3065
Journal of Cell Science 107, 3065-3076 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Fusion between large phagocytic vesicles: targeting of yeast and other
particulates to phagolysosomes that shelter the bacterium Coxiella burnetii
or the protozoan Leishmania amazonensis in Chinese hamster ovary cells
Patricia S. T. Veras1,*,†, Chantal de Chastellier2, Marie-Françoise Moreau1, Veronique Villiers3,
Monique Thibon3, Denise Mattei4 and Michel Rabinovitch1,*,‡
1Unité
d’Immunoparasitologie, Institut Pasteur, and CNRS URA 361, 2INSERM Unit 411, Laboratoire de Microbiologie, UFR de
Médecine Necker-Enfants Malades, 75730 Paris, Cedex 15, France
3Laboratoire des Rickettsiales et Chlamidiales, Institut Pasteur, 4Unité de Parasitologie Expérimentale and CNRS URA 361,
Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France
*Authors for correspondence
†Present address: Serviço de Immunologia, HUPES, Universidade Federal da Bahia, 40.140 Salvador, Bahia, Brasil
‡Present address: Rockefeller University, 1230 York Avenue, New York, NY, 10021, USA
SUMMARY
This report examines the fusion of phagocytic vesicles with
the large phagolysosome-like vacuoles induced in Chinese
hamster ovary cells by the bacterium Coxiella burnetii or
the Protozoan flagellate Leishmania amazonensis. Infection
by these organisms is compatible with cell survival and
multiplication. Fusion was inferred from the transfer of
microscopically identifiable particles from donor to target
vesicles. Donor vesicles contained heat-killed yeast,
zymosan, β-glucan or latex beads taken up by the host cells.
Yeast and zymosan were also coated with Concanavalin A
to increase their uptake by the cells (Goldman, R., Exp. Cell
Res. 104, 325-334, 1977). Particle localization, routinely
ascertained by phase-contrast microscopy, was confirmed
by confocal laser fluorescence and by transmission electron
microscopy.
Coxiella vacuoles admitted all the particles tested and
transfer took place whether the particles were given to the
cells prior to or after infection. Transfer of uncoated or
Concanavalin-A-coated yeast or zymosan was dependent
on the number of particles ingested and on the incubation
period (between 2 and 24 hours). Furthermore, the transfer
step was quite efficient, since over 85% of the particles
ingested entered Coxiella vacuoles at all particle to cell
ratios examined. The fraction of uncoated or Concanavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at
higher particle loads. In addition, only rarely did latex
beads enter these vacuoles. The models proposed may be
useful for the delineation of biochemical and molecular
mechanisms involved in the fusion of large phagocytic
vesicles and the modulation of the latter by cellular and
pathogen-derived signals.
INTRODUCTION
tive information on the fusion between late phagocytic vesicles
in mammalian cells.
Fusion of phagocytic vesicles has been inferred from the
colocalization in single vesicles of particles separately internalized (Oates and Touster, 1978). Since vesicles that contain
rapidly degradable particles are short lived, non-degradable or
only slowly degradable particles may be preferable for fusion
studies. However, such particles are often enclosed in relatively tight vacuoles, making it difficult to quantify particle
colocalization in intact cells. Fusion is more easily detected if
at least one of the partner vesicles is sufficiently spacious to
accommodate the particles contributed by the other. This
condition is fulfilled by the rather stable and large phagolysosome-like vesicles induced by certain intracellular pathogens
(Veras et al., 1992). In this study we have used two such
organisms, the protozoan flagellate Leishmania amazonensis,
Phagocytosis begins with the adhesion of a particle to the
surface of a cell, followed by cytoskeleton-dependent
spreading of the plasma membrane and the underlying cell
cortex over the particle (Greenberg and Silverstein, 1993).
Once the pouch is sealed, the particle finds itself within a moderately acidified vesicle known as a phagosome (Mellman et
al., 1986). Phagosomes fuse with early endosomes, prelysosomes and lysosomes (Desjardins et al., 1994; Harding and
Geuze, 1992; Lang et al., 1988; Mayorga et al., 1991; Rabinowitz et al., 1992; Steinman and Moberg, 1994). Maturation
of phagosomes to phagolysosomes thus involves multiple
fusion events, some of which have been reproduced in isolated
vesicle preparations (Pitt et al., 1992a,b). However, possibly
because of methodological difficulties, there is little quantita-
Key words: Chinese hamster ovary (CHO) cells, Coxiella burnetii,
Leishmania amazonensis, phagocytosis, phagolysosomes, vesicle
fusion
3066 P. S. T. Veras and others
agent of cutaneous leishmaniasis (Wilson, 1993) and the
bacterium Coxiella burnetii, agent of Q fever (Williams, 1991;
Williams et al., 1992). Dividing macrophage-like and other
cells infected with these pathogens can survive, phagocytose
and multiply in culture (Baca and Paretsky, 1983; Chang,
1980; Roman et al., 1986).
C. burnetii and L. amazonensis are sheltered in
macrophages within large, easily identified, rather stable,
acidified and hydrolase-rich vacuoles (Alexander and
Russell, 1992; Antoine and Prina, 1992; Burton et al., 1978;
Maurin et al., 1992a). Furthermore, both vacuoles were
shown to fuse with lysosomes containing marker molecules
or colloids (Akporiaye et al., 1983; Alexander and Russell,
1992; Rabinovitch et al., 1985; Shepherd et al., 1983). There
is far less information on the vacuoles that shelter L. amazonensis in cells other than macrophages. We have shown by
light microscopic cytochemistry that horseradish peroxidase
was transferred to some L. amazonensis and C. burnetii
vacuoles in CHO cells. Furthermore, these vacuoles appeared
to be acidified, since they rapidly concentrated Neutral Red
added to the culture medium (Veras and Rabinovitch, unpublished results).
We have previously shown that yeast-derived particles can
be transferred, presumably by vesicle fusion, to the vacuoles
induced by L. amazonensis in host macrophages. Transfer was
vectorial (from donor vesicles to parasite vacuoles) and apparently selective, since other particles such as latex beads did not
enter Leishmania vacuoles (Veras et al., 1992). We postulated
that the selectivity reflected the recognition of the yeast by
macrophage phagocytic receptors. In the present study we
compared the transfer of yeast and other particles to the
vacuoles induced by L. amazonensis or by phase II C. burnetii
in Chinese hamster ovary (CHO) cells. These cells do not
express Fc, complement (CR3), β-glucan and mannose/fucose
receptors that are found in macrophages and are thus more
restricted in the range of particles they can phagocytose
(Greenberg and Silverstein, 1993).
We report here that all particles tested (heat-killed yeast,
zymosan, β-glucan or latex beads) were delivered with high
efficiency, most likely by vesicle fusion, to the vacuoles
occupied by C. burnetii. Yeast and zymosan were also treated
with Concanavalin A to increase their uptake by the cells
(Goldman, 1977). Transfer occurred whether CHO cells
received the particles prior to or after infection. In contrast,
transfer of particles to Leishmania vacuoles was not only less
frequent but the efficiency of particle transfer was much lower.
The models described are potentially useful in studies of the
mechanisms of the fusion of large vesicles and its control by
pathogen- or host cell-derived signals, as well as by effector
molecules or pharmacological agents.
MATERIALS AND METHODS
Cell culture
CHO cells were provided by B. Goud from the Pasteur Institute. Cells
were grown in T75 flasks at 37°C in a 6% CO2 atmosphere in RPMI
1640 medium supplemented with 10% FCS, 2 g/l sodium bicarbonate, 20 mM HEPES, 2 mM glutamine and 20 µg/ml gentamicin
(complete medium). Cells detached with trypsin-EDTA (Gibco) were
plated on 12 mm coverslips distributed in 24-well plates. Each well
contained 0.5 ml of RPMI supplemented with 5% FCS, 20 mM
HEPES, 2 mM glutamine (assay medium) and, unless otherwise
noted, plates were kept at 34°C in an air atmosphere. In the absence
of added bicarbonate and CO2 in the culture medium, CHO cells multiplied, albeit at a lower rate.
Coxiella burnetii
Non-confluent 1-2 day cultures of Vero cells were maintained at 37°C
in MEM supplemented with 5% FCS, 2.2 g/l sodium bicarbonate and
25 mM HEPES. The flasks were washed in HBSS, and suspensions
of C. burnetii phase II, Nine Mile strain, were added in 30 ml of fresh
medium. After incubation for 5 days at 35°C, glass beads were added
to the medium and the cultures were shaken to disrupt the cells. The
medium containing broken cells and bacteria was distributed in 5 ml
samples, which were frozen and kept at −80°C. Coxiella phase I
organisms, obtained from infected animals, are highly infective for
humans. However, phase II organisms of the Nine Mile strain carried
in cell cultures are avirulent and killed by normal human serum. In
addition, reversion to phase I has not been documented (Hackstadt,
1990; Williams, 1991).
Leishmania amazonensis
Amastigotes of L. amazonensis LV79 (strain designation
MPRO/BR/72/M1841), serially transferred into the foot pads of 2025 g body weight female Balb/c mice (Animal Facility of the Pasteur
Institute), were harvested as described (Rabinovitch et al., 1986).
Parasites were suspended in RPMI 1640 supplemented with 5% FCS,
50 µg/ml gentamicin and 25 mM MOPS ((3-N-morpholino) propane
sulfonate), pH 7.3, kept at 34°C in an air atmosphere and used within
48 hours.
Particles
Dehydrated bakers’ yeast (Saccharomyces cerevisiae) and zymosan
A were from Sigma Chemical Co. Yeast particles were suspended in
Ca2+- and Mg2+-free PBS, autoclaved, and washed 3 times with sterile
PBS by centrifugation. Zymosan A (Z) particles were suspended in
PBS, incubated for 30 minutes in a boiling water bath and washed
twice with sterile PBS. Both particles were resuspended in PBS at
2.5×108 per ml and stored at −20°C. Prior to addition to the cultures,
heat-killed yeast (Y) or Z stocks were disaggregated by passage
through 26G × 1/2 inch needles at least 20 times. For fluorescence
studies Z was derivatized with 1 mg/ml fluorescein isothiocyanate
(Sigma Chemical) (Z-FITC) in 0.1 M K2CO3, overnight at 4°C,
quenched with 50 mM NH4Cl, extensively washed with PBS and
stored in the same buffer at −20°C. To increase ingestion of Y and Z
by fibroblasts (Goldman, 1977) particles were treated with 5 to 100
µg/ml Concanavalin A (Con A) (Sigma Chemical Co, St Louis, MO)
for 2 hours at room temperature, followed by 16 hours at 4°C, washed
3 times in PBS and stored at −20°C. β-Glucan particles were prepared
from bakers’ yeast as described (Veras et al., 1992), washed 4 times
with sterile PBS, suspended in PBS at 1.5×108 particles/ml and stored
at −20°C. Polystyrene carboxylated beads (0.8 µm and 2.02 µm; 10%,
v/v) were from Dow Diagnostics (Indianapolis, IN), and fluorescent
non-carboxylated beads (1 µm; 2.5%, v/v) from Polysciences (Warrington, PA). Beads were directly diluted 1:10 or 1:100 in assay
medium and added to the cultures at a final dilution of 1:200 and
1:1,000.
Infection of CHO cells with C. burnetii
CHO cells were plated on coverslips for 4 or 24 hours. The medium
was removed and 0.2 ml of Coxiella cell lysates added per well. The
plates were centrifuged for 30 to 60 minutes at 360 g at 34°C or, alternatively, incubated for 2 hours at 35°C. RPMI assay medium (0.3 ml)
was added and the plates kept in an air atmosphere. The medium was
changed 4 hours later and after overnight incubation the cultures were
washed twice and the medium was replaced again.
Targeted fusion of phagocytic vesicles 3067
Infection of CHO cells with L. amazonensis
CHO cells plated as above were infected with an estimated multiplicity of 4 to 5 amastigotes per cell in assay medium, washed the
next day and further incubated in the same medium for 1 to 2 days at
34°C in an air atmosphere.
Particle uptake by uninfected or infected CHO cells
In most experiments particles were added to the cultures 2 days after
infection with C. burnetii or with L. amazonensis. Cultures were
washed in HBSS supplemented with 0.3 g/l sodium bicarbonate and
gentamicin-free assay medium was added. Particles were added at the
following estimated multiplicities. Y and Z, 20 to 50 per CHO cell;
Y-Con A and Z-Con A, 10 to 20 per CHO cell; and β-glucan particles,
80 to 100 per CHO cell. Latex beads were added to a final dilution of
1:500 to 1:1,000. Cells were incubated with the particles in bicarbonate-free assay medium in an air atmosphere at 34°C. When
indicated, cells received particles in assay medium supplemented with
2.0 g/l sodium bicarbonate in 6% CO2 atmosphere at 34°C. Uptake of
Z or Z-Con A in bicarbonate-free medium was similar to that in cells
grown in bicarbonate-sufficient medium. However, whereas transfer
of particles to Coxiella vacuoles was unaffected by the absence of
bicarbonate, transfer of these particles to Leishmania parasitophorous
vacuoles (PVs) was enhanced by bicarbonate in a concentrationdependent fashion (results not shown). After overnight incubation
with the particles, cultures were washed 5 to 8 times, chased for the
time periods indicated in Results, washed in PBS and fixed in 1% glutaraldehyde in PBS. In other experiments CHO cells were plated on
coverslips and incubated with the particles prior to infection. One day
after plating, cultures were incubated with the particles and, 18 hours
later, cells were chased for 2 to 4 hours in particle-free medium. The
cells were then infected with either Coxiella or Leishmania and fixed
after 24 or 48 hours. Coverslips bearing the cells were inverted over
60% glycerol in water onto standard microscope slides. Preparations
were examined by phase-contrast and Nomarski differential interference microscopy. Infected cells were scored for uptake of Y or Z, for
the numbers of particles taken up and for the numbers of particles
transferred to Coxiella or to Leishmania vacuoles. Whenever possible,
between 100 and 200 infected cells that contained particles were
scored per coverslip. Fewer cells were scored when particles were
taken up by only a small proportion of infected cells.
Confocal laser fluorescence microscopy
CHO cells infected with Coxiella or Leishmania were incubated with
Z-FITC or Z-Con A-FITC, or with 1.0 µm fluorescent latex beads, for
8 to 24 hours and fixed for 30 minutes at room temperature in 4%
paraformaldehyde in PBS. Fixed coverslips were washed three times
with PBS and nonspecific fluorescence was quenched with 50 mM
NH4Cl. Cells were stained by turning the coverslips over 10 µl of 50
ng/ml propidium iodide, washed twice in PBS and mounted in 0.05%
p-phenylenediamine in 50% glycerol. Labelled cells were analysed
under a ×63 apochromatic oil immersion lens in a Confocal Laser
Scanning Microscope (Wild-Leitz Instruments, Heidelberg,
Germany) that uses an argon-krypton laser operating in multi-line
mode, at excitation wavelengths of 488 nm and 567 nm. Emission
filters of 535 nm and 610 nm were used for fluorescein and propidium
iodide, respectively. From each field 16 optical sections taken at
intervals of 0.5 to 0.6 µm were collected and stored.
Electron microscopy
Semi-confluent cultures of CHO cells plated on T25 tissue culture
flasks were infected with Coxiella or Leishmania for 2 days and
incubated with Z (2×107/ml), Z-Con A (1.5×107/ml), 1 or 2 µm latex
beads (final concentration of 1:200). Coxiella- or Leishmania-infected
cells were incubated with the particles for 4 and 18 hours, respectively, and were chased for 2 and 4 hours, respectively, prior to
fixation. Cells were first fixed in 2.5% glutaraldehyde (Sigma
Chemical Co.) in 0.1 M cacodylate buffer, pH 7.2, containing 0.1 M
sucrose, 5 mM Ca2+ and 5 mM Mg2+. Cells were washed overnight
in the same buffer, post-fixed for 1 hour at room temperature with 1%
osmium tetroxide in 0.1 M cacodylate buffer and scraped off the
culture dishes with a rubber policeman. Cells were then concentrated
in agar, and treated for 1 hour at room temperature with 1% uranyl
acetate in veronal buffer at a final pH of 5.0. Samples were dehydrated
in a graded series of acetone or ethanol, when latex beads were
present, and embedded in Epon. Thin sections were stained with 2%
uranyl acetate and lead citrate.
RESULTS
CHO cells infected with C. burnetii
Early after infection, CHO cells plated on glass coverslips
exhibited numerous small vacuoles containing the bacteria.
Two to three days later over 90% of the cells displayed one
or two large vacuoles enclosing variable numbers of bacteria,
and numerous small vesicles that contained a few organisms.
The large vacuoles, which came to occupy most of the cell
volume, were most often disc-shaped or elliptical, sometimes
lobulated (Hechemy et al., 1993) and, in nearly all cells, some
part of the vacuolar perimeter was in close contact with a cell
nucleus (e.g. Fig. 1B). As the number of bacteria increased,
Coxiella vacuoles became optically denser and shiny when
viewed with Nomarski optics. However, the bacterial density
in the vacuoles of different cells or even within the same cell
was quite variable. In living cells examined under phasecontrast microscopy Coxiella displayed Brownian movement,
indicating that the fluid within the vacuoles is of low
viscosity. Infected cells continued to multiply with time in
culture.
Observation of thin sections under the electron microscope
showed that in small vacuoles the bacteria were tightly apposed
to the vacuolar membrane. In contrast, bacteria were randomly
distributed in the enlarged vacuoles and rarely remained in
close contact with the vacuolar membranes. As judged from
their morphological appearance, most bacteria appeared to be
intact. The large Coxiella vacuoles also contained important
amounts of amorphous material and membrane debris of
unknown origin.
Transfer of particles to Coxiella vacuoles
In the usual experiments, two days after infection, cultures
were washed and the particles added to the medium. Sixteen
hours later cultures were washed four times, incubated for
several hours in particle-free medium, and fixed. Under phasecontrast microscopy, phagocytosed Y or Z particles that had
not entered Coxiella vacuoles were more refractile than those
that had been transferred to the vacuoles (Fig. 1D).
Uncoated heat-killed yeast or β-glucan particles (Fig. 1A,B)
or Z (not shown) were taken up by a small number of Coxiellainfected cells and most of the ingested particles were transferred to the Coxiella vacuoles. Uptake and subsequent transfer
of Y or Z particles were markedly increased by treatment with
Con A (Fig. 1C,D). Latex beads of 0.8 or 2 µm diameter were
also taken up and transferred to the vacuoles after overnight
incubation (Fig. 1E,F). The transfer of FITC-derivatized
zymosan (Z-FITC) or Z-Con A (Z-Con A-FITC) as well as of
fluorescent 1.0 µm latex beads was confirmed by confocal laser
3068 P. S. T. Veras and others
Fig. 1. Transfer of different
particles to Coxiella vacuoles
in CHO cells. Cultures
infected with Coxiella were
incubated with the particles
overnight. Stars indicate
Coxiella vacuoles and nuclei
are labeled N. (A) Heat-killed
yeast. (B) β-Glucan. (C) and
(D) Z-Con A particles in
Coxiella vacuole (arrow) and
in the cytoplasm (arrowhead).
(E) 0.8 µm latex beads.
(F) 2.0 µm latex. (A) and (E)
Nomarski optics. (B), (C),
(D) and (F) Phase-contrast.
Bars, 10 µm.
fluorescence microscopy. Fig. 2A,B documents the colocalization of particles and propidium iodide-stained Coxiella in
0.5 µm optical sections of paraformaldehyde fixed cells. In
these sections essentially all of the particles are contained
within the bacteria vacuoles.
Conclusive evidence for the transfer of Z and latex beads to
Coxiella vacuoles was provided by transmission electron
microscopy. In thin sections Coxiella vacuoles often contained
small numbers of Z or higher numbers of Z-Con A particles.
The Z particles contained in these vacuoles were not surrounded by membrane (Fig. 3A,B). Fig. 4A,B shows several
latex beads randomly distributed within a Coxiella vacuole.
The beads were often (Fig. 4A) but not always (Fig. 4B)
rimmed with amorphous material. As with Z particles, individual beads were not enveloped by membrane.
Kinetics of the transfer of Z or Z-Con A to
established Coxiella vacuoles
The proportion of CHO cells that took up and transferred Z
and Z-Con A to Coxiella vacuoles, as well as the numbers of
particles phagocytosed and delivered to the vacuoles varied
considerably from experiment to experiment. However, within
the same experiment, the uptake and transfer of Z particles
were directly related to the particle load and to the time of
contact between the cells and the particles. Similar results were
obtained with Y particles (not shown).
The effect of varying the incubation time on the transfer of
Z and Z-Con A (100 µg/ml) was first examined. In the experiment shown in Fig. 5A, Z particles were added at twice the
concentration of Z-Con A (3.6×106 vs 1.8×106). Z particles
were slowly taken up and by 22 hours only 14% of the cells
Targeted fusion of phagocytic vesicles 3069
Fig. 2. Confocal laser fluorescent micrographs of Coxiella-infected CHO cells incubated overnight with Z-Con A-FITC or 1 µm fluorescent
latex beads. Paraformaldehyde-fixed cells were stained with propidium iodide. Stars indicate Coxiella vacuoles. (A) Transfer of Z-Con A-FITC
(arrows). (B) Transfer of latex beads (arrowheads). Optical sections of 0.5-0.6 µm. Bars, 5 µm.
contained the particles. In 75% or more of these cells, most of
the particles were found within Coxiella vacuoles (Fig. 5A).
Comparison of Fig. 5B and A shows that, whereas at each time
point many more cells took up Z-Con A than Z, the proportion
of cells that transferred particles to Coxiella vacuoles was
nearly the same. Mainly because Z-Con A was taken up more
efficiently than Z, the numbers of Z-Con A transferred per 100
CHO cells increased much faster than the numbers of Z (Fig.
5C). Estimates of the fraction of phagocytosed particles that
entered Coxiella vacuoles (‘transfer efficiency’) revealed that
at all time periods, for both Z and Z-Con A particles, 80 to
98% of the particles phagocytosed ended up in the vacuoles
(not shown).
In the next experiment the incubation period was set at 7.5
hours and the number of particles added was varied by a factor
of 4. Fig. 6A shows that the percentage of CHO cells that
phagocytosed Z increased only slightly with the number of
particles offered. In contrast, the percentage of cells that took
up Z-Con A increased sharply with the particle load (Fig. 6B).
Furthermore, at all particle loads, 80% or more of CHO cells
that took up Z or Z-Con A transferred at least one particle to
a Coxiella vacuole (Fig. 6A,B).
The effect of varying the particle load on the number of Z
or Z-Con A transferred per 100 CHO cells is demonstrated in
Fig. 6C. Transfer of Z-Con A to the vacuoles increased more
sharply with particle load than the transfer of Z. Since most Z
particles taken up were transferred, the difference most
probably reflects the lower uptake of Z when compared to ZCon A.
Fig. 7 shows that the percentage of CHO cells with transfer
of at least one particle to a Coxiella vacuole was markedly
enhanced as the concentration of Con A used to coat the
particles was increased from 20 to 100 µg/ml.
Transfer of particles taken up by CHO cells prior to
infection with Coxiella
In the preceding experiments cells were first infected with
Coxiella and then incubated with the particles. Although target
vacuoles were presumably stable during the assay, donor
vesicles can be assumed to have undergone maturation. It is
thus possible that the maturation of donor vesicles is different
in non-infected and infected cells. In addition, infection
reduces the numbers of lysosomes and may affect cellular
transduction mechanisms (Baca and Paretsky, 1983).
In the next experiments CHO cells received the particles
overnight, were incubated for 3 hours in particle-free medium
and then infected with Coxiella. Cultures were fixed one or
two days later to allow development of large vacuoles. In
three separate experiments, definite transfer of Z, Z-Con A
and latex beads to the vacuoles was observed. In a typical
experiment, 51.0 ± 5.9% (3) and 98.0 ± 0.9% (3) (means ±
s.e.m., no. of coverslips) of cells fixed, respectively, one and
two days after infection displayed transfer of at least one ZCon A particle to a Coxiella vacuole. Transfer efficiencies
were, respectively, 81.0 ± 4.2 (3) and 98.0 ± 1.0 (3). Since
at the time of infection particles should have reached their
final compartments, these results indicate that mature
particle-containing vesicles did fuse with Coxiella vacuoles.
The difference in transfer at one and two days may be biased
by the small size of Coxiella vacuoles one day after infection
and by the difficulty in visualizing a few bacteria in Z-containing vesicles.
CHO cells infected with L. amazonensis
After overnight infection with Leishmania amastigotes CHO
cells contained a variable number of small parasitophorous
vacuoles (PVs), which progressively increased in size and in
Fig. 3. Thin sections of infected CHO cells with transfer of Z particles to Coxiella
vacuoles (stars). Coxiella (C), Coxiella vacuolar membrane (CVM), Plasma membrane
(PM), zymosan (Z). (A) Transfer of a Z particle to a Coxiella vacuole. Bars, 1 µm.
(B) Absence of membrane around Z particle. Bars, 0.5 µm.
Fig. 4. Thin sections of Coxiella-infected CHO cells after uptake of latex beads (LB).
Coxiella vacuolar membrane (CVM). (A) Large amounts of LB are transferred to
Coxiella vacuoles (star). Bars, 1 µm. (B) Membranes not seen around LB. Bars, 0.5 µm.
3070 P. S. T. Veras and others
Targeted fusion of phagocytic vesicles 3071
60
60
40
40
20
20
0
0
60
60
40
40
20
20
0
40
20
20
0
4
100
8
12
16
20
100
B
80
80
60
60
40
40
20
20
0
0
0
0
Z transferred per 100 CHO
8 12 16 20 24
time (hours)
C
Zymosan-Con A
300
200
40
Zymosan (×106)
80
400
60
0
80
500
60
0
100
4
80
24
B
0
80
Zymosan
100
% CHO with transfer (s)
8 12 16 20
time (hours)
100
A
2
4
6
8
10
Zymosan-Con A (×106)
800
C
Zymosan-Con A
600
400
Zymosan
200
0
0
4
8 12 16 20
time (hours)
0
24
Fig. 5. Uptake by CHO cells and transfer of Z or Z-Con A to
Coxiella vacuoles as a function of time. Two days after infection
cultures received an estimated 30 Z or 15 Z-Con A particles/cell for
the times indicated on the abscissae. Cultures were fixed and infected
cells were scored with a phase-contrast microscope for particle
uptake (squares). Infected cells, which contained particles, were also
scored for transfer to Coxiella vacuoles (circles). Three coverslips
per point. Bars indicate standard errors. (A) Uptake and transfer of Z.
(B) Uptake and transfer of Z-Con A. (C) Z (open triangles) and ZCon A (filled triangles) transferred to at least one Coxiella vacuole
per 100 infected CHO cells.
the number of parasites they contained. Two days later more
than half of the cells displayed one or two large vacuoles that
could coexist with a few small, ‘tight’ vacuoles (Fig. 8A).
Amastigotes were typically aligned on the inner surface of the
PVs (Fig. 8B) as they do in macrophages, and, in some
vacuoles, parasites were clustered in rosette-like arrangements
(not shown).
Transfer of particles to Leishmania vacuoles
As in the experiments with Coxiella, CHO cells were
incubated with the particles prior to or after infection with
Leishmania amastigotes. In both instances transfer was found
4
8
12
particles (×106)
16
Fig. 6. Uptake of Z or Z-Con A and their transfer to Coxiella
vacuoles in CHO cells as a function of particle numbers. Cultures
containing equal numbers of cells received the numbers of particles
indicated in the abscissae. (A) Uptake and transfer of Z. (B) Uptake
and transfer of Z-Con A. (C) Numbers of Z (open triangles) or ZCon A (filled triangles) transferred to at least one Coxiella vacuole
per 100 infected CHO cells.
% CHO with transfer ( )
0
100
600
80
500
400
60
300
40
200
20
100
0
0
0
20 40 60 80 100 120
Con A (µg/ml)
Z transferred per 100 CHO (h)
% CHO with uptake of
Zymosan-Con A (h)
100
4
% CHO with uptake of
Zymosan-Con A (h)
0
Z transferred per 100 CHO
% CHO with uptake
of Zymosan (h)
80
% CHO with transfer (s)
80
100
% CHO with transfer (s)
100
A
% CHO with transfer (s)
% CHO with uptake
of Zymosan (h)
100
Fig. 7. Percentage of infected CHO cells with transfer of Z (circles)
and numbers of Z transferred per 100 Coxiella-infected CHO cells
(squares) as a function of the concentration of Con A used to coat the
particles.
3072 P. S. T. Veras and others
Fig. 8. Transfer of particles to
the parasitophorous vacuoles
of Leishmania-infected CHO
cells. Infected cells were
incubated overnight with the
particles. Phase-contrast. Stars
indicate parasitophorous
vacuoles and black arrows
point to Leishmania
amastigotes. (A) and (B)
Zymosan particles. White
arrows indicate Z within PVs
and white arrowheads point to
particles located in the
cytoplasm. (C) 0.8 µm, and
(D) 2.0 µm latex beads. The
majority of the beads are
found outside the large
vacuoles. Bars, 10 µm.
Fig. 9. Confocal laser fluorescent micrographs of Leishmania-infected cells incubated with Z-Con A-FITC or 1 µm fluorescent latex beads.
Paraformaldehyde-fixed cells stained with propidium iodide. Stars indicate Leishmania vacuoles. Small arrows point to Leishmania
amastigotes. (A) Transfer of Z-Con-A-FITC (large arrow). (B) Transfer of latex beads (arrowhead). Optical sections of 0.5-0.6 µm. Bars, 5 µm.
to be quantitatively different from that to Coxiella vacuoles.
Indeed, transfer was found in fewer infected cells and a
smaller proportion of ingested particles was transferred to
PVs. Fig. 8A,B shows small and medium-sized PVs containing a few untreated Z particles. The same cells display
particles (arrowhead) that were not transferred to the
Targeted fusion of phagocytic vesicles 3073
vacuoles. Furthermore, the large majority of 0.8 or 2 µm
beads ingested by infected cells remained outside the PVs
(Fig. 8C,D). These observations were confirmed by laser
confocal microscopy (Fig. 9A,B).
In the EM studies many thin sections displayed Leishmania
PVs and either Z or latex-containing vacuoles. In contrast to
the observations in Coxiella-infected cells, Leishmania
amastigotes and the particles usually remained in separate
vacuoles. In a few cases, small numbers of Z particles (Fig.
10A) or latex beads (Fig. 10B) were found within a Leishmania PV. In these instances, the beads were also covered with
an amorphous material (Fig. 10B).
CHO cells were cultivated in medium supplemented with bicarbonate at
35°C in a 6% CO2 atmosphere. Two days after infection cells were incubated
for 9 hours with numbers of Z-Con A as indicated. Cells were washed and
incubated for an additional 24 hours in particle-free medium prior to fixation.
Quantitative observations of the transfer of Z or ZCon A to Leishmania PVs
Although the uptake of Z or Z-Con A by Coxiella- or Leishmania-infected cells was similar (not shown), the percentage
of cells with transfer, the numbers of particles transferred,
and the efficiency of transfer, were all consistently lower in
Leishmania-infected cells (Fig. 11A,B,C). In addition,
whereas the efficiency of transfer of Z or Z-Con A to Coxiella
vacuoles was high and relatively independent of particle
loads (Fig. 6A,B), the efficiency of the transfer of Z to Leishmania PVs was higher than that of Z-Con A (Fig. 11C). Furthermore, transfer of Z-Con A was reduced at increased
particle loads (Table 1). Similar results were obtained in cells
loaded with Z or Z-Con A prior to Leishmania infection (not
shown).
Table 1. Transfer of Z-Con A to Leishmania
parasitophorous vacuoles in Leishmania-infected cells
Particle load
% CHO cells with transfer
Total Z-Con A
Z-Con A transferred/CHO cell
Efficiency of transfer
5×106
2×107
16.61±2.40 (3)
3.18±0.31 (3)
1.31±0.09 (3)
41.81±3.31 (3)
11.22±1.22 (3)
8.11±2.68 (3)
1.44±0.12 (3)
21.85±6.30 (3)
Fig. 10. Thin sections of Leishmania (L)infected CHO cells after uptake and
transfer of Z and latex beads (LB)
particles. Stars indicate Leishmania
vacuoles, plasma membrane (PM),
parasitophorous vacuole membrane
(PVM). Bars, 1 µm. (A) Transfer of Z.
(B) Transfered LB shows amorphous
material on the surface. Partial sections of
two beads shown outside the vacuole.
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
3074 P. S. T. Veras and others
% CHO with transfer
100
A
80
60
40
20
0
Zymosan
Z transferred per CHO
8
6
2
0
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
Zymosan
100
efficiency of transfer
Z-Con A
B
4
80
60
40
20
0
C
Zymosan
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
AAA
Z-Con A
Z-Con A
Fig. 11. Percentage of CHO cells with transfer (A), numbers of
particles transferred to target vacuoles (B) and efficiency of transfer
(C) of Z or Z-Con A to Leishmania- (filled bars) or Coxiella(hatched bars) vacuoles. Each bar gives the average of 3
experiments. Standard errors are indicated.
DISCUSSION
We made use of the quantal and vectorial transfer of particles
to the spacious phagolysosome-like vacuoles induced by C.
burnetii or L. amazonensis in CHO cells to detect and to
measure the fusion between large phagocytic vesicles.
Transfer, first detected by light microscopy, was confirmed by
laser confocal fluorescence and by electron microscopy.
Particles were transferred to Coxiella vacuoles without
apparent selectivity. Transfer of Y or Z, both poorly phagocytosed, and of Con A-treated Y or Z, both well taken up, was
quite efficient, since most of the ingested particles entered
Coxiella vacuoles within a broad range of incubation periods
and of particle to cell multiplicities. The high rate and capacity
for fusion may explain why donor vesicles did not accumulate
around Coxiella vacuoles in CHO cells as is the case in Leishmania-infected macrophages (Veras et al., 1992). Transfer
occurred whether particles were ingested prior to or after
infection. In contrast, Leishmania vacuoles in CHO cells
admitted fewer untreated or Con-A-treated yeast-derived
particles and latex beads. Furthermore, the numbers of yeastderived particles transferred to the vacuoles did not increase
with particle load, indicating the low transfer capacity to these
vacuoles.
In the light of these results, it is interesting to compare the
transfer of particles to Leishmania PVs in CHO and
macrophages. We have previously reported that Z particles
were transferred to PVs in macrophages, and as in the present
observations in CHO cells, such transfer was more efficient at
low particle to cell ratios (Veras et al., 1992). In addition,
transfer of latex beads to PVs was not detected in macrophages
by light microscopy, whereas it was rarely observed in Leishmania-infected CHO cells by confocal laser fluorescence
microscopy and by transmission electron microscopy. Thus
Leishmania PVs appear to display similar selectivity of
particles transfer in the two cell types.
These results raise a number of questions that merit discussion. How mature are donor vesicles at the time they fuse with
the target vacuoles? What are the signals involved in targeting
the particles to the parasite vacuoles? Do pathogen-associated
or secreted signals control the fusion of Coxiella and Leishmania vacuoles with donor vesicles?
Stage of maturation of the donor vesicles
Donor vesicles in Coxiella- or Leishmania-infected cells have
not been characterized. In the post-load experiments, the time
available for maturation of donor vesicles, i.e. the time
between particle ingestion and transfer, is unknown. However,
in the pre-load experiments, after receiving the particles, cells
were washed and incubated for 3 hours prior to infection. This
could constitute a minimum time available for maturation of
the donor vesicles. In addition, infection by Leishmania or
Coxiella reduces the numbers of secondary lysosomes in host
cells (Barbieri et al., 1990; Maurin et al., 1992b) and this could
affect the maturation of donor vesicles.
Signals involved in the targeting of the particles to
Coxiella or Leishmania vacuoles
Lysosomal targeting of exogenous macromolecules and
particles has been associated with signals encoded in the
cytosolic domains of specific receptors engaged by the ligands
(Joiner et al., 1990; Trowbridge et al., 1993). We postulated
that macrophage-specific receptors that recognize Z may be
involved in transfer of the particles to Leishmania PVs (Veras
et al., 1992). Since these receptors are not expressed in CHO
cells, additional receptors for Z and Z-Con A may be required
for particle transfer to PVs. The mechanisms involved in the
opsonization by Con A also need to be defined. Ingestion of
yeast may be triggered by crosslinking of cell surface glycoproteins or glycosaminoglycans by particle-bound Con A, a
tetramer. Such a mechanism gains support from the observation that succinyl-Con A, which is divalent and cannot
crosslink cell surface binding sites, induced less Z uptake by
Coxiella- or Leishmania-infected cells (Veras and Rabinovitch, unpublished results). Alternatively, Con A, which is
a glycoprotein, may be recognized by cell surface lectins
(Drickamer and Taylor, 1993). Finally, untreated or Con-Atreated yeast particles could be opsonized by molecules
contained in fetal calf serum or derived from the cells, such as
fibronectin, laminin, or mannose-binding proteins (Grinnell
and Geiger, 1986; McCulloch and Knowles, 1993).
Targeted fusion of phagocytic vesicles 3075
Differences in the transfer of particles to Coxiella
and to Leishmania vacuoles
We have shown that more particles were transferred to
Coxiella than to Leishmania vacuoles and that the efficiency
of the transfer is higher for the former than for the latter
pathogen (Fig. 11A,B,C). These differences could arise from
one or more of the following mechanisms: (1) structural
features of the two systems, such as the fraction of the cell
volume occupied by Coxiella or Leishmania vacuoles, the distribution of cytoskeletal elements required for translocation of
particle-containing vesicles in infected cells, or obstructions to
the movement of donor vesicles (Luby-Phelps, 1994). (2)
There could be compositional differences between Coxiella
and Leishmania vacuoles, involving externally displayed
molecules required for recognition or fusion of the vesicles,
such as GTP-binding proteins, ARFs or annexins (Gruenberg
and Emans, 1993; Zerial and Stenmark, 1993). (3) The
pathogens could affect host cell transduction mechanisms and
thus differentially modify the composition of the particle-containing vesicles (cf. Descoteaux and Turco, 1993). (4) Parasites
could express or produce molecules that may positively or negatively control the interaction of their vacuoles with particlecontaining vesicles. Since Coxiella and Leishmania vacuoles
are not similarly prone to fuse with large phagocytic vesicles,
it is possible to ask which of these two vacuoles, if either,
expresses the constitutive behavior of ‘normal’ phagolysosomes. Does the lack of discrimination and intense fusion
activity of Coxiella vacuoles arise from a fusion-inducing
bacterial molecule? Conversely, is the reduced and more discriminating fusion displayed by Leishmania vacuoles due to an
inhibitory Leishmania-derived factor? Current experiments in
this laboratory show that Leishmania and Coxiella can share
the same vacuoles in doubly infected cells (Veras and Rabinovitch, unpublished). These mixed vacuoles may permit the
analysis of these pathogen-associated fusion signals.
In a more general vein, the results reported here emphasize
the usefulness of Leishmania- or Coxiella-infected ‘non-professional phagocytes’ for the study of biochemical and
molecular mechanisms of fusion between large phagocytic
vesicles. Although, our experiments were performed with CHO
cells, the transfer of Z-Con A to Coxiella vacuoles could also
be detected in L929, Cos, Vero and HeLa cells. In contrast,
transfer of Z-Con A to Leishmania vacuoles was observed only
in Vero and HeLa cells, not in Cos or L929 (Veras and Rabinovitch, unpublished results). It should now be possible to
examine particle transfer in cells that do not express or overexpress regulatory molecules involved in the function of the
cytoskeleton, in intracellular trafficking or in membrane fusion.
This paper is dedicated by M. R. to the memory of Zanvil A. Cohn.
This work was supported by the Institut Pasteur, the CNRS and
INSERM. P.S.T.V. was the recipient of a predoctoral fellowship from
CNPq, Brazil. Raymond Hellio provided advice and help in laser
confocal fluorescence microscopy. Bernard Bruneau provided
technical assistance in the electron microscopy studies and Christophe
Soubert printed the light microscopy micrographs. The authors are
grateful to M. Barcinsky, C. Roth and C. Rougeot for critically
reviewing the manuscript.
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(Received 6 June 1994 - Accepted 18 July 1994)