TheMycobacterium tuberculosisPhagosome in Human Macrophages Is Isolated from the Host Cell Cytoplasm

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Abstract
Knowledge of whetherMycobacterium tuberculosisresides within a relatively impermeable membrane-bound vacuole or is free within the cytoplasm within its host cell is central to an understanding of the immunobiology of this intracellular parasite but is a matter of controversy. To explore this issue, we assessed the accessibility of medium-size protein molecules (Fab fragments of 50,000 Da) toM. tuberculosiswithin human macrophages. We infected the macrophages with wild-type or green fluorescent protein-expressingM. tuberculosis, microinjected Fab fragments directed against a major surface antigen ofM. tuberculosisinto the host cell, and assayed the accessibility of the bacteria to the Fab fragments by both immunofluorescence microscopy and immunogold electron microscopy. Whereas microinjected intact immunoglobulin G molecules against cytoplasmic early endosomal antigen 1 readily stained this antigen, microinjected Fab fragments againstM. tuberculosisdid not stain the bacterium within its phagosome. In contrast, microinjected Fab fragments againstListeria monocytogenes, an intracellular bacterium known to permeabilize its phagosomal membrane, strongly stained this bacterium. Our study shows thatM. tuberculosisresides in an isolated phagosome that is relatively impermeable to cytoplasmic constituents.

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