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

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.

Gingivitis is a highly prevalent oral condition that can be studied in humans via the 21‐d experimental gingivitis model, which allows for investigations into the induction and resolution of gingivitis. In this study, we used the autolysis of saliva as a source of peptides to predict the activity of human proteases in saliva during induction and resolution of inflammation. Healthy volunteers, with no remarkable oral or systemic conditions, were recruited into the study and stimulated saliva samples were collected at days 0, 21, and 35 of experimental gingivitis. Plaque and gingival indices were recorded to ensure clinical induction and resolution. Saliva was auto‐digested at 37°C for 18 h before identification of peptides by mass spectrometry. Protease prediction was carried out using Proteasix in silico with the identified peptides. A comparison of day 0 to days 21 and 35 showed changes in predicted protease activity. Correlation network analysis revealed that at day 21 the proteases became less connected and showed a potential for a dysregulated system; by day 35 the connectivity was returning towards similar conditions at day 0. This study demonstrates that changes in predicted proteases are apparent even in saliva collected from donors experiencing inflammation around three teeth.