Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria.

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Metallobiology of Tuberculosis.

Transition metals are essential constituents of all living organisms, playing crucial structural and catalytic parts in many enzymes and transcription factors. However, transition metals can also be toxic when present in excess. Their uptake and efflux rates must therefore be carefully controlled by biological systems. In this chapter, we summarize the current knowledge about uptake and efflux systems in Mycobacterium tuberculosis for mainly three of these metals, namely iron, zinc, and copper. We also propose questions for future research in the field of metallobiology of host-pathogen interactions in tuberculosis.