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

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 - Cancer Research UK. Grant Number: FC001060 Dutch Scientific Organization. Grant Number: 91713303 Tenovus Scotland Wellcome Trust. Grant Numbers: 109357/Z/15/Z, FC001060 Medical Research Council. Grant Number: FC001060

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 - Streptococcus

KW - Mycobacterium

KW - rhamnose polysaccharide

KW - biosynthesis

KW - antimicrobial

KW - nucleotide sugars

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 -