Intercellular Spread of Burkholderia and Novel Therapeutic Strategies
Burkholderia pseudomallei (Bp), the etiologic agent of melioidosis, is increasingly recognized as a significant cause of morbidity and mortality in humans. Treatment requires up to nine months of antibiotic therapy and relapse is common. Clinical management is complicated by the fact that Bp is often resistant to multiple classes of antibiotics through both intrinsic and acquired mechanisms. Even with rigorous and appropriate antibiotic therapy, mortality rates are as high as 50%. Melioidosis is endemic in tropical and sub-tropical regions throughout the world. In addition to affecting local residents, individuals at risk include travelers and US military personnel deployed to tropical regions. The closely related pathogen, Burkholderia mallei (Bm), causes equine glanders and can also afflict humans. Although exceedingly rare, human glanders presents in forms similar to melioidosis with high rates of mortality. The lethal nature of these infections and the paucity of effective antibiotics or vaccines highlight the importance of understanding virulence mechanisms and developing means to protect populations at risk. This need is amplified by concerns regarding the potential use of these pathogens as biological weapons. Bm was, in fact, used for biowarfare in both World Wars I and II.
Intercellular spread is a hallmark of Burkholderia pathogenesis and represents an attractive therapeutic target as it is required for virulence. We have shown that efficient cell-cell spread depends on the ability of the bacterium to perform critical intracellular lifecycle steps, including invasion, endosome escape, intracellular motility, and membrane fusion. A striking feature of Burkholderia is their ability to fuse plasma membranes, forming multinucleated cells (MNCs). We have discovered that cell fusion represents the primary means of intercellular spread during infection of cell monolayers in vitro, and we have proposed a model that is similar to mechanisms used by enveloped viruses. Studies in our laboratory implicate a type VI secretion system (T6SS-5) as a key determinant in this process, and VgrG-5, a trimeric protein at the tip of the T6SS-5 apparatus. There remain significant unknowns about the mechanism of cell fusion and other critical intracellular lifecycle steps that promote cell-cell spread, including the full inventory of bacterial and host factors that participate in these processes. Furthermore, our data indicate that many important virulence determinants are highly conserved between Bp and Bm, suggesting the possibility of developing broadly acting therapeutic interventions, including small molecule inhibitors.
We developed a high-throughput cell-based phenotypic assay that comprehensively assesses the Burkholderia intercellular lifecycle and used it to screen 1] a saturating transposon mutant library in Burkholderia thailandensis, a BSL2 surrogate, to identify bacterial factors required for Burkholderia intercellular spread, 2] an siRNA host kinase library for host factors involved in facilitating Burkholderia intercellular spread, and 3] a small molecule library comprising ~220,000 small molecules to identify compounds capable of disrupting intercellular spread by Burkholderia. We identified several novel bacterial factors, and one host factor, which are necessary for efficient intercellular spread of Burkholderia. In addition, our small molecule screen uncovered 91 small molecules that inhibit cell-cell spread by Bt, 27 of which also disrupt the intercellular lifecycles of Bp and/or Bm. Among these were burkfloxacin, a fluoroquinolone analog with potent intracellular bactericidal activity, and an FDA-approved drug. We found that this FDA-approved drug inhibits Burkholderia-mediated membrane fusion by targeting the secretion activity, but not expression, of the type VI secretion system-5 (T6SS-5), a critical virulence determinant. Bacterial metabolism of this drug is required for activity and ensures selective toxicity. These results suggest that burkfloxacin and this FDA-approved drug should be advanced as potential therapeutic countermeasures for melioidosis, and demonstrate the utility of cell-based phenotypic screening for select agent drug discovery.