B. pseudomallei lifecycle [42].…”

“…Recently, plaque formation has been used as a high-throughput screen for inhibitors of the intracellular B . pseudomallei lifecycle [ 42 ].…”

Telescope: an interactive tool for managing large-scale analysis from mobile devices

“…Telescope is capable of leveraging common and familiar technologies that do provide a user-friendly interface to manage jobs from any mobile device without compromising flexibility for advanced users. For example, Telescope allows users to track with their smartphones any bioinformatics tools (e.g., GATK 14 ) or jobs submitted by 4 specific platforms (e.g., Galaxy Project 14,15 ), displaying in real-time the partial outputs, warnings, and error messages associated with each job. Telescope includes the following functionality:…”