Demonstration of spread by Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayers

Abstract

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process. ß

Prison Break: Pathogens' Strategies To Egress from Host Cells

“…Apart from cell death-mediated mechanisms (see "Apoptosis as a Nonlytic Exit Strategy" and "Unconcerted Egress from the Vacuole and the Host Cell"), there is evidence for an alternative strategy of intercellular spreading for cytosolic M. marinum and M. tuberculosis in epithelial cell layers (27,32) as well as in amoebae (69). RD1, the major virulence locus of pathogenic mycobacteria that is absent in attenuated strain M. bovis BCG, has been implicated in cell lysis, egress, and dissemination (42,75,82,152).…”

Elastin, a Novel Extracellular Matrix Protein Adhering to Mycobacterial Antigen 85 Complex

“…In addition, in vitro tissue culture model studies combined with fluorescence techniques have directly observed the binding and invasion of alveolar epithelial cells by M. tuberculosis (5,6). A dramatic reduction in the number of M. tuberculosis cells adhering to the extracellular matrix (ECM) 2 after preincubation of bacteria with fibronectin (Fn) was also reported (7).…”

β‐Defensin Gene Expression during the Course of Experimental Tuberculosis Infection

“…We do not know the significance of this high mBD production at the beginning of the infection; however, considering that the usual human infecting dose is very low and that bacteria can be endocytosed by epithelial cells [14], in which they induce the production of b-defensins that efficiently kill bacteria, it is plausible to propose that b-defensins could participate in the early elimination of bacilli. This bactericidal activity might be more efficient in persons who apparently do not have infection and could also participate in the establishment of latent infection.…”