Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

Deletion of the Burkholderia pseudomallei Class II Fructose 1,6-bisphosphate Aldolase (bpsl0798) has a Complex Pleiotropic Effect on both Metabolism and Virulence   (#172)

Clare L Moran 1 , Chris Jenkins 2 , Aleksandra Debowski 1 , Keith Stubbs 3 , Phil Ireland 2 , Mitali Sarkar-Tyson 1
  1. School of Biomedical Sciences, University of Western Australia, Perth, Western Australia, Australia
  2. Dstl, Porton Down, Salisbury, United Kingdom
  3. School of Molecular Sciences, University of Western Australia, Perth, Western Australia, Australia

The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis, a severe illness that can arise following exposure to contaminated environments. Understanding the metabolic reactions that sustain B. pseudomallei replication within the host could expand the limited pool of targets for antimicrobial drug discovery. Fructose 1,6-bisphosphate aldolases are enzymes that catalyse both cleavage and synthesis of fructose-1,6-bisphosphate, a crucial metabolic intermediate of central carbon metabolism. Disruption of fructose-1,6-bisphosphate activity has been demonstrated to reduce both viability and virulence in other bacterial pathogens. This work investigates the Class II fructose 1,6-bisphosphate aldolase (bpsl0798) of B. pseudomallei strain K96243 via characterization of unmarked deletion mutant BpsΔbpsl0798. Prior work conducted with this mutant strain revealed it to be avirulent in a murine infection model, though the underlying mechanism remained unclear. This study demonstrates that in comparison to wild-type B. pseudomallei, BpsΔbpsl0798 exhibits a significant carbon-source dependent growth defect in media containing succinate, galactose, or glycerol. However, no growth defects were identified in media containing glucose, or fructose indicating that alternative glycolytic pathways of B. pseudomallei may bypass bpsl0798 activity. The ability of BpsΔbpsl0798 to infect cells in vitro was investigated using RAW264.7 macrophages and A549 epithelial cell lines. Within the RAW264.7 model, infection with BpsΔbpsl0798 resulted in significantly reduced cytotoxicity, relative to the wild-type strain. Reductions in cytotoxicity were also observed in the A549 model. These in vitro results were supported by immunofluorescence staining of cell monolayers, where BpsΔbpsl0798 infected monolayers were observed to lack the characteristic cytopathic effects of wild-type B. pseudomallei infection. This phenotype was attributed in part to the significantly increased sensitivity of BpsΔbpsl0798 to oxidative stress, as demonstrated via H2O2 survival assays. Future work aims to determine the transcriptional profile of the BpsΔbpsl0798 mutant strain, which will facilitate the identification of metabolic pathways that may be contributing to B. pseudomallei pathogenesis.