TY - JOUR
T1 - Streptococcal dTDP-L-rhamnose biosynthesis enzymes
T2 - functional characterization and lead compound identification
AU - van der Beek, Samantha L
AU - Zorzoli, Azul
AU - Çanak, Ebru
AU - Chapman, Robert N
AU - Lucas, Kieron
AU - Meyer, Benjamin H
AU - Evangelopoulos, Dimitrios
AU - de Carvalho, Luiz Pedro S
AU - Boons, Geert-Jan
AU - Dorfmueller, Helge C
AU - van Sorge, Nina M
N1 - Funding Information:
The authors would like to acknowledge Dr Olawale Raimi (University of Dundee) for assistance with BLI and Dr Dejere Abate Negatu and Dr Thomas Dick for sharing the unpublished results concerning the compound F8. We are grateful for funding to HCD and his laboratory from Tenovus Scotland, Wellcome Trust and Royal Society (grant number 109357/Z/15/Z.), FEBS and the University of Dundee. This work was supported by a VIDI grant (91713303) from the Netherlands Organization for Scientific Research (NWO) to N.M.v.S and S.v.d.B. The authors declare no conflict of interest. Work in L.P.S.C.’s lab is funded by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001060), the UK Medical Research Council (FC001060) and the Wellcome Trust (FC001060).
Funding Information:
The authors would like to acknowledge Dr Olawale Raimi (University of Dundee) for assistance with BLI and Dr Dejere Abate Negatu and Dr Thomas Dick for sharing the unpublished results concerning the compound F8. We are grateful for funding to HCD and his laboratory from Tenovus Scotland, Wellcome Trust and Royal Society (grant number 109357/Z/15/Z.), FEBS and the University of Dundee. This work was supported by a VIDI grant (91713303) from the Netherlands Organization for Scientific Research (NWO) to N.M.v.S and S.v.d.B. The authors declare no conflict of interest. Work in L.P.S.C.?s lab is funded by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001060), the UK Medical Research Council (FC001060) and the Wellcome Trust (FC001060).
Publisher Copyright:
© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd
PY - 2019/4
Y1 - 2019/4
N2 - 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.
AB - 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.
KW - Anti-Bacterial Agents/isolation & purification
KW - Biosynthetic Pathways
KW - Hydro-Lyases/genetics
KW - Inhibitory Concentration 50
KW - Nucleoside Diphosphate Sugars/biosynthesis
KW - Racemases and Epimerases/genetics
KW - Streptococcus/drug effects
KW - Thymine Nucleotides/biosynthesis
UR - https://www.scopus.com/pages/publications/85060883972
U2 - 10.1111/mmi.14197
DO - 10.1111/mmi.14197
M3 - Article
C2 - 30600561
SN - 0950-382X
VL - 111
SP - 951
EP - 964
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 4
ER -