Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy

The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.

Research Highlights
► Observation of the intracellular lipolysis in real time in M. smegmatis.
► Proof of involvement of intracellular lipases in the process of LIBs degradation.
► Lipid consumption account for at least 25% of the energy required in cell growth.
► No lipid consumption in dormant cells suggesting a role in the reactivation process.
► Lipid storage contributes to survival of mycobacteria in a non- replicating state.