Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

Investigating Anthranilate Synthase in the Tryptophan Biosynthetic Pathway as a Target for Antimicrobial Drug Design against Burkholderia pseudomallei (#132)

Sharita Ganeshan 1 , Mitali Sarkar-Tyson 1 , Keith Stubbs 2 , Christopher Jenkins 3 , Phil Ireland 3
  1. Marshall Centre for Infectious Diseases Research and Training, School of Biomedical Sciences, University of Western Australia, Perth, WA, Australia
  2. School of Molecular Sciences, University of Western Australia, Perth, WA, Australia
  3. Defence Science and Technology Laboratory, Porton Down, Salisbury, United Kingdom

Burkholderia pseudomallei, a gram-negative environmental bacterium, is the causative agent of melioidosis- a life-threatening disease that is an endemic in at least 45 countries worldwide across tropical areas including Northern Australia and South East Asia.1 Despite the few effective antibiotic treatments available, mortality rates of melioidosis remain high. Therefore, there is a need to identify new targets for the development of therapeutics against B. pseudomallei with novel modes of action. Tryptophan is incorporated into polypeptide chains of enzymes and proteins and is an essential biosynthetic precursor within the bacterial cell. Although tryptophan can be obtained from certain surrounding environments, when not available, the survival and replication of many bacteria require the ability to synthesize L-tryptophan as a substrate for protein synthesis.2 Targeting tryptophan biosynthesis in various intracellular bacteria including Mycobacterium tuberculosis has resulted in decreased survival.3,4

This research investigates targeting the anthranilate synthase (AS) enzyme complex, responsible for the two-step biosynthesis of anthranilate, an intermediate in the biosynthesis of L-tryptophan in B. pseudomallei. AS consists of the TrpE and TrpG subunits; TrpG supplies the glutamine amidotransferase activity, which is then utilized in the reaction catalyzed by TrpE to produce anthranilate. To understand the role of the trpE and trpG genes of B. pseudomallei, the in vitro characterization of the deletion strains Bps∆trpE, Bps∆trpG, and the double mutant strain Bps∆trpEG, was executed. When grown in minimal media conditions, all mutant strains were observed as tryptophan auxotrophs, with complementation of auxotrophy evident via exogenous tryptophan supplementation. Furthermore, TrpE demonstrated ability to synthesize anthranilate directly from high concentrations of ammonia as revealed through study of Bps∆trpG under standard-high and low-ammonia growth conditions. Investigation of infection in RAW 264.7 murine macrophages demonstrate that although all mutant strains retain the ability to infect and survive in the macrophages, Bps∆trpG and Bps∆trpEG lack the ability to spread cell to cell. Additionally, the capability of Bps∆trpE to spread is directly impacted by the availability of L-glutamine in the macrophage environment, indicating the importance of TrpG. These studies suggest genes in the tryptophan biosynthetic pathway are potentially promising antivirulence targets in B. pseudomallei.

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  3. Zhang, Y. J. et al. Tryptophan biosynthesis protects mycobacteria from CD4 T-cell-mediated killing. Cell 155, 1296-1308, doi:10.1016/j.cell.2013.10.045 (2013).
  4. Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, VK., et al. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase. Nat. Chem. Biol 13, 943–950, doi:10.1038/nchembio.2420 (2017).