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.

The host type I interferon (IFN) response protects against a Legionella pneumophilia infection. Other pathogens inhibit type I IFN-mediated cell signaling; however, the interaction between this signaling pathway and L. pneumophila has not been described. This study demonstrates that L. pneumophila inhibits the IFN-β signaling pathway in an IFN-β dose-dependent manner. However, L. pneumophila does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-β to L. pneumophila-infected macrophages limited bacterial growth in a nitric oxide (NO)-dependent pathway since inhibiting NO production via a chemical inhibitor increased bacterial growth. Finally, this study demonstrates that the type IV secretion system of L. pneumophila is required to inhibit IFN-β-mediated cell signaling. In conclusion, this study describes a novel host cell signaling pathway inhibited by L. pneumophila, which improves the survival of bacteria in infected macrophages.