Publication date: Jan 02, 2025

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on Mtb-containing vacuoles (MCVs) remain only partially understood. We employed time-lapse confocal microscopy to investigate the recruitment of LC3B (LC3), a key autophagy marker, to MCVs at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. We show that approximately 70% of MCVs exhibited LC3 recruitment but that was lost in about 40% of those MCVs. The LC3 recruitment to MCVs displayed a high variability in timing that was independent of the size of the MCV or the bacterial burden. Most notably, the LC3-positive MCVs did not acidify, indicating that LC3 recruitment does not necessarily lead to the formation of mature autophagolysosomes. Interferon-gamma pre-treatment did not affect LC3 recruitment frequency or autophagosome acidification but increased the susceptibility of the macrophage to Mtb-induced cell death. LC3 recruitment and lysotracker staining were mutually exclusive events, alternating on some MCVs multiple times thus demonstrating a reversible aspect of the autophagy response. The LC3 recruitment was associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. ATG7 knock-down did not impact membrane repair, suggesting that autophagy is not directly involved in this process but is coregulated by the membrane damage of MCVs. In summary, our findings provide novel insights into the dynamic and variable nature of LC3 recruitment to the MCVs over time during Mtb infection. Our data does not support a role for autophagy in either cell-autonomous defense against Mtb or membrane repair of the MCV in human macrophages. In addition, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for investigating the damage and repair processes of phagosomal membranes.

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