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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.

SUBMITTER: Rodriguez-Rivera FP

PROVIDER: S-EPMC5345120 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Visualization of mycobacterial membrane dynamics in live cells.

Rodriguez-Rivera Frances P FP Zhou Xiaoxue X Theriot Julie A JA Bertozzi Carolyn R CR

Journal of the American Chemical Society 20170223 9

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 ...[more]

PMID: 28075574

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Project description:We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments-capsid, tegument, and envelope-fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.

Dataset Information

Visualization of mycobacterial membrane dynamics in live cells.

Publications

Visualization of mycobacterial membrane dynamics in live cells.

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