Visualization of mycobacterial membrane dynamics in live cells

Abstract

Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose–fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein–trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

“…B) Structures of trehalose, TMM and TDM and their interconversion by Ag85. C) 6‐FITC‐Tre . D) DMN‐Tre .…”

Mycobacterium Tuberculosis and Interactions with the Host Immune System: Opportunities for Nanoparticle Based Immunotherapeutics and Vaccines

“…M. tuberculosis is surrounded by a characteristically thick and waxy cell envelope containing interconnected polymers of mycolic acids (MAs), arabinogalactan (AG) and peptidoglycan (PG) (17,18). This unique envelope renders M. tuberculosis hydrophobic; a trait primarily attributed to the presence of the MAs which are long chain fatty acids of up to 90 carbon atoms in length (19).…”

“…This unique envelope renders M. tuberculosis hydrophobic; a trait primarily attributed to the presence of the MAs which are long chain fatty acids of up to 90 carbon atoms in length (19). The hydrophobic membrane acts as a permeability barrier towards various hydrophilic and lipophilic compounds making M. tuberculosis inherently resistant to antibiotics (17,18,20,21). However, the inherent resistance cannot solely be attributed to the impermeable membrane since experiments have shown that drugs are able reach cytotoxic levels within cells (20).…”

Visualizing glycans on single cells and tissues–Visualizing glycans on single cells and tissues

“…FRAP measurements by Edidin’s group using class I MHC molecules revealed that mutants with reduced N-linked glycans have increased lateral diffusion as compared with wild-type and that a large mobile fraction of diffusing glycoproteins enabled bleached regions to become repopulated with fluorescent molecules [24]. In contrast to these pioneering studies, contemporary FRAP imaging of the dynamics of glycolipids within the cell envelope of mycobacterial membranes exploits the power of metabolically incorporated analogues [26]. …”