Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments

Abstract

Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes.

FIG. 1

Kinetics of B. abortus replication. (A) Monolayers of HeLa cells were inoculated with attenuated strain 19 or virulent strains 544 and 2308 for 4 h at 37°C. After gentamicin treatment, the number of CFU of brucellae were determined. Values are averages ± standard errors for triplicate samples. (B) Quantification of extracellular and intracellular bacteria was performed by double immunofluorescence staining after 1 h of incubation at 37°C with 10⁸ CFU of attenuated strain 19 or virulent strain 2308. Experiments were done in triplicate and repeated at least three times (approximately 500 intracellular and extracellular bacteria were counted per strain).

FIG. 2

B. abortus replication assessed by confocal microscopy. After fixation, permeabilization, and immunolabelling of bacteria, HeLa cells were observed by analyzing reflection and fluorescence of cells and bacteria, respectively. Superimposed images of reflection and fluorescence are presented. (A, B, and C) Cells were infected with strain 2308 and observed at 4, 24, and 72 h p.i., respectively. (D, E, and F) Cells were infected with strain 19 and observed at the above-mentioned times, respectively. Bar, 10 μm.

FIG. 3

Cathepsin D in B. abortus vacuoles analyzed by double immunofluorescence. HeLa cells were immunostained to localize both Brucella (A and C) and cathepsin D (B and D). Double immunofluorescence micrographs show that strain 2308 does not colocalize with cathepsin D (A and B), whereas in strain 19-infected cells few remaining bacteria or bacterial degradation products colocalize with the lysosomal marker (C and D) as indicated by arrows. Bar, 10 μm.

FIG. 4

B. abortus 2308 is associated with autophagosome-like structures. At 24 h p.i., HeLa cells were processed for electron microscopy. An electron micrograph shows two multimembranous autophagosomal structures (large arrows), one of which contains the bacterium (b). Three small arrows indicate the presence of ribosomes. Double arrows show the presence of a phagosome containing one bacterium (6) surrounded by a single membrane with no ribosomes. Bar, 1 μm.

FIG. 5

Modulation of autophagy affects B. abortus 2308 infection. HeLa cells were incubated with or without 3-methyladenine (+3MA; 10 mM) or wortmannin (W; 10 nm) or without FCS-glutamine for 15 min before bacterial inoculation. Then the cells were infected with strain 2308 for 1 h with or without drugs or without FCS, washed, and further incubated with fresh cell culture medium supplemented with gentamicin for 23 h. Cells were lysed, and the number of CFU were estimated. Inhibition of autophagy by 3-methyladenine or wortmannin significantly (P < 0.01%) reduces the number of isolated viable intracellular strain 2308, while starvation conditions without FCS increase the bacterial yield at 24 h postinoculation. Columns represent the averages of triplicate treatments ± standard deviations.