Abstract
SummaryBiosynthesis 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 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 lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.
Highlights
SummaryBiosynthesis 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
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-keto6-deoxyglucose-3,5-epimerase enzymes respectively
Ri03 could inhibit growth of S. mutans, S. equi subsp. zooepidemicus (Group C Streptococcus, GCS) and Mycobacterium tuberculosis with similar efficacy. These results demonstrate that rhamnose biosynthesis inhibitors can directly interfere with bacterial viability and could form a new class of antibiotics targeting nucleotide sugar production
Summary
SummaryBiosynthesis 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. The typical cell wall architecture of a Gram-positive bacterium consists of a thick peptidoglycan layer that is decorated with wall teichoic acids, proteins and capsular polysaccharides. Examples of rhamnose cell wall polysaccharides are the group A carbohydrate (GAC) of Group A Streptococcus (GAS; Streptococcus pyogenes) and the serotype-determining polysaccharides, referred to as rhamnose-glucose polysaccharides (RGP), of Streptococcus mutans. The GAC and RGP share structural similarities; both consist of an α-1,2-/α-1,3-linked polyrhamnose backbone with alternating N-acetylglucosamine side chains for GAS and glucose or galactose side chains for S. mutans (Heymann et al, 1964; Linzer et al, 1985; Huang et al, 1986; Pritchard et al, 1986; St Michael et al, 2017) (Fig. 1). For S. mutans, the type of side chain as well as their linkage to the rhamnan backbone determines the S. mutans
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