Uptake-independent killing of macrophages by extracellular aggregates of Mycobacterium tuberculosis is ESX-1 and PDIM-dependent

Mycobacterium tuberculosis ( Mtb ) infection is initiated by inhalation of small numbers of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular replication of Mtb leads to death of the infected macrophages, release of bacterial aggregates, and rapid growth of the extracellular aggregates on host-cell debris. Here, we show that extracellular Mtb aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use single-cell time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytoplasmic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb type VII secretion system ESX-1, 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 ESX-1-mediated secretion of the EsxA/EsxB virulence factors is not required for uptake-independent killing of macrophages after contact with extracellular Mtb aggregates. In the absence of EsxA/EsxB secretion, killing is mediated by the 50-kDa isoform of the ESX-1-secreted protein EspB, while blocking secretion of both EsxA/EsxB and processed EspB reduces killing to background levels. Treatment with a small-molecule 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.