Intracellular Trafficking ofBrucella abortusin J774 Macrophages

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Abstract
Brucella abortusis a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport ofB. abortusin macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing liveB. abortuswere reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of theBrucellabacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization ofB. abortuswith LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that theB. abortusbacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3′-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate thatB. abortusbacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.