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

Beek, Samantha L. van der; Zorzoli, Azul; Çanak, Ebru; Chapman, Robert N.; Lucas, Kieron; Meyer, Benjamin; Evangelopoulos, Dimitrios; Carvalho, Luiz Pedro S. de; Boons, Geert‐Jan; Dorfmueller, Helge C.; Sorge, Nina M. van

doi:10.1111/mmi.14197

Cited by 44 publications

(34 citation statements)

References 54 publications

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“…L-rhamnose biosynthetic pathways are also essential in S. pyogenes [38] and Mycobacterium smegmatis [39]. Several of these genes have, however, been mutated in S. mutans, including rmlBC [40], rmlD [38], rgpE [30,41], rgpF [41,42], and rgpG [43]. Inconsistencies between our observations and others could be related to the nature of our Tn-seq selection and/or the presence of undetected suppressor mutations that bypass essentiality in strains that carry RGP gene deletions in other studies.…”

Section: L-rhamnose Glycopolymers Are Critical For S Mutans Cell DIVmentioning

confidence: 99%

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

et al. 2020

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A recent genome-wide screen identified~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9 Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9 Smu with a constitutively active singleguide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9 Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9 Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

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Section: L-rhamnose Glycopolymers Are Critical For S Mutans Cell DIVmentioning

confidence: 99%

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

et al. 2020

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“…We have shown that a complete loss of the RGP structure has significant consequences for the organism in nearly all aspects required for pathogenesis in the oral cavity and potential to cause disease. The rhamnose biosynthesis pathway is an attractive antimicrobial target (50) and an alternative to targeting the machinery involved in cell wall polysaccharide synthesis. Rhamnose biosynthesis, as a target, might also be extended to life-threatening streptococcal species.…”

Section: Figmentioning

confidence: 99%

Disruption of l -Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

et al. 2020

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The rhamnose-glucose cell wall polysaccharide (RGP) of Streptococcus mutans plays a significant role in cell division, virulence, and stress protection. Prior studies examined function of the RGP using strains carrying deletions in the machinery involved in RGP assembly. In this study, we explored loss of the substrate for RGP, l-rhamnose, via deletion of rmlD (encoding the protein responsible for the terminal step in l-rhamnose biosynthesis). We demonstrate that loss of rhamnose biosynthesis causes a phenotype similar to strains with disrupted RGP assembly (ΔrgpG and ΔrgpF strains). Deletion of rmlD not only caused a severe growth defect under nonstress growth conditions but also elevated susceptibility of the strain to acid and oxidative stress, common conditions found in the oral cavity. A genetic complement of the ΔrmlD strain completely restored wild-type levels of growth, whereas addition of exogenous rhamnose did not. The loss of rhamnose production also significantly disrupted biofilm formation, an important aspect of S. mutans growth in the oral cavity. Further, we demonstrate that loss of either rmlD or rgpG results in ablation of rhamnose content in the S. mutans cell wall. Taken together, these results highlight the importance of rhamnose production in both the fitness and the ability of S. mutans to overcome environmental stresses. IMPORTANCE Streptococcus mutans is a pathogenic bacterium that is the primary etiologic agent of dental caries, a disease that affects billions yearly. Rhamnose biosynthesis is conserved not only in streptococcal species but in other Gram-positive, as well as Gram-negative, organisms. This study highlights the importance of rhamnose biosynthesis in RGP production for protection of the organism against acid and oxidative stresses, the two major stressors that the organism encounters in the oral cavity. Loss of RGP also severely impacts biofilm formation, the first step in the onset of dental caries. The high conservation of the rhamnose synthesis enzymes, as well as their importance in S. mutans and other organisms, makes them favorable antibiotic targets for the treatment of disease.

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“…This is because RmlA is an enzyme in the TDP-L-rhamnose pathway. TDP-L-rhamnose is used for the synthesis of cell wall carbohydrates (24)(25)(26) and for the decoration of WTA in L. monocytogenes (27,28). Therefore, we extracted WTA from the sigB and rsbU mutants and compared the apparent molecular mass with the WTA extracted from the rmlA mutant.…”

Section: S2cmentioning

confidence: 99%

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation

et al. 2020

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Listeria monocytogenes is a Gram-positive firmicute that causes foodborne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation, we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains single nucleotide polymorphisms (SNPs) compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor SigB, and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-l-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-l-rhamnose but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant) showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together, these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted. IMPORTANCE Biofilms are an important mode of growth in many settings. Here, we looked at small differences in the genomes of the bacterium Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

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

Cited by 44 publications

References 54 publications

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

Disruption of l -Rhamnose Biosynthesis Results in Severe Growth Defects in Streptococcus mutans

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation

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