Asymmetric trehalose analogues to probe disaccharide processing pathways in mycobacteria

Parker, Hadyn L.; Tomás, Ruben M F; Furze, Christopher M.; Guy, Collette S.; Fullam, Elizabeth

doi:10.1039/d0ob00253d

Cited by 19 publications

(40 citation statements)

References 24 publications

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“…In these studies, we observed that the asymmetric epimeric analogs, galactotrehalose and mannotrehalose, showed an 3 and 12-fold reduction in binding affinity respectively. These findings are compatible with our recent studies in Mycobacterium smegmatis which, unexpectedly, showed that 6-azidogalactotrehalose is incorporated into the mycobacterial cell envelope with a similar efficiency as 6-azido-trehalose via the M. smegmatis LpqY-SugABC transporter (12,13). This result indicates that Mtr LpqY is able to tolerate epimerization of the hydroxy group at the 4-position, whereas epimerization at the 2-position is less favored.…”

Section: Substrate Specificity Of Mtr Lpqysupporting

confidence: 92%

“…As expected, given the lack of genes encoding for phosphotransferase systems in Mtb, Mtr LpqY did not recognize trehalose-6-phosphate. Modified trehalose derivatives have been developed as tools to probe trehalose processing pathways in mycobacteria; however, up until now, the structural basis for the selective recognition of these analogs was unknown (11)(12)(13). Notably, our STD NMR studies in combination with MST analyses support the uptake of the 6-azido-trehalose analog by the mycobacterial LpqY-SugABC transporter and explain the reduced affinity for this chemically modified substrate.…”

Section: Discussionmentioning

confidence: 61%

“…Next, we probed whether modified trehalose analogs known to be imported by mycobacteria, including 2 H-trehalose, 2-azido-2-deoxy-α,α 0 -trehalose (2-azido-trehalose), 4-azido-4-deoxy-α,α 0 -trehalose (4-azido-trehalose), 6-azido-6deoxy-α,α 0 -trehalose (6-azido-trehalose), α-D-mannopyranosyl-(1→1)-α-D-glucopyranoside (mannotrehalose), and α-D-galactopyranosyl-(1→1)-α-D-glucopyranoside (galactotrehalose) (see SI methods for synthetic details), are substrates of Mtr LpqY (11)(12)(13)(14). All of these disaccharides influenced the melting temperature of Mtr LpqY but to a lesser extent (Fig.…”

Section: Substrate Specificity Of Mtr Lpqymentioning

confidence: 99%

“…Despite the importance of trehalose uptake in mycobacteria, the molecular details that govern how this disaccharide are recognized and whether alternative sugars are substrates for this recycling system remain unresolved. Some understanding into the substrate preference of this mycobacterial ABC-transporter can be obtained from studies which found that modified trehalose analogs retaining the α1-1-glycosidic linkage are actively imported by the LpqY-SugABC recycling system and metabolically incorporated into the trehalose-mycolates located within the cell envelope (11)(12)(13). Whether the mycobacterial LpqY-SugABC transporter is able to facilitate the import of alternative, more diverse, sugars is not yet known.…”

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confidence: 99%

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Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter

Furze

Delso

Casal

et al. 2021

Journal of Biological Chemistry

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The Mycobacterium tuberculosis ( Mtb ) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.

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Section: Substrate Specificity Of Mtr Lpqysupporting

confidence: 92%

Section: Discussionmentioning

confidence: 61%

Section: Substrate Specificity Of Mtr Lpqymentioning

confidence: 99%

mentioning

confidence: 99%

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Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter

Furze

Delso

Casal

et al. 2021

Journal of Biological Chemistry

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“…Recently our understanding of the role of the Mtb carbohydrate importers has improved and substrates for some of these systems have been identified ( Fenn et al, 2019 , Fullam et al, 2016 , Kalscheuer et al, 2010b , Parker et al, 2020 , Furze et al, 2021 , Lowery et al, 2015 ). Interestingly, it has been shown that the Mtb LpqY-SugABC transporter specifically recycles trehalose from the mycobacterial cell envelope and is essential for Mtb virulence ( Kalscheuer et al, 2010b , Furze et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC

et al. 2021

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Mycobacterium tuberculosis ( Mtb ) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: Δ uspC or the entire transport complex: Δ uspABC . Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an N in -C in orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and Δ uspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.

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Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation

Marton,

Bhatt,

Sagona

et al. 2023

Biomacromolecules

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Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

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Asymmetric trehalose analogues to probe disaccharide processing pathways in mycobacteria

Abstract: Chemoenzymatic synthesis of azido-functionalised asymmetric trehalose analogues that are resistant to enzymatic degradation to probe carbohydrate processing pathways in mycobacteria.

Cited by 19 publications

References 24 publications

Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter

Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter

Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC

Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation

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