Galectin-3 recruitment at the Mycobacterium tuberculosis-containing phagosome is critical in macrophage but dispensable in epithelial cells

Abstract

Mycobacterium tuberculosis (Mtb) virulence relies in part on its ability to induce phagosomal membrane rupture, enabling bacterial access to the host cell cytosol. This process is largely mediated by the ESX-1 secretion system, which is present in Mtb but absent from the vaccine strain BCG. Galectin-3 (Gal3), a {beta}-galactoside-binding lectin, is recruited to damaged endomembranes and functions as a cytosolic sensor of membrane disruption. However, the kinetics and quantitative features of Gal3 recruitment to Mtb-containing vacuoles have remained poorly characterized. Here, we performed a longitudinal quantitative imaging study of Gal3 recruitment in human macrophages over a five-day infection period. Gal3 was recruited to mycobacteria-containing vacuoles shortly after infection with both live and heat-killed Mtb. Differences between the two conditions emerged from day 1 post-infection and persisted until macrophage death. A similar kinetic profile was observed with recombinant BCG::ESX-1, whereas parental BCG failed to induce Gal3 recruitment, confirming the requirement for ESX-1-dependent membrane damage. Cytoplasmic Gal3 levels were higher in bystander macrophages than in infected cells and were comparable to non-infected controls, suggesting that diffuse cytoplasmic Gal3 is associated with cells lacking intracellular mycobacteria. Functional studies revealed that Gal3 silencing enhanced long-term intracellular Mtb replication, demonstrating a role for Gal3 in restricting bacterial growth. Importantly, Gal3 recruitment was not observed in alveolar epithelial cells. This cell-type specificity was confirmed in a microfluidic alveolus-on-chip model. Together, these findings identify sustained Gal3 recruitment to the mycobacteria-containing vacuole as a robust quantitative marker of ESX-1-dependent phagosomal rupture and Mtb virulence.