Trafficking and release of mycobacterial lipids from infected macrophages

Analysis of infected macrophages revealed that lipid-containing moieties of the mycobacterial cell wall are actively trafficked out of the mycobacteri al vacuole. To facilitate the analysis of vesicular trafficking from mycoba cteria-containing phagosomes, surface-exposed carbohydrates were labeled wi th hydrazide-tagged markers. The distribution of labeled carbohydrate/lipid moieties and subsequent interaction with cellular compartments were analyz ed by immunoelectron microscopy and by fluorescence microscopy of live cell s. The released mycobacterial constituents were associated with several int racellular organelles and were enriched strikingly in tubular endocytic com partments. Subcellular fractionation of infected macrophages by density gra dient electrophoresis showed temporal movement of labeled bacterial constit uents through early and late endosomes. Thin layer chromatography analysis of these subcellular fractions confirmed their lipid nature and revealed fi ve dominant bacteria-derived species. These mycobacterial lipids were also found in extracellular vesicles isolated from the medium and could be obser ved in un-infected 'bystander' cells. Their transfer to bystander cells cou ld expand the bacteria's sphere of influence beyond the immediate confines of the host cell.