Asymmetric trehalose analogues to probe disaccharide processing pathways in mycobacteria

The uptake and metabolism of the disaccharide trehalose by Mycobacterium tuberculosis is essential for the virulence of this pathogen. Here we describe the chemoenzymatic synthesis of new azido-functionalised asymmetric trehalose probes that resist degradation by mycobacterial enzymes and are used to probe trehalose processing pathways in mycobacteria

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

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodelling 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, STD NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays and the synthesis of trehalose analogues. 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 analogues, and specifies a framework that can be exploited for the design of new anti-tubercular agents and/or diagnostic tools

Tools to explore carbohydrate metabolism pathways in Mycobacteria

Mycobacterium tuberculosis (Mtb) survives intracellularly in a nutrient deprived environment, within the human host, for decades. The nutrients that Mtb uses to survive are not well understood and, in particular, the pathways that are involved in carbohydrate uptake and metabolism are not well defined. Mtb has a highly unique cell-wall comprising of complex carbohydrates and lipids. Trehalose, a disaccharide, is an essential component of trehalose-lipids within the Mtb cell-wall, which play a key role in virulence. Recent studies have shown that trehalose provides an energy source to Mtb through its uptake and recycling by the LpqY-SugABC transporter before being metabolised through various pathways. To further understand how Mtb utilises trehalose, novel ‘unnatural’ trehalose analogues were synthesised to evaluate how modifications might alter uptake, toxicity and stability. Four trehalose analogues; mannotrehalose (ManGlc), galactotrehalose (GalGlc) and 6-azido derivatives were chemoenzymatically synthesised by trehalose synthase (TreT). The trehalose analogues were found to be imported into the cell and potentially incorporated into the cell envelope in M. smegmatis and M. bovis BCG. The analogues are well tolerated by mycobacteria as they did not inhibit mycobacterial growth or influence trehalose processing proteins within the proteome. However, the trehalose analogues were resistant to utilisation by trehalase, TreS and OtsA, the main trehalose processing pathways in mycobacteria, and therefore could not be used to support growth in minimal media. Combined these results present novel insights into how trehalose is processed by mycobacteria and provide the tools for enzymatically stable, trehalose-based probes with potential applications in the diagnostic or treatment of Mtb. In addition, the differential radial capillary action of ligand assay was optimised for the high-throughput detection of carbohydrate binding proteins. The development of an Mtb overexpression ORFeome is ongoing and could be used to identify novel trehalose binding proteins or pathways in mycobacteria

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