Burkholderia pseudomallei type III secretion system cluster 3 ATPase BsaS, a chemotherapeutic target for small-molecule ATPase inhibitors

Abstract

Melioidosis is an infectious disease of high mortality for humans and other animal species; it is prevalent in tropical regions worldwide. The pathogenesis of melioidosis depends on the ability of its causative agent, the Gram-negative bacterium Burkholderia pseudomallei, to enter and survive in host cells. B. pseudomallei can escape from the phagosome into the cytosol of phagocytic cells where it replicates and acquires actin-mediated motility, avoiding killing by the autophagy-dependent process, LC3 (microtubule-associated protein light chain 3)-associated phagocytosis (LAP). The type III secretion system cluster 3 (TTSS3) facilitates bacterial escape from phagosomes, although the mechanism has not been fully elucidated. Given the recent identification of small-molecule inhibitors of the TTSS ATPase, we sought to determine the potential of the predicted TTSS3 ATPase, encoded by bsaS, as a target for chemotherapeutic treatment of infection. A B. pseudomallei bsaS deletion mutant was generated and used as a control against which to assess the effect of inhibitor treatment. Infection of RAW 264.7 cells with wild-type bacteria and subsequent treatment with the ATPase inhibitor compound 939 resulted in reduced intracellular bacterial survival, reduced escape from phagosomes, and increased colocalization with both LC3 and the lysosomal marker LAMP1 (lysosome-associated membrane protein 1). These changes were similar to those observed for infection of RAW 264.7 cells with the bsaS deletion mutant. We propose that treatment with the ATPase inhibitor compound 939 decreased intracellular bacterial survival through a reduced ability of bacteria to escape from phagosomes and increased killing via LAP. Therefore, small-molecule inhibitors of the TTSS3 ATPase have potential as therapeutic treatments against melioidosis.