Simple SummaryTuberculosis is a life-threatening disease caused by infection with mycobacteria. These bacteria can grow inside the cells of our immune system, particularly in macrophages, one of the first cells responding to an infection. Macrophages activate several defense mechanisms to combat the infection, but mycobacteria are notorious for escaping these mechanisms. One of the macrophage defense mechanisms is autophagy, a vital process that keeps cells in a healthy condition by clearing worn-out cell parts or microbial invaders. Stimulating autophagy is explored as a potential strategy to improve the treatment of tuberculosis, but this will require a better understanding of how mycobacteria interact with the autophagy machinery. In this study, we infected zebrafish larvae with a mycobacterial species closely related to the human tuberculosis pathogen. Zebrafish larvae are transparent and ideally suited for microscopic imaging of the early stages of the infection process. Therefore, we could observe that mycobacteria, when taken up by macrophages, reside in vesicles formed by the autophagy machinery. We found that these vesicles dynamically change shapes and that they could serve as a temporary hiding place for mycobacteria, facilitating the spreading of the infection to other tissues.AbstractExisting drug treatment against tuberculosis is no match against the increasing number of multi-drug resistant strains of its causative agent,Mycobacterium tuberculosis(Mtb). A better understanding of how mycobacteria subvert the host immune defenses is crucial for developing novel therapeutic strategies. A potential approach is enhancing the activity of the autophagy machinery, which can direct bacteria to autophagolysosomal degradation. However, the interplay specifics between mycobacteria and the autophagy machinery must be better understood. Here, we analyzed live imaging data from the zebrafish model of tuberculosis to characterize mycobacteria-autophagy interactions during the early stages of infection in vivo. For high-resolution imaging, we microinjected fluorescentMycobacterium marinum(Mm) into the tail fin tissue of zebrafish larvae carrying the GFP-LC3 autophagy reporter. We detected phagocytosedMmclusters and LC3-positiveMm-containing vesicles within the first hour of infection. LC3 associations with these vesicles were transient and heterogeneous, ranging from simple vesicles to complex compound structures, dynamically changing shape by fusions betweenMm-containing and empty vesicles. LC3-Mm-vesicles could adopt elongated shapes during cell migration or alternate between spacious and compact morphologies. LC3-Mm-vesicles were also observed in cells reverse migrating from the infection site, indicating that the autophagy machinery fails to control infection before tissue dissemination.