Infect Immun, May 1998, p. 2387-2392, Vol. 66, No. 5
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Virulent Brucella abortus Prevents
Lysosome Fusion and Is Distributed within Autophagosome-Like
Compartments
Javier
Pizarro-Cerdá,1
Edgardo
Moreno,1
Veronique
Sanguedolce,2
Jean-Louis
Mege,2 and
Jean-Pierre
Gorvel1,*
Centre d'Immunologie INSERM-CNRS de
Marseille-Luminy, 13288 Marseille Cedex 9,1 and
Centre National de Référence des Rickettsioses,
Centre Hospitalier Universitaire la Timone, 13385 Marseille,2 France
Received 3 November 1997/Returned for modification 26 January
1998/Accepted 6 February 1998
 |
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.
| |
TEXT |
The genus Brucella
consists of facultative intracellular gram-negative bacteria that
infect humans and animals (4), causing brucellosis, a
disease that occurs worldwide and is endemic in many underdeveloped
countries (39). During the course of infection, professional
phagocytes are the first target for Brucella invasion (4, 23, 27, 34), and the bacteria can survive within these
cells. Despite their tropism for the sexual organs, these bacteria can
also be found in bones, joints, the eyes, and the brain
(14). In vitro, Brucella abortus replicates in
both the epitheloid HeLa and the fibroblastic Vero cell lines (9,
17). Smooth virulent B. abortus strains replicate
intracellularly more efficiently than smooth attenuated strain 19 or
rough strains. Inhibition of phagosome-lysosome fusion has been
proposed as a mechanism used by B. abortus for intracellular
survival (16). It has been suggested that in trophoblasts of
pregnant ruminants (1) as well as in Vero cells, pathogenic
B. abortus replicates within the rough endoplasmic reticulum
(RER) (9), and transfer from phagosomes to RER was proposed
to be a limiting step in Brucella infection (10,
11). However, the identity of compartments where the bacteria
replicate or are destroyed is still a matter of debate.
In this study, we established a protocol of infection based on the
inoculation of 500 bacteria per HeLa cell for 4 h. Log-phase cultures of virulent smooth B. abortus 544 and 2308 (provided by J.-M. Verger, INRA, Nouzilly, France) and attenuated
smooth B. abortus 19 (Professional Biological Co., Denver,
Colo.) were prepared by incubating 5 × 1010 CFU in 5 ml of tryptic soy broth for 15 h at 37°C. Subconfluent monolayers of HeLa cells cultured in 24-well tissue culture plates were
inoculated with bacteria diluted to 108 CFU/ml in Dulbecco
minimal essential medium (DMEM; GIBCO, Paisley, Scotland) supplemented
with 10% fetal calf serum (FCS) and 2 mM glutamine without antibiotics
(cell culture medium). Plates were centrifuged for 10 min at 1,000 rpm
at room temperature and placed in a 5% CO2 atmosphere at
37°C. After 4 h, wells were washed five times with DMEM and
further incubated with DMEM supplemented with 5% FCS-100 µg of
gentamicin (Sigma, Saint Quentin Fallavier, France) per ml to kill
remaining extracellular brucellae. The number of intracellular viable
B. abortus CFU was determined at different times
postinfection (p.i.) after the monolayers were washed twice with DMEM
and once with phosphate-buffered saline (PBS) (pH 7.4) and treated for
10 min with 1 ml of 0.1% Triton X-100 (Sigma) in deionized water
(13). Lysates were serially diluted and plated on tryptic
soy agar dishes for quantitation of CFU.
The intracellular and extracellular brucellae and the lysosomal marker
cathepsin D were analyzed by confocal microscopy after immunofluorescence labelling (10, 22). Cells were
extensively washed to remove nonadherent bacteria prior to fixation for
15 min with 3% paraformaldehyde in PBS, washed once in PBS, and
incubated for 10 min with PBS-NH4Cl (50 mM) to quench free
aldehyde groups. Extracellular bacteria were detected by incubation of
the cells for 30 min with serum (diluted 1/10,000 in PBS-10% horse
serum) from a B. abortus-infected cow (30) and
revealed by donkey Texas red-conjugated anti-cow immunoglobulin G (IgG)
antibodies (Immunotech, Marseille, France). For detection of
intracellular bacteria, cells were further permeabilized with 0.05%
saponin (Sigma, St. Louis, Mo.), incubated for 30 min with serum (used
at 1/5,000 dilution in PBS-10% horse serum) from a B. abortus-infected goat, and revealed with donkey fluorescein
isothiocyanate-conjugated anti-goat IgG antibodies (Immunotech).
Cathepsin D was revealed with rabbit anti-cathepsin D antibodies
(obtained from S. Kornfeld, St. Louis, Mo.) revealed with donkey cyanin
5-conjugated anti-rabbit IgG antibodies (Immunotech). Samples mounted
in Mowiol (25) were observed with a Leica TCS 4DA confocal
laser scanning microscope by analyzing cells and bacteria by reflection
and fluorescence, respectively. For electron microscopy, HeLa cells
infected with B. abortus 2308 were fixed with 2%
glutaraldehyde in 0.1 M cacodylate buffer for 30 min at 4°C,
postfixed in OsO4, and processed for embedding in Epon 812 resin.
After an initial decrease in the number of bacteria between 4 and
8 h p.i. (data not shown), the number of strain 2308 and strain
544 bacteria increased slightly from 8 to 24 h (Fig.
1A). Then replication became faster
between 24 and 36 h, and the highest rate of bacterial growth was
detected between 36 and 48 h p.i. Between 4 and 48 h p.i.,
the percentage of the input inoculum recovered ranged from 0.003 to
1.62% for strain 2308 and from 0.004 to 1.41% for strain 544. In
contrast, strain 19 replicated very slowly (Fig. 1A). Indeed, the
percentage of recovered input inoculum changed from 0.003 to only
0.03% in the same period of time. Between 48 and 72 h p.i., the
growth of strains 2308 and 544 dropped dramatically due to the
breakdown of the plasma membrane of heavily infected cells and contact
of released bacteria with gentamicin in the incubating medium. To
determine whether the deficient replication of strain 19 within cells
was due to failure in bacterial adherence or to a low penetration rate,
the immunofluorescence assay for detecting extracellular and
intracellular bacteria, described above, was performed. Figure 1B shows
that both the attenuated and the pathogenic strains adhered to the cell
surface and penetrated. However, the efficiency of invasion (Fig. 1B) of the pathogenic strain was greater than that of the attenuated strain, since at 4 h postinoculation almost all strain 2308 bacteria were found within cells, while approximately 50% of strain 19 bacteria remained extracellular (Fig. 1B).
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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).
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Next we explored the intracellular traffic of the various
Brucella strains. At 4 h p.i., from 1 to 3 intracellular bacteria from strain 2808 and strain 19 were detected in
each infected cell (Fig. 2A and D). At
24 h p.i., cells infected with virulent strain 2308 showed a
significant increase in the number of intracellular bacteria in the
perinuclear region (Fig. 2B). In contrast, in cells infected with
strain 19, a small number of intact bacteria together with bacterial
degradation products scattered throughout the cytoplasm were observed
(Fig. 2E and F). At 72 h p.i., cells infected with virulent
strains were filled with intracellular bacteria. In addition, some
heavily infected cells broke down, leading to a release of free
bacteria in the cell culture medium (Fig. 2C).
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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.
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Indirect methods have suggested that pathogenic Brucella is
capable of avoiding phagosome-lysosome fusion in macrophages
(16). Therefore, to directly assess this hypothesis in
nonprofessional phagocytic HeLa cells, we used double
immunofluorescence (25). At 48 h p.i., cathepsin D, a
well-known marker for lysosomes, was colocalized with phagosomes
containing strain 19 and its degradation products (Fig. 3C and
D), indicating that phagosomes had fused with lysosomes. In contrast, intracellular strain 2308 never
colocalized with cathepsin D (Fig. 3A and B), indicating that those
bacteria avoided fusion with lysosomes.
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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.
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|
Since pathogenic B. abortus has been reported to replicate
within the RER (9), we analyzed the characteristics of the
Brucella intracellular compartment by electron microscopy.
Electron micrographs show that at 24 h p.i., bacteria from strain
2308 were localized in two different membrane structures (Fig.
4). Some bacteria were found in
phagosomes surrounded by a single membrane, and others were found in
compartments resembling multimembranous autophagosomes (12)
that were characterized by the presence of ribosomes (Fig. 4). To
further investigate the possible involvement of autophagy in B. abortus infection, we tested the action of autophagic inhibitors. Several inhibitors of the autophagic machinery have been identified. Both 3-methyladenine and wortmannin inhibit autophagy by blocking phosphatidylinositol 3-kinase activity (5, 31). In contrast, autophagy can be increased by limiting amounts of amino acids (32), and maximal levels of autophagy can be reached in as
little as 20 min after amino acid withdrawal (32). For
autophagic inhibition, cells were incubated for 15 min with cell
culture medium in the presence of 3-methyladenine (10 mM) or
wortmannin (100 nM). For autophagic activation, cells were grown in
serum-glutamine-free cell culture medium for 15 min. Cells were
inoculated with strain 2308 bacteria, incubated for 1 h with drugs
or without FCS-glutamine, washed twice, and further incubated with cell
culture medium supplemented with 100 µg of gentamicin per ml. At
24 h postinoculation, cells were lysed and CFU were estimated as
described below. In the presence of autophagic inhibitors, fewer strain
2308 CFU were recovered from treated cells than from untreated cells
(Fig. 5). The low bacterial yields
observed were not due to the inhibition of bacterial replication by
irreversible effects of drugs on HeLa cells, since monolayers infected
after drug withdrawal showed viable bacterial counts similar to those
of nontreated control cells (data not shown). Moreover, equal numbers
of intracellular bacteria were found in drug-treated and untreated
cells during the first hour of inoculation (data not shown), suggesting
that drugs were acting on a critical step involving the accessibility
of bacteria to the autophagic vacuoles or the intracellular replication
compartment. When autophagy was increased by depleting cell culture
medium of FCS and glutamine for 15 min and cells were infected for
1 h in the presence of the starving cell culture medium, an
increase in the number of CFU recovered 24 h after infection was
observed (Fig. 5). These results suggest that autophagy is important
for the bacteria to reach their final intracellular multiplication compartment.
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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.
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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.
|
|
Our results, together with the results of previous studies (9, 11,
16), demonstrate that virulent Brucella need to evade
lysosome fusion to multiply inside professional and nonprofessional phagocytes. Other pathogenic bacteria are known to avoid fusion with
lysosomes as has been demonstrated with Mycobacterium avium and Mycobacterium tuberculosis (6-8).
Legionella is another intracellular pathogen that is well
adapted to life inside phagosomes. As for Mycobacterium, the
phagosome containing Legionella is not acidified (20,
37), and at later stages of infection, vacuoles are found in the
vicinity of the RER (19, 38).
Our results indicate that the efficiency of intracellular replication,
but not of adherence to cells, correlates with the virulence of various
B. abortus strains as in other cell types. Indeed, strain
2308 has been shown to persist longer in mice than strain 19 (3,
36) and replicates more efficiently than rough strain 45/20 or
strain 19 in bovine mammary gland macrophages (18) and
murine peritoneal macrophages (21). Strain 544 has also been
shown to survive intracellularly better than strain 45/20
(33). It is clear that the attenuated strain 19 can invade cells but multiplies poorly because the bacteria degrade after the
Brucella-containing phagosomes fuse with lysosomes. In
contrast, virulent strain 2308 possesses mechanisms to escape lysosomal degradation. This characteristic seems to be independent of cell surface adherence, since strain 19 bacteria were able to adhere at
higher levels than strain 2308 bacteria (Fig. 1B). This difference in
adherence cannot yet be related to a difference in the compositions of
the outer membrane components, since those of smooth virulent strain
2308 and smooth attenuated strain 19 are very similar (9, 15, 24,
30). The O-chain of the lipopolysaccharide (LPS) has been
implicated as a key moiety for intracellular survival, as smooth
strains are more resistant to intracellular killing in phagocytic cells
(22, 29) and more resistant to the action of lysosomal
bactericidal substances (24). However, as previously proposed (28), the O-chain of LPS cannot be the only factor correlated with intracellular survival, since the pathogenic strains 2308 and 544 and the attenuated strain 19 behave quite differently in
HeLa cells despite their indistinguishable smooth LPS (2, 15). The decreased virulence of strain 19 has been attributed to
its inability to metabolize erythritol (35), the only
genetic defect characterized in this attenuated strain. We cannot rule out the possibility that changes in erythritol metabolism are responsible for the observed difference in intracellular replication between virulent and attenuated strains. However, other
yet-unknown defects might be involved, since pathogenic
erythritol-negative Brucella strains have been
previously identified (26). More work is needed to determine
which components in strain 19 are responsible for its increased
adherence. Therefore, virulence seems to reflect the ability of
pathogenic Brucella to persist and replicate within specific
intracellular compartments.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from INSERM (4N004B) and
institutional grants from INSERM and CNRS, LNFCC des Bouches du Rhône. J. Pizarro-Cerdá received a fellowship from CNRS.
We thank J. Ewbank and S. Méresse for critically reading the
manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre
d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France. Phone: 33-4-91-26-94-66. Fax:
33-4-91-26-94-30. E-mail: gorvel{at}ciml.univ-mrs.fr.
Editor: P. J. Sansonetti
| |
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Infect Immun, May 1998, p. 2387-2392, Vol. 66, No. 5
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
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