Intracellular Trafficking ofBrucella abortusin J774 Macrophages

Abstract

Brucella abortus is 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 of B. abortus in 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 live B. abortus were 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 the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria 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 that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.The facultative intracellular parasite Brucella abortus causes abortion and infertility in cattle and undulant fever in humans. The bacterium is endemic in many underdeveloped countries and responsible for large economic losses and chronic infections in human beings (30). Brucella infects its hosts through mucosae and wounds and initially is incorporated into professional phagocytes where it survives and reproduces (14). Afterwards, the bacterium infects several types of nonprofessional phagocytic cells including those of endocardium, brain, joints, and bones. Brucella has a special tropism for reproductive organs, causing a high rate of abortion in pregnant animals (28).The intracellular survival of Brucella has been documented for several cell types. According to multiple observations, B. abortus is incorporated into phagosomes and remains in membrane-bound compartments until the host cell dies. In nonprofessional phagocytes, Brucella is located in structures that resemble the endoplasmic reticulum (ER) (6). Recent evidence indicates that Brucella is transported through the autophagic pathway before accumulating in the ER (22, 23).Macrophages are particularly important for the survival and spreading of Brucella during infection ...

A Brucella abortus cstA mutant is defective for association with endoplasmic reticulum exit sites and displays altered trafficking in HeLa cells

“…counting showed no difference with the wild-type strain. This is not very surprising since previous data reported proliferation outside ER-derived compartments (Arenas et al, 2000;Bellaire et al, 2005 (ERGIC) or autophagosomes, as proposed previously (Pizarro-Cerdá et al, 1998). ERGIC would be an interesting candidate since it was already shown that (i) Rab2, a Rab GTPase associated with ERGIC, is recruited on the BCVs and that (ii) the GAPDH/COPI/Rab2/PKC i/l complex, involved in retrograde transport between the ER and Golgi apparatus, is required for bacterial proliferation (Fugier et al, 2009).…”

The analysis of the intramacrophagic virulome ofBrucella suisdeciphers the environment encountered by the pathogen inside the macrophage host cell

“…is different in HeLa cells and macrophages. Indeed, results obtained on intracellular trafficking of B. abortus in J774 macrophages report that only a few bacteria are localized in autophagosomes and ER-like structures (55). However, partial analysis of the virulome of B. melitensis has been performed in HeLa cells, and some amino acid biosynthesis pathways have been shown to be necessary (6,56).…”

Roles of Brucella abortus SpoT in morphological differentiation and intramacrophagic replication

“…Phagosomes containing virulent B. abortus are reluctant to fuse with lysosomes, whereas those with dead B. abortus colocalize with endocytic compartments in the early stage of infection in macrophages (Arenas et al, 2000). To test the ability of B. abortus to target properly within macrophages early in infection, interaction of the mutants with the endocytic pathway was quantified by immunofluorescence localization of LAMP-1, a membrane protein of late endosomes and lysosomes (Chen et al, 1988).…”