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, Nina M van Sorge (Lead / Corresponding author)

Research output: Contribution to journalArticle

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 Mycobacterium tuberculosis with an IC50 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 a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria. This article is protected by copyright. All rights reserved.

LanguageEnglish
JournalMolecular Microbiology
DOIs
Publication statusE-pub ahead of print - 1 Jan 2019

Fingerprint

Rhamnose
Enzymes
Streptococcus mutans
Mycobacterium tuberculosis
Interferometry
Hydro-Lyases
Bacteria
Streptococcus pyogenes
Streptococcus
Point Mutation
Cell Wall
Inhibitory Concentration 50
Polysaccharides
Virulence
Nucleotides
thymidine diphosphate rhamnose
Lead
Anti-Bacterial Agents
Growth

Cite this

van der Beek, Samantha L ; Zorzoli, Azul ; Çanak, Ebru ; Chapman, Robert N ; Lucas, Kieron ; Meyer, Benjamin H ; Evangelopoulos, Dimitrios ; de Carvalho, Luiz Pedro S ; Boons, Geert-Jan ; Dorfmueller, Helge C ; van Sorge, Nina M. / Streptococcal dTDP-L-rhamnose biosynthesis enzymes : functional characterization and lead compound identification. In: Molecular Microbiology. 2019.
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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 Mycobacterium tuberculosis with an IC50 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 a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria. This article is protected by copyright. All rights reserved.",
author = "{van der Beek}, {Samantha L} and Azul Zorzoli and Ebru {\cC}anak and Chapman, {Robert N} and Kieron Lucas and Meyer, {Benjamin H} and Dimitrios Evangelopoulos and {de Carvalho}, {Luiz Pedro S} and Geert-Jan Boons and Dorfmueller, {Helge C} and {van Sorge}, {Nina M}",
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year = "2019",
month = "1",
day = "1",
doi = "10.1111/mmi.14197",
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journal = "Molecular Microbiology",
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Streptococcal dTDP-L-rhamnose biosynthesis enzymes : functional characterization and lead compound identification. / van der Beek, Samantha L; Zorzoli, Azul; Çanak, Ebru; Chapman, Robert N; Lucas, Kieron; Meyer, Benjamin H; Evangelopoulos, Dimitrios; de Carvalho, Luiz Pedro S; Boons, Geert-Jan; Dorfmueller, Helge C; van Sorge, Nina M (Lead / Corresponding author).

In: Molecular Microbiology, 01.01.2019.

Research output: Contribution to journalArticle

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T1 - Streptococcal dTDP-L-rhamnose biosynthesis enzymes

T2 - Molecular Microbiology

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 - This article is protected by copyright. All rights reserved.

PY - 2019/1/1

Y1 - 2019/1/1

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 Mycobacterium tuberculosis with an IC50 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 a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria. This article is protected by copyright. All rights reserved.

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 Mycobacterium tuberculosis with an IC50 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 a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria. This article is protected by copyright. All rights reserved.

U2 - 10.1111/mmi.14197

DO - 10.1111/mmi.14197

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JO - Molecular Microbiology

JF - Molecular Microbiology

SN - 0950-382X

ER -