Uptake‐independent killing of macrophages by extracellular Mycobacterium tuberculosis aggregates

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular Mtb replication induces the death of the infected macrophages and the release of bacterial aggregates. Here, we show that these aggregates can evade phagocytosis by killing macrophages in a contact‐dependent but uptake‐independent manner. We use time‐lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytosolic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb ESX‐1 secretion system, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface‐exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that blocking ESX‐1‐mediated secretion of the EsxA/EsxB virulence factors does not eliminate the uptake‐independent killing of macrophages and that the 50‐kDa isoform of the ESX‐1‐secreted protein EspB can mediate killing in the absence of EsxA/EsxB secretion. Treatment with an ESX‐1 inhibitor reduces uptake‐independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this anti‐phagocytic mechanism could prevent the propagation of extracellular bacteria within the lung.