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

Autophagy can act as a defense mechanism for macrophages infected by intracellular pathogens. Mycobacterium tuberculosis (Mtb) is known to both induce and repress autophagic responses, such as xenophagy and LC3-associated phagocytosis (LAP) which both involve the recruitment of LC3 to the Mtb-containing vacuole (MCV). However, the dynamics of MCV interaction with xenophagy or LAP are unclear. Here, using time-lapse confocal microscopy, we present a comprehensive spatio-temporal analysis of the LC3 recruitment to the MCVs during the infection of macrophages. The results revealed frequent LC3 recruitment in the form of large tubule-vesicular structures to the MCV, characteristic of xenophagy, and demonstrated that Mtb could efficiently escape from this signal. We found that the main driver of the LC3 recruitment is the initial macrophage bacterial burden before a second phagocytosis event. We also assessed the potential bactericidal properties of the LC3 recruitment and observed that interferon-gamma treatments did not affect the LC3 recruitment frequency. Additionally, no sign of acidification in the formed autophagosome with or without interferon-gamma treatment was observed. Interestingly, the time-lapses using the acidification probe lysoview revealed that the LC3 recruitment happened shortly after a drop in acidity, a typical sign of membrane damage that is a well-known trigger for autophagy. However, LC3 subsequent loss of signal or escape could also be followed by a restoration of acidification in the vacuole, thus showing restoration of membrane integrity. In conclusion, we show that LC3 recruitment to the MCV correlates with subsequent membrane repair. However, the LC3 recruitment did not show bactericidal properties, questioning its cell intrinsic role in controlling the Mtb infection in macrophages.