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 wildtype 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.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE

“…In addition, each of the pBHR1:: P glmS2 :: bopE TC, pBHR1:: bapA TC, pBHR1:: bapB TC and pBHR1:: bapC TC constructs was transferred into a B . pseudomallei bsaS mutant [ 30 ] by conjugation. Transconjugants were designated ΔbsaS [ bopE TC], ΔbsaS [ bapA TC], ΔbsaS [ bapB TC] and ΔbsaS [ bapC TC], respectively.…”

“…Following SDS-PAGE, proteins were transferred to PVDF membranes (Merck Millipore, USA) and the membranes probed by immunoblotting with rabbit anti-BopE 78-261 antiserum [ 27 ] as described previously [ 30 ]. Antibody binding was analyzed using Amersham ECL Western Blotting Detection Reagent (GE Healthcare, NSW, Australia), and chemiluminescent signals detected with X-ray film (Kodak, NY, USA).…”

“…The TTSS3 gene cluster encodes a range of TTSS components, including structural proteins, translocators, chaperones, transcriptional regulators and effector proteins [ 13 , 25 ]. The TTSS3 genes bsaQ ( BPSS1543 ), bsaU ( BPSS1539 ), bsaN ( BPSS1546 ), bicA ( BPSS1533 ), bsaZ ( BPSS1534 ), bipB ( BPSS1532 ), bipD ( BPSS1529 ), bopA ( BPSS1524 ), bopB ( BPSS1514 ), bopE ( BPSS1525 ), bapA ( BPSS1528 ) and bapC ( BPSS1526 ), have been characterized to some extent [ 13 , 18 , 26 – 30 ]. However, to date, few putative TTSS3 effectors have been confirmed as secreted by wild-type B .…”

Identification of chlamydial T3SS inhibitors through virtual screening against T3SS ATPase

“…Recently, one of the inhibitors described in the work of Swietnicki et al . was also demonstrated to inhibit T3SS ATPase of another intracellular pathogen, B. pseudomallei . Addition of this inhibitor at 10 μ m concentration blocked secretion of BopE effector protein, prevented bacteria from escaping phagosomes, and significantly reduced their survival in mammalian cells.…”

“…Some data, however, suggest that effector unfolding and export are fueled by proton motive force at the membrane, rather than ATPase . Regardless of what the precise function of T3SS ATPase is, several studies demonstrated the necessity of functional ATPase for proper T3SS functioning and full virulence in several pathogens, including E. coli , Burkholderia pseudomallei , Yersinia pestis , and Yersinia enterocolitica …”