Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Samantha L. van der Beek, Azul Zorzoli, Ebru Çanak, Robert N. Chapman, Kieron Lucas, Benjamin H. Meyer, Dimitrios Evangelopoulos, Luiz Pedro S. de Carvalho, Geert-Jan Boons, Helge C. Dorfmueller (Lead / Corresponding author), Nina M. van Sorge (Lead / Corresponding author)

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Abstract

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC 50 of 120-410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

Original languageEnglish
Pages (from-to)951-964
Number of pages14
JournalMolecular Microbiology
Volume111
Issue number4
Early online date1 Jan 2019
DOIs
Publication statusPublished - Apr 2019

Keywords

  • Streptococcus
  • Mycobacterium
  • rhamnose polysaccharide
  • biosynthesis
  • antimicrobial
  • nucleotide sugars

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    van der Beek, S. L., Zorzoli, A., Çanak, E., Chapman, R. N., Lucas, K., Meyer, B. H., Evangelopoulos, D., de Carvalho, L. P. S., Boons, G-J., Dorfmueller, H. C., & van Sorge, N. M. (2019). Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification. Molecular Microbiology, 111(4), 951-964. https://doi.org/10.1111/mmi.14197