A Bifunctional Chemical Reporter for in Situ Analysis of Cell Envelope Glycan Recycling in Mycobacteria.

In mycobacteria, the glucose-based disaccharide trehalose cycles between the cytoplasm, where it is a stress protectant and carbon source, and the cell envelope, where it is released as a byproduct of outer mycomembrane glycan biosynthesis and turnover. Trehalose recycling via the LpqY-SugABC transporter promotes virulence, antibiotic recalcitrance, and efficient adaptation to nutrient deprivation. The source(s) of trehalose and the regulation of recycling under these and other stressors are unclear. A key technical gap in addressing these questions has been the inability to trace trehalose recycling in situ, directly from its site of liberation from the cell envelope. Here we describe a bifunctional chemical reporter that simultaneously marks mycomembrane biosynthesis and subsequent trehalose recycling with alkyne and azide groups. Using this probe, we discovered that the recycling efficiency for trehalose increases upon carbon starvation, concomitant with an increase in LpqY-SugABC expression. The ability of the bifunctional reporter to probe multiple, linked steps provides a more nuanced understanding of mycobacterial cell envelope metabolism and its plasticity under stress.

Transient drug-tolerance and permanent drug-resistance rely on the trehalose-catalytic shift in Mycobacterium tuberculosis.

Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.

A chemical reporter strategy for detecting and identifying O-mycoloylated proteins in Corynebacterium.

Protein O-mycoloylation is a unique post-translational lipidation that was recently discovered in Corynebacterium. We describe an alkyne-modified trehalose monomycolate chemical reporter that can metabolically tag O-mycoloylated proteins in C. glutamicum, enabling their detection and identification through click chemistry.