Spatio-temporal analysis of LC3 association to Mycobacterium tuberculosis phagosomes in human macrophages

Autophagy plays a crucial role in the host response to Mycobacterium tuberculosis (Mtb) infection, yet the dynamics and regulation of autophagy induction on mycobacterial phagosomes remain partially understood. In this study, we employed time-lapse confocal microscopy to investigate in real time the recruitment of LC3B (LC3), a key autophagy marker, to Mtb-containing vacuoles (MCVs) at the single cell level with our newly developed workflow for single cell and single MCV tracking and fluorescence quantification. The results reveal that approximately 70% of MCVs exhibited LC3 recruitment but 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. In addition, interferon-gamma (IFN-{gamma}) pre-treatment did not affect LC3 recruitment frequency or autophagosome maturation, but increased the susceptibility of the macrophage to Mtb-induced cell death. Instead, LC3 recruitment and lysotracker staining were mutually exclusive events alternating on some MCVs multiple times showing a new reversible aspect of this autophagy response. It also suggested a role of autophagy in membrane repair of the MCV. Consistently, LC3 recruitment was strongly associated with galectin-3 and oxysterol-binding protein 1 staining, indicating a correlation with membrane damage and repair mechanisms. However, knockdown of ATG7 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 and autophagy to the MCVs over time during Mtb infection. Our data suggests that there is no major role of autophagy in cell autonomous defense against Mtb nor membrane repair of the MCV in human macrophages. However, the combined dynamics of LC3 recruitment and Lysoview staining emerged as promising markers for future research focused on directly investigating the damage and repair processes of phagosomal membranes.