1 Unité d'Immunophysiologie et Parasitisme Intracellulaire, Institut
Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
2 INSERM U411, UFR de Médecine Necker-Enfants Malades, Paris,
France
3 Laboratoire de Cinémicrographie INSERM, Le Vésinet, France
4 Unité de Biologie des Interactions Cellulaires, Institut Pasteur,
Paris, France
* Author for correspondence (e-mail: jantoine{at}pasteur.fr )
Accepted 14 March 2002
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Summary |
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Key words: Leishmania, Promastigote, Amastigote, Macrophage, Phagosome, Phagolysosome, Parasitophorous vacuole
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Introduction |
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The morphologies of mature parasite-harbouring compartments, known as
parasitophorous vacuoles (PVs), vary depending upon the Leishmania
species. Large communal PVs (L. amazonensis, L. mexicana) and tight
individual PVs (L. major, L. donovani) have been identified. In spite
of their very different aspects, they share some properties and features (for
a review, see Antoine et al.,
1998). PVs are acidic compartments containing certain lysosomal
enzymes. They are surrounded by a membrane enriched with late
endosomal/lysosomal proteins, such as rab7p, macrosialin, lamp-1, lamp-2 and
vacuolar H+-ATPase, and with molecules of the antigen-presentation
machinery (MHC class II and H-2M molecules) in IFN-
-treated
macrophages.
Compared with our knowledge of the events following internalization of
inert particles such as latex beads (for reviews, see
Desjardins, 1995;
Garin et al., 2001
), the
biogenesis of Leishmania-harbouring PVs is still poorly understood.
However, it has been recently described that the formation of PVs occurs
differently according to the stage of the parasites internalized, at least in
terms of kinetics (Desjardins and
Descoteaux, 1997
; Dermine et
al., 2000
). Thus, it has been shown that, after the phagocytosis
of cultured stationary phase L. donovani promastigotes by the
macrophage-like cells J774, the parasites are transiently located in
phagosomes with poor fusogenic properties towards late endocytic compartments
(Desjardins and Descoteaux,
1997
). In contrast, after their internalization, amastigotes are
found in compartments that rapidly fuse with late endocytic organelles
(Lang et al., 1994b
;
Dermine et al., 2000
). These
distinctive features of the early phagosomes could be linked to the
stage-specific expression of a high molecular weight glycolipid, the
lipophosphoglycan (LPG), on the plasma membrane of the promastigotes, which
may modify the fusion capacity of the phagosomal membrane, at least
temporarily. (Desjardins and Descoteaux,
1997
; Scianimanico et al.,
1999
; Dermine et al.,
2000
). Furthermore, it has been shown in these studies that most
of the phagosomes containing LPG-bearing promastigotes display an impaired
recruitment of the small GTPase rab7p, which is involved in the homotypic
fusions of late endosomes or lysosomes and in the heterotypic fusions of late
endosomes with lysosomes (Scianimanico et
al., 1999
). A long-lasting (more than 1 hour) accumulation of
filamentous (F) actin has also been noted around these phagosomes
(Holm et al., 2001
). It is
suspected that these anomalies reflect the transient lack of phagosome
maturation or are involved in the maintenance of this property. This fusion
restriction lasts several hours, which may allow the parasites to initiate
their differentiation into amastigotes, which are more adapted to the
lysosomal compartment. Such a proposal is consistent with the fact that L.
major promastigotes that are unable to synthesize LPG survive poorly
within mouse peritoneal macrophages
(Späth et al., 2000
).
The generality of this model was recently questioned by a study showing
that stationary phase LPG-deficient L. mexicana promastigotes bind to
and multiply within mouse peritoneal macrophages as efficiently as, or even
more efficiently than, wild-type promastigotes. This indicates that, at least
for this Leishmania species, LPG is not a determining factor for the
differentiation of promastigotes into amastigotes
(Ilg, 2000). Consequently, the
biological role of the transient restriction of fusion described for
phagosomes containing L. major or L. donovani is not
obvious, but it suggests that each Leishmania species has developed
its own establishment strategy for mammalian macrophages
(Turco et al., 2001
).
We used different parasite-host cell combinations to study the maturation
of Leishmania phagosomes. Previous studies on this topic were carried
out on unselected and thus heterogeneous (especially in terms of virulence)
stationary phase promastigotes. In contrast, we mainly focused on the early
events following the phagocytosis of metacyclic promastigotes, which are
pre-adapted to the encounter with mammals, in particular to intracellular
conditions. We used immunofluorescence confocal microscopy and quantitative
analyses to determine whether the kinetics of association of late
endosomal/lysosomal molecules to Leishmania-harbouring phagosomes
varies according to the Leishmania species or the parasitic stage put
into contact with the macrophages. We examined the association with phagosomes
of the following molecules: the TfR, which, at steady state, is localized in
early sorting and recycling endosomes (for a review, see
Gruenberg and Maxfield, 1995);
EEA1, a rab5p effector that is mainly detected on the cytosolic side of early
endosomes (Mu et al., 1995
);
rab7p, which appears to control the aggregation and fusion of late endocytic
structures/lysosomes (Chavrier et al.,
1990
; Bucci et al.,
2000
); macrosialin, a macrophage-specific membrane glycoprotein
belonging to the lamp family and mainly expressed in late endosomes
(Rabinowitz et al., 1992
;
Holness et al., 1993
); lamp-1,
a major protein constituent of late endosomal and lysosomal membrane (for a
review, see Hunzinker and Geuze,
1996
); cathepsins B and D, two acid hydrolases mainly concentrated
in macrophage lysosomes (Rodman et al.,
1990
; Claus et al.,
1998
); and MHC class II molecules, which are localized within
antigen-presenting cells, in compartments called MIIC that have all of the
characteristics of the late endosomes or lysosomes (for a review, see
Geuze, 1998
).
We conclude that young phagosomes containing L. amazonensis promastigotes or amastigotes or L. major promastigotes rapidly acquire a competence to fuse with late endosomes/lysosomes.
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Materials and Methods |
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Amastigotes of L. amazonensis strain LV79 (MPRO/BR/1972/M1841) and
of L. major strain NIH173 (MHOM/IR/-/173) were purified from the feet
of infected nude mice as described previously
(Antoine et al., 1989).
Metacyclic promastigotes of these two Leishmania strains were
obtained from amastigotes cultured at 26°C
(Courret et al., 1999
). They
were purified by negative selection using the peanut agglutinin (NIH173)
(Vector Laboratories, Burlingame, CA) or the monoclonal antibody (mAb) 3A1
(LV79) (Sacks et al., 1985
;
Courret et al., 1999
). L.
amazonensis stationary phase promastigotes submitted to the same cycle of
washings and centrifugations as metacyclic promastigotes during their
purification, but not incubated with the mAb 3A1, were also prepared.
Macrophage infections
Macrophages were obtained from BALB/c mice by in vitro differentiation of
bone marrow precursor cells in 24-well plates containing 12 mm diameter round
glass coverslips for light microscopy and scanning electron microscopy
studies, or in 35 mm culture dishes for transmission electron microscopy
studies. For microcinematography, precursors were deposited in 60 mm cultures
dishes containing 34x34 mm square coverslips. Cells were cultured in
RPMI 1640 medium (Seromed, Berlin, Germany) supplemented with 10% (v/v)
heat-inactivated fetal calf serum (Dutscher, Brumath, France), 50 U/ml
penicillin, 50 µg/ml streptomycin and 20% L-929 fibroblast-conditioned
medium. After 5 days at 37°C in a 5% CO2/95% air atmosphere,
non-adherent cells were removed and adherent macrophages were further
incubated in culture medium containing only 3% of conditioned medium. At this
step, cells were treated or not with rIFN- (10-25 U/ml, Genentech, San
Francisco, CA) for 18-24 hours before the addition of the parasites.
Macrophages were infected at a stationary phase promastigote-, metacyclic
promastigote- or amastigote-host cell ratio of 3:1 to 5:1. The parasites and
macrophages were quickly brought into contact by centrifugation at 20°C
(130 g, 5 minutes). Cultures were then incubated at 34°C (L.
amazonensis infections) or 37°C (L. major infections) for 30
minutes before being washed with Dulbecco's phosphate-buffered saline (PBS,
Seromed) to remove free parasites. Macrophages were fixed either immediately
(time point 30 minutes) or after various times (between 1 and 48 hours
post-infection). In some experiments, cultures were centrifuged as above after
the addition of the parasites and only incubated at 34 or 37°C for 5
minutes before fixation (time point 10 minutes). In this case, the cultures
were not washed before fixation. Control experiments ensured that
centrifugation of the parasites did not modify the characteristics of the
early events analysed in this study.
Loading of macrophages with endocytic tracers
Before infection, macrophages were incubated either for 2 hours at 37°C
with 2-3 mg/ml anionic, lysine fixable fluorescein dextran (FDex, average
Mr 10,000, Molecular Probes, Eugene, OR), or for 30
minutes at 37°C with 25 µg/ml horseradish peroxidase (HRP, RZ 3, Sigma
Chemical Co., St Louis, MO). Cells were then thoroughly washed with cold PBS
and chased for 140-160 minutes or overnight (17-20 hours) in tracer-free
medium.
Antibodies and fluorescent reagents
The mAb 3A1, a mouse IgG2b specific to the LPG of L. amazonensis
log phase promastigotes (Courret et al.,
1999), was provided by D. L. Sacks (Laboratory of Parasitic
Diseases, NIAID, Bethesda, MD). A rabbit immune serum raised against L.
mexicana leishmanolysin was obtained from P. Overath [Max Planck
Institute for Biology, Tübingen, Germany
(Bahr et al., 1993
)]. The mAb
RI7 217.1.3, a rat IgG2a specific for the mouse transferrin receptor [TfR
(Lesley et al., 1984
)], was a
gift from D. Ojcius (Institut Pasteur, Paris, France). Rabbit immune sera
specific for EEA1 and rab7p were provided by M. Zerial (Max Planck Institute
for Molecular Cell Biology and Genetics, Dresden, Germany). Before use, the
anti-rab7p immune serum was adsorbed on a L. amazonensis amastigote
lysate. Hybridoma cells secreting the FA/11 mAb, a rat anti-mouse macrosialin
IgG2a (Smith and Koch, 1987
),
were obtained from G. Koch (MRC Laboratory of Molecular Biology, Cambridge,
UK). The anti-mouse lamp-1 (CD107a) mAb 1D4B, a rat IgG2a
(Chen et al., 1985
), and the
biotin-conjugated anti-mouse I-Ad/I-Ed mAb 2G9 [rat
IgG2a (Becker et al., 1992
)]
were purchased from Pharmingen (San Diego, CA). Rabbit IgG specific to rat
cathepsin B or D and crossreacting with mouse cathepsin B or D were obtained
from B. Wiederanders (Friedrich-Schiller University, Jena, Germany) and H.
Kirschke (University of Halle, Halle, Germany). A Leishmania-specific
immune serum was prepared from L. amazonensis-infected BALB/c mice.
The mAb JES6-1A12, a rat IgG2a specific to mouse IL-2
(Abrams et al., 1992
), and a
normal rabbit serum, were used as controls for the specific primary Abs or
immune sera described above. Primary Abs associated with cell preparations
were detected by use of the following conjugates: fluorescein-labeled
F(ab')2 fragments of donkey anti-rat or anti-rabbit Ig; Texas
Red-labeled F(ab')2 fragments of donkey anti-mouse Ig
(Jackson ImmunoResearch Laboratories, West Grove, PA); and fluorescein-labeled
ExtrAvidin (Sigma). Alexa Fluor 488-phalloidin (Molecular Probes) was used to
stain F-actin.
Fluorescence microscopy
Macrophages were fixed with paraformaldehyde and then permeabilized
(Lang et al., 1994a). They
were labeled with primary Abs and fluorescent conjugates according to standard
procedures (Lang et al.,
1994a
). After simple immunolabelings, nucleic acids were stained
with propidium iodide (Lang et al.,
1994a
). Cell preparations were mounted in Mowiol (Calbiochem, San
Diego, CA) before observation under an Axiophot Zeiss epifluorescence
microscope or under a LSM 510 Zeiss confocal microscope (Carl Zeiss
Microscopy, Jena, Germany). Confocal microscopy images were acquired and
analysed by use of the 2.5 version of the LSM 510 software before being
exported to Adobe PhotoShop (Mountain View, CA).
Transmission electron microscopy
Infected macrophages exposed to HRP were fixed for 1 hour at room
temperature with 2.5% glutaraldehyde in 0.1 M sodium cacodylate, HCl buffer,
pH 7.2, containing 0.1 M sucrose. Cells were washed overnight at 4°C with
sucrose-containing cacodylate buffer and incubated with
3,3'-diaminobenzidine tetrachlorhydrate and H2O2
(Malmgren and Olsson, 1977)
before post-fixation with osmium and Epon embedding. Sections were examined
with a Jeol 100CXII electron microscope (Jeol Ltd., Akishima, Japan).
Scanning electron microscopy
Macrophages were exposed to L. amazonensis metacyclic
promastigotes (five parasites/host cell) for 10 minutes at 34°C. Cells
were then fixed overnight at 4°C with 2.5% glutaraldehyde in 0.1 M sodium
cacodylate, HCl buffer, pH 7.2, containing 0.1 M sucrose. After three washes
with sucrose-containing cacodylate buffer, cells were dehydrated in ethanol
and processed for scanning electron microscopy according to standard
protocols. Cells were examined at the Centre Inter-Universitaire de
Microscopie Electronique (CIME) Jussieu (Paris, France).
Time-lapse microcinematography
Coverslips with macrophages were mounted in observation microchambers known
as Rose's chambers (Rose,
1954), which were then placed on the stage of a thermostated
(34°C) Zeiss inverted microscope linked to an automated 16 mm Arriflex
camera. The camera was programmed to take one or two pictures per second for
30-60 minutes. The grabbing sequences started when the promastigotes were
placed in the Rose's chambers. The images were analyzed on a NAC
projector.
Online supplemental material
Movies 1 and 2 (see
http://jcs.biologists.org/supplemental
) correspond to Fig. 1A and B
and contain QuickTime sequences depicting the phagocytosis of metacyclic
promastigotes by macrophages. Images were captured every 0.5 seconds over the
course of 367 seconds (Movie 1) or 125 seconds (Movie 2).
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Results |
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Entry of L. amazonensis metacyclic promastigotes into
macrophages
After the parasites bound to the macrophages, either long tubular
pseudopods tightly encircling their cell body or their flagellum
(Fig. 1A,C) or ruffles
(Fig. 1B,D) were formed. Very
often, parasites were phagocytosed with the cell body entering first
(Fig. 1; see also Movies 1 and
2 at
http://jcs.biologists.org/supplemental
), but ingestion starting by the flagellum was also observed (data not shown).
In some cases, the parasites first interacted with the macrophages via their
flagellum (Fig. 1B, time points
0 to 15 seconds) but they rapidly turned around
(Fig. 1B, time point 16
seconds) and finally entered macrophages by the cell body
(Fig. 1B, time points 37
seconds to 61 seconds). We also observed the lateral attachment of the
parasites to the macrophage cell surface. In these cases, the plasma membrane
folds wrapped themselves around the parasites, and the different parts of the
latter were simultaneously internalized (data not shown). Complete
phagocytosis took about 3-9 minutes.
Polymerization of macrophage actin is a transient event during
metacyclic promastigote or amastigote phagocytosis
The metacyclic promastigotes and amastigotes of L. amazonensis
were internalized by an actin-dependent process as shown by the accumulation
of F-actin around the parasites during and after phagocytosis. To determine
whether the duration of F-actin accumulation varied with the parasitic stage,
macrophages were fixed at different times after infection, and F-actin was
stained with fluorescent phalloidin. At 10 minutes, about 40-55% of
metacyclics or amastigotes were surrounded by F-actin. At this stage, most of
the F-actin+-parasites were still in the process of phagocytosis.
After completion of the internalization process, the percentage of
F-actin+-parasites rapidly dropped to reach 5-10% at 1-2 hours
post-infection (Fig. 2A). At
the early time point, F-actin appeared generally as a thick ring around the
promastigote cell bodies and amastigotes or adopted the shape of a sleeve
around the promastigote flagella (Fig.
2B,C). With promastigotes, F-actin was often concentrated around
either the cell body or the flagellum and less frequently around both parts.
This suggests that F-actin sequentially polymerizes and depolymerizes along
the parasites during their internalization. Presence of F-actin in the ruffles
formed at the entry point of some parasites was also noted (data not
shown).
|
As with L. amazonensis, F-actin was detected around L. major metacyclic promastigotes in the process of phagocytosis. Thereafter, parasite-associated F-actin disappeared with a kinetics slower than that observed for L. amazonensis. Thus, 10 minutes, 30 minutes and 2 hours after adding parasites, 77.2, 48.0 and 24.7% of them were F-actin+, respectively. This, apparently, did not slow down the recruitment of endosome/lysosome `markers' into phagosomes (see below).
Kinetics of PV formation following metacyclic promastigote or
amastigote phagocytosis
All the parasites still present in the cultures were completely
internalized 30-60 minutes after the addition of L. amazonensis
promastigotes to macrophage monolayers pre-exposed or not to IFN-. Most
of them (about 70%) were elongated, with the cell body directed towards the
macrophage nucleus and the flagellum directed towards the periphery
(Fig. 3, group 1;
Fig. 4A). About 3% of the
parasites were in the opposite direction
(Fig. 3, group 3). The
remainder (about 30%) displayed no clear orientation and were in vacuoles
already located near to the macrophage nucleus
(Fig. 3, group 2). At this step
of the infectious process, most of the parasites were located in very long
(several tens of microns), narrow compartments, the membrane of which adopted
their exact shape (see below). The lumen of these organelles could not
normally be seen under the light microscope
(Fig. 4A). Similar compartments
were observed after infection of macrophages with L. major metacyclic
promastigotes (data not shown). At 5 hours post-infection, most of the
parasites had lost their long flagella and were located in smaller
compartments gathered around the macrophage nucleus
(Fig. 4C).
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|
After amastigote phagocytosis, parasites were initially localized in peripheral, tight, ovoid vacuoles that then rapidly reached the macrophage cell center (Fig. 4E). With L. amazonensis, which induces the formation of huge PVs, we noted that the time elapsed before the enlargement process began varied according to the parasitic stage used. The first signs of dilatation were seen about 12-18 hours after infection with metacyclic promastigotes and 2 hours after infection with amastigotes (Fig. 4F). At later times, large vacuoles containing numerous parasites began to appear. They displayed a similar size in macrophages infected for 18/24 hours with metacyclics or for 5 hours with amastigotes (Fig. 4D,G).
Kinetics of endosome/lysosome `marker' recruitment into phagosomes
containing initially metacyclic promastigotes
It has been reported that huge communal PVs housing L. mexicana or
L. amazonensis are formed by the fusion of small individual vacuoles
between them and with compartments of the endocytic pathway (for a review, see
Antoine et al., 1998). To
determine whether the different kinetics of PV formation described above were
linked to the capacity of the early phagosomes to fuse with endocytic
compartments, we studied the acquisition by these organelles of various
soluble and membrane molecules known to be preferentially associated with
early endosomes or with late endosomes/lysosomes (TfR, EEA1, rab7p,
macrosialin, lamp-1, cathepsins B and D, and MHC class II molecules). We
initially focused on the association of these molecules with phagosomes formed
after metacyclic entry.
In macrophages that had been pre-treated with IFN-, no more than
10-15% of the phagosomes formed after internalization of L.
amazonensis metacyclics displayed the early endosome `markers' TfR and
EEA1, even at the earliest time points (10-30 minutes)
(Fig. 5A,B). In contrast, at 30
minutes, about 95% of these compartments already contained macrosialin and
lamp-1, about 75 and 90% of them had acquired cathepsins B and D, respectively
and 70% were rab7p-positive (Fig.
5A,B; Fig. 6A-D).
In macrophages expressing MHC class II molecules, about 50% of the
phagosomes/phagolysosomes contained class II at 30 minutes post-infection. At
this infection stage, the soluble and membrane `markers' detected in
phagosomes were both closely associated with the promastigotes and surrounded
the entire parasites, including the flagella
(Fig. 6). Later on,
phagolysosomes were virtually all positive for macrosialin and lamp-1 from 1
hour post-infection (Fig. 5A,B;
Fig. 6E). The percentage of
rab7p+ and of class II+ parasite-containing organelles
gradually increased, reaching 100% at 48 hours post-infection. About 60-80% of
the phagosomal compartments were cathepsin-positive between 2 and 18 hours, a
time period during which enzymes were clearly detected in the lumen of the
still small PVs (Fig. 5A,B;
Fig. 6F). The percentage of
cathepsin+-compartments dropped considerably at 48 hours
(Fig. 5A,B) but this can easily
be explained by the fact that soluble molecules like these enzymes are not
retained in the large PVs during fixation.
|
|
As the results described above were obtained using IFN--pre-treated
macrophages as host cells, we next examined whether the IFN-
treatment
could modify the kinetics of PV maturation. Comparison of
Fig. 5A, B and C clearly
indicates that, at the dose used, IFN-
had no effect on the analyzed
parameters.
The metacyclic promastigotes used in the preceding experiments were
prepared from stationary phase promastigotes by negative selection using the
mAb 3A1 (Courret et al.,
1999). To see whether the treatment applied to the parasites could
influence the biogenesis of the phagosomal compartments, a side-by-side
comparison of phagosomes containing initially either stationary phase or
metacyclic promastigotes of L. amazonensis was undertaken.
Fig. 7 shows that phagosome
formation, measured by the appearance in the phagosomal membrane or lumen of
the transferrin receptor, rab7p, lamp-1 or cathepsin B, was strictly similar
in both conditions of infection. Study of the association of EEA1, macrosialin
and MHC class II molecules ended at the same conclusion (data not shown).
|
Likewise, similar data were obtained when metacyclic promastigotes of L. major, a species that does not induce the formation of large PVs, were used to initiate infection (Fig. 8). This suggests that the rapid acquisition of late endosome/lysosome `markers' by promastigote-containing organelles is a Leishmania species-independent process.
|
As negative controls, promastigote-infected macrophages were incubated with an irrelevant mAb or normal rabbit serum instead of specific reagents. We also incubated purified metacyclic promastigotes with macrophage organelle-specific Abs. No staining or a very weak background staining could be detected on these preparations (data not shown).
Fusion of FDex- or HRP-loaded late endocytic compartments with
phagosomes harbouring initially metacyclic promastigotes
Previous data clearly showed that, at 30 minutes post-infection with
metacyclic promastigotes, most parasite-containing organelles displayed
molecules of the late endocytic compartments. We demonstrated that the
recruitment of these molecules occurred through the fusion of late endocytic
compartments with phagosomes as follows. Before infection with metacyclics,
macrophages were pre-incubated with FDex for 2 hours, washed and then
incubated for 2-3 hours or overnight to allow the preferential accumulation of
the fluorescent molecules in late endosomes or lysosomes, respectively.
Regardless of the time of chase, FDex rapidly appeared in compartments
containing initially metacyclic promastigotes. For example, at 30 minutes,
90-100% and 80-85% of these organelles were fluorescent after infection with
L. amazonensis and L. major metacyclics, respectively
(Fig. 9A). At this stage,
phagosome-associated FDex was localized around the cell body and the flagellum
of the parasites and adopted their exact shape
(Fig. 9B,C). The percentage of
fluorescent parasite-containing organelles descreased slightly at later times
of infection, which could be due to a redistribution of fluorescent molecules
or, in the case of L. amazonensis- containing compartments, to the
loss of soluble FDex from the enlarging organelles during fixation. At this
stage, FDex associated with the small individual PVs was clearly localized in
the lumen of the organelles (Fig.
9D,E).
|
Very similar results were obtained with macrophages that had been pre-incubated for 30 minutes with HRP, chased for 160 minutes or overnight and then infected with L. amazonensis metacyclic promastigotes. Electron microscopy analysis of these cells showed that HRP was present in the lumen of the parasite-containing compartments 30 minutes after infection (Fig. 10A). Typical images of fusion of HRP-loaded late endosomes/lysosomes with phagosomes were observed (Fig. 10B,C). As controls, macrophages that had not been incubated with HRP were infected and then processed as above. No staining could be detected under these conditions.
|
Kinetics of endosome/lysosome `marker' acquisition by phagosomes
formed after amastigote ingestion
PV maturation assessed by the acquisition of early endosome, late endosome
or lysosome `markers' was studied after the internalization of L.
amazonensis amastigotes. The kinetics of `marker' recruitment were rather
similar to those we measured after the ingestion of metacyclic promastigotes
(Fig. 11). However, small
differences were noted (compare Figs
5 and
7 with
Fig. 11). The percentage of
rab7p+ phagocytic compartments was slightly higher after amastigote
internalization. For example, at 30 minutes and 12 hours post-infection, 90%
and 100% of the amastigote-harbouring compartments displayed rab7p in their
membrane (Fig. 11;
Fig. 12A), respectively,
whereas, at the same times after infection with metacyclics, rab7p was
detected on only 70 and 75% of the parasite-containing compartments,
respectively. Similar results were obtained for lamp-1 and macrosialin with
both parasitic stages (Fig.
11; Fig. 12B),
except that the percentages of positive compartments were higher at 10 minutes
post-infection with amastigotes, indicating a slightly faster acquisition of
the molecules. Likewise, class II molecules were acquired more quickly after
infection with amastigotes. In contrast, although most amastigote-harbouring
phagosomes had cathepsin B in their lumen at 30 minutes post-infection
(Fig. 11, Fig. 12C), this enzyme, at
later times, was more difficult to detect in these organelles than in
compartments formed after phagocytosis of metacyclics. This is probably
because of an earlier enlargement of the PVs which, during fixation, lose a
part of their soluble content. Negative controls performed as described
previously for promastigote-infected macrophages gave only weak background
staining (data not shown).
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![]() |
Discussion |
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Most of the studies on parasite entry into macrophages have concerned the
multiple receptor-ligand systems involved in the binding and internalization
of promastigotes and amastigotes (Alexander
and Russell, 1992; Guy and
Belosevic, 1993
; Love et al.,
1993
; Peters et al.,
1995
) and only a few reports have looked at the phagocytic events
(for a review, see Chang,
1983
; Rittig et al.,
1998
). In particular, it is not yet clear whether
Leishmania, which are strongly polarized cells, are bound
preferentially by a pole or not. Likewise, it is not known whether the primary
binding sites are the first to be internalized. Our microcinematographic data
indicate that the first interactions between L. amazonensis
metacyclic promastigotes and mouse bone-marrow-derived macrophages occur
through the parasite flagellum, the cell body or the entire parasite. We also
found that the binding of the flagellum can be followed by phagocytosis
starting by the cell body. Scanning electron microscopy of similar cell
preparations further indicated that 10 minutes after adding parasites to
macrophage monolayers, flagella are free or have established contacts with
macrophage plasma membranes, but the only partial internalization processes
observed involved parasite bodies. The fact that shortly after promastigote
internalization (30-60 minutes), most of the parasites (
70%) are
elongated with their cell bodies directed towards the macrophage nucleus and
their flagella directed towards the plasma membrane also suggests that
promastigote phagocytosis mainly occurs in a polarized manner with the cell
body entering macrophages first. Although unlikely, a re-orientation of the
parasites after phagocytosis or of the parasite-containing phagosomes just
after their formation could also explain this preferential polarity of the
intracellular parasites. Similar findings were obtained with L. major
metacyclics as well as with unselected L. amazonensis stationary
phase promastigotes. This indicates that this phenomenon is not linked to a
particular Leshmania species and is not the consequence of the
treatments used to obtain homogeneous metacyclics. Furthermore, identical
patterns were noted in macrophages that had been pre-treated with IFN-
and those that had not. This supports the view that binding, phagocytosis and
the first steps of phagosome biogenesis are not greatly influenced by this
cytokine, at least at low concentrations. Our data are consistent with earlier
publications that showed that L. donovani promastigotes predominantly
enter mouse or hamster macrophages by their posterior end
(Pulvertaft and Hoyle, 1960
;
Akiyama and Haight, 1971
).
This method could not be used to determine whether amastigotes also bind
and are phagocytosed in a polarized manner because the polarity of this
parasitic stage is not as easy to assess by light microscopy as that of
promastigotes. A quantitative electron microscopic study will be needed to
examine this point. It is noteworthy that, within PVs, parasites are bound to
PV membrane through their posterior pole
(Benchimol and De Souza, 1981;
Antoine et al., 1998
;
Courret et al., 2001
). It
would thus be very interesting to determine whether this particular area of
the parasite plasma membrane is also used as a primary binding site for
promastigotes and amastigotes with the macrophage plasmalemma or whether this
interaction is engaged in the first steps of phagocytosis.
Morphologically distinct phagocytic events were observed after promastigote
binding, including the formation of tubular pseudopodia in close contact with
the parasites, as already noted by others
(Chang, 1979;
Rittig et al., 1998
), and
ruffles. The fact that, shortly after internalization, most of the parasites
are in very long, close-fitting phagosomes that exactly follow their outline
suggests that ingestion mainly occurs by a zipper mechanism sequentially
engulfing the different parts of the parasites. Otherwise, phagocytosis
following ruffle extension could be at the origin of the rare phagosomes
displaying a distinct lumen, early after their formation (such a phagosome is
visible in Movie 1). It is not yet known whether different receptor-ligand
interactions are at the origin of the various phagocytic mechanisms observed.
In any case, these mechanisms as well as amastigote phagocytosis are dependent
upon actin polymerization, as shown by the rapid appearance of F-actin around
the parasites. Actin is then rapidly shed from the newly formed phagosomes
harbouring either L. amazonensis metacyclic promastigotes or
amastigotes and at 30 minutes post-infection no more than 10-20% of the
phagosomes are still surrounded by F-actin. The data concerning amastigote
infections are consistent with those of Love et al., who showed that
periphagosomal actin is rapidly lost when parasites are internalized
(Love et al., 1998
). In
contrast, we found no evidence of a lasting accumulation of actin around
promastigote-containing compartments (more than 60 minutes) as is the case, in
J774 macrophages, for L. donovani-harbouring phagosomes
(Holm et al., 2001
). These
authors found that the persistence of actin is LPG-dependent, which is
difficult to reconcile with our data showing that the kinetics of
periphagosomal actin dissociation are similar after ingestion of L.
amazonensis promastigotes or amastigotes, which express LPG or are devoid
of LPG, respectively (Courret et al.,
2001
). Whether this discrepancy is due to the different
Leishmania species/host cells used in these experiments is unclear
but it is interesting to note in this respect that we found a higher
percentage of L. major metacyclic promastigotes surrounded by actin
at the three time points examined after adding parasites to macrophages,
namely 10 minutes, 30 minutes and 2 hours.
After internalization, all of the promastigotes are located in
membrane-bound compartments, including those that are in a phagosome not
detected by phase-contrast microscopy. Indeed, at 30 minutes to 1 hour
post-infection, all parasites are delineated by the integral membrane proteins
lamp-1 and macrosialin, indicating the presence of an endosomal/lysosomal
membrane. These data do not agree with previous descriptions of cytosolic
promastigotes within phagocytic cells
(Akiyama and Haight, 1971;
Rittig et al., 1998
). Even if
such a localization could possibly occur, for example after the rupture of
phagosomes, it must be extremely rare and must be formally proven. The
formation of very long phagosomes, the membrane of which tightly follows the
outline of promastigotes, has never been described before. Nevertheless, these
results are reminiscent of the early stages of infection of fibroblasts or
epithelial cells by Trypanosoma cruzi trypomastigotes, which use a
very different mode of entry (Hall et al.,
1992
). It is currently not known how the integrity of these
slender compartments containing highly motile parasites is maintained, but
their binding to cytoskeletal elements could be involved. At later time points
(2-5 hours), the phagosomal membrane remains tightly associated with the
parasites but the compartments become shorter. This reduction in size
correlates with the progressive loss of the flagellum, suggesting that the
parasite remodeling is accompanied by the removal of phagosomal membrane.
Our most important finding is that promastigote-containing phagosomes can
fuse with late endocytic compartments very quickly after their formation, as
shown by (1) the rapid acquisition by these organelles of both soluble and
membrane molecules mainly associated with late endosomes/lysosomes, namely
cathepsin B, cathepsin D, macrosialin, lamp-1 and rab7p; and (2) the transfer
of the content of late endosomes/lysosomes previously loaded with FDex or HRP
in the lumen of these organelles. This fusion capacity is consistent with the
fact that F-actin is rapidly shed from the newly formed phagosomes. It is
noteworthy that, compared with the other molecules that are acquired almost
synchronously, except for MHC class II molecules, rab7p is present on a higher
percentage of phagosomes at the earliest time point examined (10 minutes).
This implies that rab7p is recruited sooner, and possibly by another way, for
instance from the cytosol or from organelles other than late endocytic
compartments. The presence of rab7p on a high percentage of early phagosomes
(70 to 80%) is consistent with their ability to fuse with late endocytic
compartments as recent data indicate that this protein is important for late
endosome/lysosome fusion events
(Méresse et al., 1995;
Papini et al., 1997
;
Bucci et al., 2000
). Whereas
rab7p associates only with latex bead phagosomes transiently (between 10 and
200 minutes post-internalization)
(Scianimanico et al., 1999
),
the percentage of parasite-containing compartments with rab7p on their surface
progressively increases with time and reaches about 100% at 48 hours. It is
not yet known whether this occurs by a deregulation of rab7p function but
these results suggest that PVs can retain their ability to fuse with late
endocytic compartments for a long time, which could provide the parasites with
a means of getting nutrients. The persistent expression of membrane-associated
rab proteins has been described for phagosomes harboring other intracellular
microorganisms. For example, Mycobacterium bovis BCG phagosomal
compartments retain rab5p on their surface and, perhaps as a consequence of
that, the capacity to fuse with early endosomes. However, these phagosomes do
not acquire rab7p and are unable to fuse with late endocytic compartments
(Via et al., 1997
).
The slower kinetics of class II molecule association with parasite-containing compartments could be due to the fact that these molecules are located in subsets of late endosomes/lysosomes (MIIC) with different fusion capacities towards phagocytic compartments. On the other hand, at all time points, only very low numbers of phagocytic compartments were found to display the early endosome `markers' TfR and EEA1. This indicates that either low or, more likely, very transient interactions occur with early endocytic compartments just after completion of phagocytosis and that they are followed by rapid recycling of early endosome-associated molecules.
The validity of our conclusions concerning PV biogenesis in
promastigote-infected macrophages can be extended to phagocytic compartments
containing different Leishmania species (L. amazonensis, L.
major) as well as to PVs present in macrophages under different states of
activation (macrophages that were or were not pre-treated with IFN-).
Finally, the characteristics of PV formation are not biased by the treatment
used to purify metacyclic forms. Indeed, we checked that phagosomes harbouring
initially either L. amazonensis metacyclic or unselected stationary
phase promastigotes behaved similarly in terms of late endosome/lysosome
`marker' acquisition.
We also observed an early association of late endosome/lysosome `markers'
with parasite-containing compartments after the internalization of
amastigotes. The recruitment of the different molecules examined was slightly
more efficient than after ingestion of promastigotes because, at 10 minutes
post-infection, a higher percentage of phagosomes displayed rab7p,
macrosialin, lamp-1 and MHC class II molecules in their membrane. At 30
minutes, the differences become blurred in terms of percentage of positive
phagosomes but the immunolabeling intensity of the different molecules was
generally slightly weaker for phagosomes/phagolysosomes harbouring initially
promastigotes. Together, these data show that, after internalization of
promastigotes and amastigotes, early phagosomes rapidly fuse with late
endocytic compartments but that the rate of fusion is higher after amastigote
internalization. This difference could be due to a lower association of rab7p
with phagocytic compartments harbouring initially metacyclic promastigotes.
This was suggested by Scianimanico et al. to explain the fusion properties of
phagosomes containing L. donovani promastigotes
(Scianimanico et al., 1999),
despite the fact that their results were clearly different from ours. These
authors showed that these organelles, in contrast to phagocytic compartments
containing amastigotes or LPG-deficient promastigotes, have limited
interaction with late endocytic organelles for several hours
(Desjardins and Descoteaux,
1997
; Scianimanico et al.,
1999
; Dermine et al.,
2000
). The origin of this discrepancy is not clear, but the very
different characteristics of the host cells used (J774 macrophages vs
bone-marrow-derived macrophages) are an important point to consider.
In conclusion, we have demonstrated that both amastigote-and
metacyclic-containing phagosomes interact with late endosomes/lysosomes in the
minutes following parasite phagocytosis. Our phase-contrast microscopy study
of L. amazonensis-infected macrophages also showed that the
enlargement of the PVs harbouring this Leishmania species is delayed
when promastigotes are used to initiate infection. Thus, as suggested by
Desjardins, Descoteaux and colleagues, PV formation may be parasite
stage-dependent, at least with certain Leishmania species. However,
in our experimental conditions, the events at the origin of these differences
seem to occur after the fusion of early phagosomes with late
endosomes/lysosomes. As the formation of the huge PVs is due to the fusion of
several individual vacuoles and to the fusion of these vacuoles with
compartments of the endocytic pathway, our data suggest that the kinetics of
PV formation can be modulated either by the release (1) shortly after
phagocytosis, of promastigote-derived molecules that inhibit these processes
[e.g. LPG as proposed previously (Dermine
et al., 2000)]; or (2) at later times of infection, of
amastigote-specific molecules that alter the balance between fusion and
fission events in favor of fusions. In this respect, it has been suggested
that the proteophosphoglycan secreted by L. mexicana amastigotes in
the PV lumen could be involved in the expansion of PVs
(Peters et al., 1997
). The
co-infection of macrophages with promastigotes and amastigotes of L.
amazonensis should allow us to determine whether the molecules
expressed/synthesized by the promastigote/intermediate stages transiently
block the enlargement of PVs in our experimental conditions.
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Acknowledgments |
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Footnotes |
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References |
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