Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation

Mycobacterium tuberculosis establishes infection, progresses towards diseas e, and is transmitted from the alveolus of the lung. However, the role of t he alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a mod el in which to examine cytotoxicity during infection with either virulent o r avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculos is strains Erdman and CDC1551 demonstrated significant cell monolayer clear ing, whereas infection with either Mycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells c orrelated to necrosis, not apoptosis, Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a signif icant reduction in the necrosis of A549 cell monolayers, This mycobacterium -induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of i nfected cells with propidium iodide demonstrated that M, tuberculosis induc ed increased permeation of A549 cell membranes within 24 h postinfection, Q uantitation of lactate dehydrogenase (LDH) release from infected cells furt her demonstrated that cell permeation was specific to M. tuberculosis infec tion and correlated to A549 cellular necrosis, Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release , These studies demonstrate that lung epithelial cell cytotoxicity is speci fic to infection by virulent mycobacteria and is caused by cellular necrosi s, This necrosis is not a direct correlate of mycobacterial growth or of th e expression of host cell factors, but is preceded by permeation of the A54 9 cell membrane and requires infection with live bacilli.