International Journal for Parasitology 33 (2003) 185–197
www.parasitology-online.com
Acidification modulates the traffic of Trypanosoma cruzi trypomastigotes
in Vero cells harbouring Coxiella burnetii vacuoles
Walter K. Andreoli, Renato A. Mortara*
Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo – UNIFESP, Escola Paulista de Medicina, Rua Botucatu,
862 6th floor, 04023-062, São Paulo, SP, Brazil
Received 19 September 2002; received in revised form 28 November 2002; accepted 3 December 2002
Abstract
We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella
burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture
trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and
tissue-culture trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic
trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote
hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2
cells facilitate the escape of both forms. Endosomal – lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous
vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of
C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus
suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.
q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved
Keywords: Trypanosoma cruzi; Coxiella burnetii; Double infection; Cell invasion; Membrane traffic; Vacuolar pH; Confocal microscopy
1. Introduction
Through the life cycle of Trypanosoma cruzi, the
causative agent of Chagas’ disease, two flagellated infective
forms can be identified. Trypomastigotes may be either
released from infected cells (designated tissue-culture
derived trypomastigotes) in the mammalian host or may
evolve from the differentiation of epimastigotes in the
rectum of triatomine vectors (metacyclic forms). Under
experimental conditions metacyclic trypomastigotes are
isolated from axenic cultures and tissue-culture trypomastigotes from the supernatant of mammalian cell cultures.
Trypomastigotes are able to invade a variety of phagocytic
and non-phagocytic cells engaging a variety of molecules
and signalling pathways (Burleigh and Andrews, 1995;
Yoshida et al., 2000).
Although morphologically similar, metacyclic and
tissue-culture trypomastigotes are quite distinct regarding
* Corresponding author. Tel.: þ55-11-5579-8306; fax: þ 55-11-55711095.
E-mail address: [email protected] (R.A. Mortara).
their infectivity towards cells, the expression of surface
components such as the sialic acid acceptor (Acosta-Serrano
et al., 2001), and the molecules mobilised during host cell
invasion (Burleigh and Andrews, 1995; Yoshida et al.,
2000). Endosomal (Wilkowsky et al., 2002) followed by
lysosomal recruitment (Meirelles and De Souza, 1983;
Tardieux et al., 1992) may occur as early events during
parasite invasion, and lysosomal markers are found in
parasitophorous vacuoles of both trypomastigote forms
(Procópio et al., 1998; Tardieux et al., 1992). After a few
hours, the activities of a low pH active haemolysin
(Andrews and Whitlow, 1989) and of transialidase (Hall
et al., 1992) disrupt the parasitophorous vacuole membrane
allowing the parasite to escape to the cytoplasm. In this
period of residence within the parasitophorous vacuole, it is
thus possible that T. cruzi parasitophorous vacuoles may
interact with other vesicular compartments like Coxiella
burnetii large cytoplasmic vacuoles, as has already been
demonstrated (Rabinovitch et al., 1999; Rabinovitch and
Veras, 1996).
The study of cell co-infection may allow the observation
0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved
doi:10.1016/S0020-7519(02)00262-X
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of the behaviour of pathogens in the presence of one
another, and provide new insights on the course of infection
and interaction of each pathogen with the endocytic
pathway (Rabinovitch et al., 1999; Rabinovitch and Veras,
1996). A now classic study is the co-infection of
Toxoplasma gondii and HIV in which it has been shown
that the intracellular replication of this protozoan is
enhanced in macrophages isolated from AIDS patients
(Biggs et al., 1995). In another study of macrophages doubly
infected with T. gondii tachyzoites and T. cruzi epimastigotes, lysosomal and endosomal pathways were analysed.
Toxoplasma gondii parasitophorous vacuoles did not
interact with lysosomes while T. cruzi parasitophorous
vacuoles clearly fused to lysosomes (Meirelles and De
Souza, 1983).
More recently, bacterial – bacterial or protozoan –bacterial double infection studies advanced with the use of cells
harbouring Coxiella burnetii vacuoles. Belonging to the
Rickettsiaceae family, C. burnetii is a strict intracellular
bacterium and causative agent of Q fever, an opportunistic
pneumonia affecting humans. Coxiella burnetii may inhabit
both phagocytic and non-phagocytic cells (Baca and
Paretsky, 1983). Once inside cells, C. burnetii forms large
vacuoles with lysosomal characteristics by acquisition of
hydrolases and surface lysosomal markers (LAMP-1 and
LAMP-2). Coxiella burnetii is a well adapted organism that
accomplishes all metabolic processes at low pH (Hackstadt
and Williams, 1981), as it has been established that C.
burnetii vacuoles maintain an acidic pH during infection
(Maurin et al., 1992). Early experiments involving CHO
cells infected with C. burnetii showed that the large
vacuoles fuse with high efficiency with inert particle
vacuoles (Veras et al., 1994). Thus, C. burnetii infected
cells provide an interesting model to examine how different
intracellular pathogens interact with endosomal-lysosomal
pathways. Leishmania (L.) amazonensis infection of CHO
cell lines containing C. burnetii clearly showed that the two
microorganisms can share the same intracellular space.
Survival and replication of L. (L.) amazonensis also occur
within C. burnetii vacuoles (Rabinovitch and Veras, 1996;
Veras et al., 1995). Coxiella burnetii and L. (L.) amazonensis are pathogens that grow within vacuoles with similar
features such as acidic pH and ability to fuse with
lysosomes. Recently, macrophages previously infected
with Mycobacterium avium were then superinfected with
other intracellular pathogens but only C. burnetii colocalised with M. avium (De Chastellier et al., 1999). The
C. burnetii vacuole microenvironment disclosed differences
in growth and survival of M. avium and Mycobacterium
tuberculosis, the latter being more susceptible to acidic
conditions (Gomes et al., 1999). Fibroblast double-infection
with C. burnetii and T. gondii also occurs, but there is a
minimal co-localisation, confirming the refractory behaviour of T. gondii parasitophorous vacuole towards fusion
(Sinai et al., 2000).
The aim of the present work was therefore to examine
how C. burnetii affects the fate of different T. cruzi
trypomastigotes in Vero cells persistently infected with
the rickettsia, from the initial invasion steps to parasitophorous vacuole formation from where the parasites may
either escape into the cytoplasm or be transferred to the
rickettsia vacuole (Scheme 1). Our results show that
metacyclic and cell-derived trypomastigotes are internalised
within parasitophorous vacuoles whose trafficking inside
Vero cells containing C. burnetii vacuoles may be pHmodulated.
2. Materials and methods
2.1. Cells and parasites
Vero cells obtained from the Instituto Adolpho Lutz (São
Paulo, SP, Brazil) were cultivated in RPMI 1640 medium
(GIBCO BRL) with 10% foetal calf serum (FCS/ CULTILAB) in a humid atmosphere with 5% CO2 at 36.58C.
Avirulent phase II C. burnetii, nine mile strain, was kindly
supplied by Dr Michel Rabinovitch from our Parasitology
Division. Semi-confluent Vero cells (grown in 75 cm2
flasks) were infected with C. burnetii (aliquots of 250 ml
containing 109 cells/ml were usually used). This aliquot was
sufficient to induce large vacuole formation after 3 days of
Scheme 1. Schematic representation of the possible destinations of T. cruzi
metacyclic (I) or tissue-culture trypomastigotes (II) inside cells harbouring
Coxiella burnetti vacuoles. (A) Parasite adhesion; (B) lysosome recruitment; (C) lysosome fusion with parasitophorous vacuole membrane; (D)
parasitophorous vacuole fusion with C. burnetti vacuole; (E) co-habitation
of T. cruzi trypomastigotes and C. burnetti; (F) parasitophorous vacuole
membrane rupture and escape of trypomastigotes into the cytoplasm. In this
illustration metacyclic trypomastigotes are preferentially transferred to the
rickettsia vacuole whereas escape into the cytoplasm is more noticeable in
tissue-culture trypomastigote trafficking.
W.K. Andreoli, R.A. Mortara / International Journal for Parasitology 33 (2003) 185–197
incubation and we defined this as acute C. burnetii infection
(Zamboni et al., 2001). Vero cells persistently infected with
C. burnetii were obtained by more than five sub-cultivation
passages of acutely infected cells in antibiotic-free Dulbecco’s modified Eagles medium (DMEM) containing glucose
(GIBCO), grown at 35.58C in the absence of CO2
(supplemented with 4.5 mM NaHCO3). These conditions
allowed large vacuole formation in approximately 80% of
the Vero cells. CHO and Lec-2 cells (kindly provided by
Sergio Schenkman from the Cell Biology division of our
Department, and originally obtained from American Type
Culture Collection (ATCC)) were cultivated on a-MEM
with ribonucleosides and ribonucleotides (GIBCO) supplemented with 5% FCS in a humid atmosphere with 5%
CO2 at 36.58C. Coxiella burnetii infection of CHO and Lec2 was performed as described for Vero cells.
Cell-derived trypomastigotes from the CL strain (Brener
and Chiari, 1963) were obtained after infection of semiconfluent Vero cells. Cells grown in 162 cm2 flasks were
infected with recent released cell-derived trypomastigotes
(108 parasites/ml). Infection proceeded overnight at 36.58C
in RPMI 1640 medium supplemented with 10% FCS. The
supernatant is then replaced with RPMI 1640 with 2% FCS
and cells were then kept at 35.58C. Trypomastigotes emerge
from Vero cells after approximately 6 days of infection.
Parasites were separated from cell debris by centrifugation
at 1000 £ g for 5 min. Infective trypomastigotes were then
isolated by centrifugation of the mixed population of
amastigotes and trypomastigotes at 2500 £ g for 5 min
and the competent parasites left the pellet after 2 h at 36.58C
(Schenkman et al., 1991). Metacyclic trypomastigotes from
the CL strain were obtained by the following procedures:
about 1 ml of blood collected by heart puncture from
previously infected albino mice is divided in 0.3 ml aliquots
and transferred to 5 ml of LIT (liver infusion tryptose)
medium containing 10% FCS and 0.2% glucose. After 10
days of growth at 288C epimastigote cultures were expanded
five-fold, and after a further 7 days in culture, parasites were
concentrated five-fold in LIT medium by centrifugation at
2500 £ g for 5 min and then 2.5 ml are placed in 35 ml of
GRACE’S medium (GIBCO) pH 6.35. Differentiation of
epimastigotes into metacyclic trypomastigotes occurs after
another 7 days at 288C.
2.2. Cell invasion assays
Semi-confluent cells containing C. burnetii vacuoles
were infected with metacyclic or cell-derived trypomastigotes in a 10:1 (parasite:cell) ratio. After 1.5, 3, 4, 6, 8, and
12 h of infection at 36.58C, DMEM medium supplemented
with 5% FCS was removed and cells washed three times
with PBS. Cells were then fixed for 1 h with 3.5%
formaldehyde in PBS, washed three times with PBS and
incubated for 15 min in 50 mM NH4Cl to quench reactive
aldehyde groups. Cells were placed in PBS/0.15% gelatin/0.1% NaN3 prior to immunofluorescence labelling. For
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the experiments of comparative infection of Vero cells
containing or not C. burnetii vacuoles fixation was done
with 2% glutaraldehyde in 0.1 M sodium phosphate buffer
pH 7.0 after 3 h of infection (Procópio et al., 1998). Invasion
index was calculated according to the formula: number of
parasites inside cells/ number cells infected £ % of infected
cells, counting 100 cells in triplicate coverslips (Procópio
et al., 1998). Parasites inside the C. burnetii vacuole were
scored based on the co-localisation of DAPI (40 ,6-diamidino-2-phenylindole dihydrochloride, Molecular Probes)
labelling of nuclei and kinetoplasts with the rickettsia, as
well as by differential interference contrast-DIC imaging.
2.3. Haemolytic and transialidase activity assays
Experiments were performed as described by Andrews
and Whitlow (1989) with minor modifications. Briefly,
female albino mice erythrocytes freshly collected were
washed three times with isotonic PBS/ 0.1% gelatin, 0.15%
CaCl2 and 1 mM MgCl2. Isolated tissue-culture and
metacyclic trypomastigotes (108/ml) were transferred to
10 mM sodium acetate buffer, pH 5.5, 0.15 M NaCl,
containing 1% glucose and the erythrocytes (107/ml).
Positive controls were obtained by treating the erythrocytes
with 0.1% saponin. Released haemoglobin was measured at
540 nm in a Labsystems Multiskan MS (Finland) ELISA
reader, in duplicate samples collected at 3, 6, and 10 h.
Transialidase activity of purified parasites was assayed as
described in (Schenkman et al., 1992). Briefly, the
enzymatic transference of sialic acid to 14[C]-lactose
generated 14[C]-sialyl – lactose that was captured in QAESephadex A-25 columns, and we used Y strain tissueculture derived trypomastigotes as positive controls
(Schenkman et al., 1992).
2.4. Interference with vacuolar pH
In order to alkalinise C. burnetii vacuoles, cells were
exposed for 1 h at 36.58C, to culture medium containing
either chloroquine (100 mM), bafilomycin A1 (50 nM) or
concanamycin A (50 nM, Sigma). Cells were then washed
three times with PBS and medium containing the parasites
was added to the flasks. For bafilomycin A1 and
concanamycin A, intravacuolar pH (see below) was
maintained above 6.5 even after 6 h after the drug was
washed from the cell supernatant (controls not shown).
2.5. Antibodies and immunofluorescence labelling
For invasion assay quantitations, cells fixed in 2%
glutaraldehyde were incubated with mAb (ascitic fluids
diluted 1:40 in PBS/0.1% gelatin, 0.15% sodium azide):
mAb 3F5 is directed against metacyclic trypomastigote
35/50 kDa mucin (Mortara et al., 1992) and mAb 3B2 is
directed against membrane antigen of cell-derived trypomastigotes (Barros et al., 1997). After 1 h, coverslips were
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washed three times with PBS and incubated 1 h with antimouse IgG-FITC conjugated (Sigma) diluted 1:50 in PBS/
0.15% gelatin/0.1% NaN3 and 10 mM DAPI. Using this
methodology only parasites outside cells are labelled with
antibodies. Invasion index was calculated as described
above. For kinetic studies on parasite transference to C.
burnetii vacuole, 3.5% formaldehyde fixed cells were
permeabilised with 0.1% saponin in PBS/0.15% gelatin/
0.1% NaN3 for 15 min, and then incubated with the
appropriate antibodies as described above. For doublelabelling assays, samples containing parasites were first
labelled with 1:30 rabbit polyclonal serum against T. cruzi
for 1 h and incubated with anti-rabbit IgG conjugated to
FITC. Then, coverslips were incubated with anti-LAMP-1
antibodies (clone H4A4 anti-human LAMP-1, and clone
UH3, anti-hamster LAMP-1, both developed in mouse were
supernatants from Development Studies Hybridoma Bank,
Iowa, USA) for 1 h and finally incubated 1 h with antimouse IgG conjugated to Cy3 (Sigma) diluted 1:50 in PBS/
0.15% gelatin/0.1% NaN3 and 10 mM DAPI. Characterisation of T. cruzi parasitophorous vacuole was performed
using mouse monoclonal antibody against EEA1 (Transduction Laboratories), mouse mAb E41120, 1:40), mAb
anti-LAMP-1 (as above), and rabbit antibodies against Rab7
(1:40, kindly supplied by Dr Marino Zerial, EMBL,
Heidelberg, Germany).
2.6. Intra-vacuolar pH determinations
Coxiella burnetii persistently infected Vero cells grown
in 75 cm2 culture flasks were harvested and allowed to
attach (105 cells/ml) overnight to 0.15 mm thick coverslips
of Delta T dishes (Bioptechs). Medium was then replaced
with Hank’s salt solution with 2% glucose, containing
10 mM of SNAFL-calcein AM (semi-naphtho-fluorescein–
calcein, acetoxymethyl ester, molecular probes). After
20 min of incubation when the pH sensitive dye had
compartimentalised into the rickettsia vacuoles, the solution
was replaced with DMEM with 2% foetal calf serum and the
probe localised within C. burnetii vacuoles. Once the loaded
cells stabilised, dual-wavelength emission (Zhou et al.,
1995) was performed using a BioRad 1024 UV confocal
system attached to a Zeiss Axiovert 100 microscope. Images
(20 cells per field) were obtained through a 63 £ 1.4 NA.
plan-apochromatic oil immersion lens from the Delta-T
dishes that kept the cells at 36.58C. Pinhole size,
photomultiplier gain and black level settings were kept the
same for both emission channels. Lasersharp software
version 3.2TC was used for image acquisition and basic
processing. Calibration experiments were performed as
described (Schramm et al., 1996) with some modifications:
MES buffer 30 mM with 2% glucose and 20 mM Nigericin
(Sigma) with pH values of 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5; the
lag time for image acquisition was about 15 min of
incubation for the first buffer and 5 min for the others,
beginning with the more acidic calibration buffer data points
to avoid cell blebbing. Emission ratios (550/640 nm) of the
dual SNAFL-calcein dyes from about 20 cells of at least five
fields were calculated with Lasersharp software.
2.7. In vivo transfer of T. cruzi metacyclic trypomastigotes
to Vero cells containing C. burnetii vacuoles
Before intravacuolar pH determination, metacyclic
trypomastigotes were loaded with 1.5 mM Hoechst 33258
(2,5’-Bi-1H-benzimidazole, 2’-(4-ethoxyphenyl)-5-(4methyl-1-piperazinyl)-trihydrochloride, a vital DNA dye
from Molecular Probes) for 30 min. The parasites were then
washed three times with PBS in order to remove excess
probe. Invasion by labelled parasites was performed during
3 h in Vero cells containing C. burnetii vacuoles grown in
Delta T dishes at 36.58C. After unattached parasites were
removed, the pH measurements were performed as
described above.
2.8. Quantitations and statistical calculations
All experiments were performed in triplicate using three
coverslips. On average, 100 cells per coverslip were
analysed. For experiments involving co-localisation of
parasites and endocytic markers, images were acquired in
a Nikon Labophot microscope through a 40 £ objective
using a CCD camera and the Leica Q win image analysis
program. Statistical calculations were done with SigmaStat
(Version 1.0, Jandel Scientific), using the t-test for
significance and standard deviations for paired data sets.
3. Results
3.1. Invasion by T. cruzi trypomastigotes in Vero cells
infected or not with C. burnetii
In order to assess whether C. burnetii infection by itself
could affect the susceptibility of Vero cells to T. cruzi
infection, we first compared tissue-culture and metacyclic
trypomastigotes invasion parameters in uninfected or C.
burnetii-persistently infected Vero cells. Cells were infected
with T. cruzi CL strain trypomastigote forms for 3 h. We
observed that the presence of C. burnetii in the cytoplasm of
Vero cells affected the invasion of both forms in C. burnetii
harbouring Vero cells (Fig. 1B) suggesting that the
rickettsia influenced the initial invasion steps. Since the
pH of acidic intracellular compartments can be raised by
inhibiting the Hþ-ATPase with drugs such as bafilomycin
A1 or concanamycin A (Dröse and Altendorf, 1997), we
examined their effect on the invasion of T. cruzi trypomastigote forms in normal or C. burnetii infected Vero cells
(Fig. 1A, B). When normal Vero cells were treated with
either drug there was a significant inhibition of the invasion
of both infective stages (Fig. 1A, B). Surprisingly, pretreatment of cells with bafilomycin A1 or concanamycin A
W.K. Andreoli, R.A. Mortara / International Journal for Parasitology 33 (2003) 185–197
189
Fig. 1. Trypomastigote invasion in Vero cells and Vero cells containing C. burnetii vacuoles is differentially affected by alkalinising agents. Infection with
metacyclic (A) or cell-derived trypomastigotes (B) in Vero cells (open bars), or Vero cells containing C. burnetii vacuoles (hatched bars) was allowed to
proceed for 3 h, using a 1:10 parasite:cell ratio. Invasion index (note the difference in scale between the two trypomastigote stages) was calculated by the
following formula: number of parasites inside cells/ number cells infected £ % of infected cells by counting 100 cells in triplicate coverslips (Procópio et al.,
1998). * denotes a statistically significant ðP # 0:05Þ difference from the appropriate uninfected Vero cell control. ** denotes that invasion index in C. burnetti
infected cells is significantly higher ðP # 0:0007Þ than in the corresponding uninfected control. Note that whereas in uninfected Vero cells (open bars)
Bafilomycin A1 (BAF A1), Conacanamycin A (Conca A), and chloroquine (CHL) inhibit the invasion of both trypomastigote stages, but when the cells are
chronically infected with C. burnetii the effect is reverse for all drugs. (C– F) Confocal fluorescence microscopy of LAMP-1 labelling of chronically infected
Vero cells C. burnetii (C,D) treated with BAF A1 (E,F). Note that in untreated cells the bacterium vacuoles have an uniform labelling with LAMP-1 (C)
contrasting to BAF A1-treated cells where labelled vacuoles display a more granular morphology (D). Bars ¼ 50 mm.
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caused an enhancement of T. cruzi infectivity in Vero cells
persistently infected with C. burnetii that was significant
ðP , 0:0007Þ for both trypomastigote stages (Fig. 1A, B).
Controls confirmed that exposure of trypomastigotes to
either drug did not alter their motility or viability (data not
shown). Similar results were obtained with another
alkalinising agent, chloroquine that raises cytoplasmic pH
and alters the distribution of lysosomes (Fig. 1). When
lysosomal distribution was examined in untreated Vero
cells, C. burnetii vacuoles display uniform labelling with
LAMP-1 but, in contrast, a dispersed granular arrangement
was observed in the lysosomal distribution after treatment
with bafilomycin A1 (Fig. 1C).
3.2. Trypanosoma cruzi metacyclic and tissue-culture
trypomastigote parasitophorous vacuole destination inside
Vero cells persistently infected with C. burnetii
To examine the possibility that the infective stages of T.
cruzi could have distinct trafficking patterns inside Vero
cells infected with C. burnetii, the kinetics of parasitophorous vacuole fusion with the rickettsia vacuole, estimated by
the rate of parasite transference, was studied from 3 to 12 h
after exposure to T. cruzi infective stages. Coverslips
containing Vero cells infected with C. burnetii were then
infected with metacyclic or tissue-culture trypomastigotes
and after 3 h, unattached parasites were removed from the
supernatant and culture medium replaced. Parasitophorous
vacuole fusion to the C. burnetii vacuole was estimated by
counting under phase contrast microscopy parasites effectively transferred to the rickettsia compartment. Metacyclic
trypomastigote parasitophorous vacuoles fused with C.
burnetii vacuoles to a higher rate than tissue-culture
trypomastigote parasitophorous vacuoles (Fig. 2A, B).
After 12 h, about 80% of internalised metacyclic trypomastigotes co-localised within C. burnetii vacuoles and the
percentage of parasite transfer to C. burnetii vacuoles
increased linearly with time (Fig. 2A). In contrast, tissueculture trypomastigote transfer to C. burnetii was less
efficient (Fig. 2B). From 6 to 12 h post-infection, there was
no significant increase in tissue-culture trypomastigote
transfer to C. burnetii vacuoles (Fig. 2B), and approximately 50% of internalised parasites were transferred to C.
burnetii vacuoles. Trypanosoma cruzi tissue-culture trypomastigotes disrupt the parasitophorous vacuole membrane
by secreting an acid-active haemolysin and engaging transsialidase activity (Andrews, 1994; Hall et al., 1992) in order
to multiply as amastigotes in the host cell cytoplasm.
To examine the possibility that metacyclic trypomastigotes remain within the parasitophorous vacuole for
longer periods than tissue-culture trypomastigotes
because of poor haemolysin (Andrews and Whitlow,
1989) activity, we directly tested low-pH haemolytic
activity of these forms. Our results confirmed that Y
(used by Andrews and Whitlow, 1989) as well as CL
strain tissue-culture trypomastigotes have higher haemo-
lytic activities than CL strain metacyclic trypomastigotes
(Fig. 3). In these experiments tissue-culture trypomastigotes clearly showed haemolytic activity, in contrast to
metacyclic trypomastigotes that displayed no detectable
activity.
It has been previously shown that trans-sialidase activity
involved in removing sialic acid from lysosomal glycoproteins lining the parasitophorous vacuole (Hall et al.,
1992) is higher in tissue-culture than in metacyclic
trypomastigotes (Pereira-Chioccola et al., 2000). We have
confirmed these results and found that trans-sialidase
activity of tissue-culture trypomastigotes is at least three
times higher than metacyclic trypomastigotes (CL strain,
data not shown). In order to test whether the sialylation of
lysosomal glycoproteins could account for some of the
observed differences in the transfer rates of trypomastigotes
from parasitophorous vacuole to C. burnetii vacuoles,
experiments were performed with CHO cells and its related
Lec-2 CMP-sialic acid defective mutant (Deutscher et al.,
1984) both persistently infected with C. burnetii. Our results
showed that metacyclic trypomastigotes are less efficiently
transferred to C. burnetii vacuoles present in Lec-2 cells
Fig. 2. Distinct kinetics of T. cruzi trypomastigote transfer to C. burnetii
vacuoles in Vero cells. Vero cells persistently infected with C. burnetii
were fixed with 3.5% formaldehyde from 3 to 12 h of infection with
metacyclic (A) or cell-derived trypomastigotes (B). The percentage of
parasites transferred to C. burnetii vacuoles corresponds to the ratio
between the total number of parasites inside the cells (of about 100 cells per
coverslisp, in triplicate) and the trypomastigotes within the bacteria
vacuoles.
W.K. Andreoli, R.A. Mortara / International Journal for Parasitology 33 (2003) 185–197
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and, after 24 h post-infection, less than 30% of the
intracellular parasites had been transferred to the bacterium
vacuoles. Comparing with CHO cells, the transference of
tissue-culture trypomastigotes was apparently also altered in
Lec-2 cells persistently infected with C. burnetii but the
differences were smaller than for metacyclic trypomastigotes (Fig. 4A). These results suggested that in sialic acid
deficient Lec-2 cells, metacyclic trypomastigotes escape
faster from the parasitophorous vacuole. To confirm these
observations, we compared LAMP-1 labelling of metacyclic trypomastigote parasitophorous vacuoles in Lec-2 and
CHO cells at different times of infection and found that
LAMP-1 þ parasitophorous vacuoles remain for up to 12 h
in CHO whereas in Lec-2 cells there is a marked decrease of
LAMP-1 labelled parasites with time (Fig. 4B).
3.3. Characterisation of T. cruzi parasitophorous vacuoles
inside Vero cells persistently infected with C. burnetii
Since parasitophorous vacuoles from the two trypomastigote infective stages show different kinetics of
fusion to C. burnetii vacuoles inside Vero cells, we
carried out a preliminary parasitophorous vacuole membrane typing with endocytic pathway markers and clear
differences were observed between parasitophorous vacuoles formed during entry of the two trypomastigote
stages. In these experiments, cytoplasmically located
parasites were only considered to be within parasitophorous vacuoles when labelling with one of the three
markers was positive. When parasite-containing vacuoles
were labelled for EEA1, LAMP-1, and Rab7 noticeable
differences in the proportion of labelled parasites and
kinetics of marker expression between the two types of
parasitophorous vacuoles were found (Fig. 5A, B). After
the initial 90 min of infection, EEA1 labelled about 35%
Fig. 3. Haemolytic activity of T. cruzi infective stages of CL and Y strains.
Erythrocyte lysis was assayed at pH 5.5 in acetate buffer. Tissue-culture
(TCT) and metacyclic trypomastigotes from CL strain (META CL) were
compared with tissue-culture trypomastigotes of the Y strain. From 6 h, the
haemolytic activities of Y and CL strains tissue-culture trypomastigotes
were significantly higher than META CL (P # 0:05, from standard
deviations of three independent experiments). SL, spontaneous lysis.
Fig. 4. Distinct kinetics of T. cruzi trypomastigote transfer to C. burnetii
vacuoles in CHO and mutant Lec-2 cells. (A) Lec-2 (closed bars) and wild
type CHO cells (open bars) show different kinetics of parasite transfer to C.
burnetii vacuoles. At 12 and 24 h the differences in metacyclic
trypomastigote transfer to C. burnetti vacuoles in CHO cells is significantly
different from Lec-2 cells ð* P # 0:05Þ, whereas no significant differences
are observed for tissue-culture trypomastigote (TCT) transfer. (B)
Metacyclic trypomastigotes escape faster from Lec-2 than CHO parasitophorous vacuole. LAMP-1 positive parasites (indicative of parasitophorous vacuole residence) were scored in cells after 6 and 12 h of infection
and it is seen that the proportion of labelled parasites is consistently lower
in Lec-2 cells.
of metacyclic trypomastigotes that were localised in the
cell cytoplasm (and not in the C. burnetii vacuole) and
while EEA1 decreased to less than 10% at 12 h Rab7, a
late endosomal marker, as well as LAMP-1 positive
parasites increased at the later stages of infection
reaching about 80% of the cytoplasmic parasites after
12 h (Fig. 5A). The pattern of EEA1 labelling of tissueculture trypomastigotes parasitophorous vacuoles was
found to be similar to that of metacyclic trypomastigotes
parasitophorous vacuoles, decaying as infection progressed (Fig. 5B). However, the trend of the parasitophorous vacuole labelling with late endosomal markers
was remarkably different (Fig. 5B). After 12 h, less than
40% of the cytoplasmic parasites could be labelled for
late endosomal markers indicating that most parasites had
already escaped from the parasitophorous vacuoles.
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When the distribution of EEA1 positive vacuoles was
examined by confocal fluorescence microscopy, most of the
labelled parasites were located at the cell periphery, in
arrangements compatible with the initial stages of invasion
(Fig. 6). By contrast, Rab7 and LAMP-1 positive parasitophorous vacuoles localised closer to the C. burnetii
vacuoles (Fig. 7).
3.4. Transference of metacyclic trypomastigote to C.
burnetii vacuoles is related to low vacuolar pH
In order to assess whether parasite transference could be
influenced by the acidic milieu found within C. burnetii
vacuoles, we performed experiments in which the C.
burnetii intravacuolar pH was modified. For this purpose,
we examined the influence of the vacuolar ATPase
inhibitors bafilomycin A1 and concanamycin A, as well as
of chloroquine on the kinetics of metacyclic and tissueculture trypomastigote transference to C. burnetii vacuoles.
Non-invasive ratiometric confocal microscopy with duallabel SNAFL – calcein-AM probes was used to measure the
intravacuolar pH. We found that C. burnetii vacuoles, in
agreement with pervious observations (Maurin et al., 1992)
have an intravacuolar pH ranging between 4.0 and 5.8, and
bafilomycin A1 raised it to values of around 6.5, persisting
Fig. 5. Characterisation of T. cruzi parasitophorous vacuoles in Vero cells
containing C. burnetii vacuoles. Metacyclic (A) or cell-derived trypomastigotes (B) parasitophorous vacuoles were labelled for EEA1, LAMP-1, and
Rab7 at 1.5 and 12 h of mixed infection. The percentage of labelled
parasites refers only to trypomastigotes remaining in the cytoplasm (Cyto)
and hence not transferred to C. burnetii vacuoles. Mean values and standard
errors were obtained from 300 cells counted on triplicate coverslips of three
independent experiments. ***denotes lack of Rab7 labelling.
Fig. 6. EEA1-positive parasitophorous vacuoles are localised at the cell
periphery. EEA1 labels parasitophorous vacuoles at the cell edge after 3 h
of infection. (A) Nomarski differential interference contrast (DIC) image;
(B) EEA1 labelling of metacyclic trypomastigote parasitophorous vacuole;
(C) merged image of EEA1 (in red) and DAPI (blue). Bar ¼ 10 mm.
W.K. Andreoli, R.A. Mortara / International Journal for Parasitology 33 (2003) 185–197
at least 6 h after the drugs were washed (data not shown).
After 6 h of parasite infection metacyclic trypomastigote
transfer to C. burnetii vacuoles was inhibited by 80%
following bafilomycin A1 or concanamycin A treatment and
by 60% in the presence of chloroquine (Fig. 8). Similar
inhibitory effects were obtained for tissue-culture trypomastigote transference to alkalinised C. burnetii vacuoles
(data not shown).
Experiments using live metacyclic trypomastigotes
stained with the vital DNA probe Hoechst 33258 revealed
an interesting pH dependence of the vacuolar transference
process. We observed that the more acidic the C. burnetii
vacuoles were, the higher was the degree of metacyclic
trypomastigote transference to the vacuoles (Fig. 9A).
Quantitation of intravacuolar parasites showed that there
was a higher proportion of metacyclic trypomastigotes
transferred to C. burnetii vacuoles with pH ranging from 4.5
to 5.0 (Fig. 9B).
4. Discussion
We have reported here that cell invasion by two distinct
T. cruzi trypomastigote forms is affected by modifications of
the cytoplasmic milieu with alkalinising agents or the
presence of large cytoplasmic vacuoles of C. burnetti. Also,
Fig. 7. LAMP-1- and Rab7-positive parasitophorous vacuoles localise
closer to the C. burnetii vacuole. After 6 h of infection, metacyclic
trypomastigote parasitophorous vacuoles are closer to the C. burnetii
vacuole. (A) Nomarski differential interference contrast (DIC) image; (B)
LAMP-1 labelling; (C) labelling for Rab7; (D) merged image of LAMP-1
(red), Rab7 (green), and DAPI (blue). Note in (D) areas of co-localisation of
LAMP1 and Rab7 compartments that appear in yellow/orange tones.
Bar ¼ 10 mm.
193
Fig. 8. Increasing C. burnetii vacuolar pH inhibits the transfer of metacyclic
trypomastigote. Vero cells chronically infected with C. burnetii were
treated with: 50 nM bafilomycin A1 (BAF A1), 50 nM concanamycin A
(Conca A), and 100 mM chloroquine (CHL) for 1 h, washed, and then
infected with metacyclic trypomastigotes for 6 h. The percentages of
intracellular parasites that co-localise with the C. burnetii vacuole are
indicated.
upon invasion, the two forms display distinct trafficking
characteristics when invading cells that contain in their
cytoplasm large C. burnetii. In control Vero cells, all
alkalinising agents reduced the invasion indexes of both
trypomastigote forms, but had the opposite effect in cells
previously infected with C. burnetti (Fig. 1). It has been
known that bafilomycin A1 not only prevents lysosome
acidification by inhibiting vacuolar Hþ-ATPase (Dröse and
Altendorf, 1997) but also inhibits the trafficking of
endosomal and lysosomal (Clague et al., 1994; Yamamoto
et al., 1998) components and may also retard the recycling
of membrane receptors like transferrin receptor (Presley
et al., 1997). Also, in previous studies Tardieux et al. (1992)
have shown that the NH4Cl-induced lysosomal redistribution inhibited tissue-culture trypomastigote invasion in
NRK cells. There are at least two not mutually exclusive
explanations for our findings. They could indicate that
lysosomal acidification is required for efficient trypomastigote invasion and/or that the putative inhibition of recycling
of both endosomal and lysosomal components by these
agents, interferes with the availability of membrane
elements required to the generation of parasitophorous
vacuoles in Vero cells.
In a similar fashion to the alkalinising agents, the
presence of the large acidic C. burnetti vacuoles in the
cytoplasm also reduced the invasion of Vero cells by the
different trypomastigote stages (Fig. 1A, B). This observation is in line with the reported data that C. burnetii
infected Vero cells represent a system in which the
occurrence of lysosomal depletion or deficiency in lysosomal activity (Akporiaye et al., 1983; Burton et al., 1978)
could inhibit the lysosomal-dependent (Tardieux et al.,
1992) invasion by trypomastigotes (Fig. 1). Depletions of
secondary lysosomes have also been described in mouse
macrophages infected with Leishmania species (Barbieri
et al., 1985). However, the cytoplasmic presence of the
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Fig. 9. Metacyclic trypomastigote parasitophorous vacuoles preferentially
fuse with more acidic C. burnetii vacuoles in chronically infected Vero
cells. Three hours after infection with metacyclic trypomastigotes (labelled
with Hoechst dye), intravacuolar pH was measured with SNAFL-calcein
AM, and the number of transferred parasites counted from at least 100 cells.
Cells were grouped into three pH ranges and the numbers of parasites/vacuole were scored for each group. Cells containing more acidic vacuoles
showed higher numbers of parasites within the vacuoles, in contrast to cells
with less acidic vacuoles that contained fewer transferred parasites (A). The
values represent typical results from three independent experiments. (B) C.
burnetiivacuoles in chronically infected Vero were labelled with SNAFL–
calcein AM and imaged by confocal microscopy. The more acidic the
intravacuolar pH, the greener the organelle appears, whereas a more
alkaline environment appear in tones towards the yellow range. (B)
SNAFL-calcein AM dual emission images (green/red) merged with images
of parasite (arrows) nuclei and kinetoplasts labelled with Hoechst dye
(blue). Bar ¼ 50 mm.
rickettsia had a dramatic, and opposite effect when vacuolar
ATPases were specifically inhibited with bafilomycin A1 or
concanamycin A, or the overall cytoplasmic pH raised with
chloroquine (Fig. 1). In all these experiments, trypomastigote invasion in Vero cells harbouring, C. burnetti was
enhanced when the vacuolar pH was raised (Fig. 1),
suggesting that raising the pH somehow overcomes the
lack of available lysosomal components required for
invasion. Exactly how this operates in C. burnetti infected
Vero cells is still unclear but we have shown that the
distribution of LAMP-1 bearing compartments (including
the rickettsia vacuole) is greatly affected upon treatment
with bafilomycin A1 and become much more dispersed in
the cytoplasm (Fig. 1C –F).
Upon invasion of Vero cells chronically infected with C.
burnetti, tissue-culture and metacyclic trypomastigotes
display distinct trafficking patterns. Metacyclic trypomastigotes have been shown to express reduced transialidase
activity (Pereira-Chioccola et al., 2000) and display almost
undetectable haemolysin activity when compared to tissueculture trypomastigotes (Fig. 3). These activities that are
related to the ability of the parasite to escape from the
parasitophorous vacuole and colonise the cytoplasm
(Andrews, 1994; Hall et al., 1992), correlate well with the
maturation pattern of the metacyclic trypomastigote parasitophorous vacuoles (Fig. 5) and the longer times that these
forms remain within their parasitophorous vacuoles rendering them more susceptible to be transferred to the C.
burnetti vacuoles (Fig. 2). By contrast, tissue-culture
trypomastigotes that display higher haemolysin (Fig. 3)
and transialidase activities (Pereira-Chioccola et al., 2000)
reside shorter periods in their parasitophorous vacuoles
(Fig. 5) and are much less efficiently transferred to the C.
burnetti vacuole (Fig. 2). When persistently infected with C.
burnetii, Lec-2 sialic acid mutant cells that were previously
shown to facilitate tissue-culture trypomastigote escape
from the parasitophorous vacuole (Hall et al., 1992),
displayed the same pattern as described above and it was
observed that metacyclic trypomastigotes were not only less
efficiently transferred to the C. burnetti vacuole but also
escaped much faster into the cytoplasm, when compared
with wild type CHO cells (Fig. 4). These observations
confirmed the role of sialylated lysosomal glycoproteins
(Hall et al., 1992) in retarding the escape of both
trypomastigote forms from the parasitophorous vacuoles.
The differences in the parasitophorous vacuole maturation
patterns exhibited by the two trypomastigote forms when
invading Vero cells colonised with C. burnetti may also be
associated with parasite ligands and the corresponding
putative host cell receptors. It has been shown that because
T. cruzi epimastigotes interact with CR3 or FcR receptors in
J774 macrophages, whereas metacyclic trypomastigotes
avoid these molecules, the resulting phagolysosomes are
strikingly different and epimastigotes are eventually
destroyed (Hall et al., 1991).
The expression of distinct repertoires of parasite surface
molecules that can take part in the invasion process may
thus be also related to the differences in parasitophorous
vacuole formation and maturation by tissue-culture and
metacyclic trypomastigotes. Tissue-culture trypomastigotes
apparently trigger invasion entry through a cytosolic
parasite oligopeptidase B (Burleigh et al., 1997), whereas
in metacyclic trypomastigotes an 82 kDa glycoprotein and a
35/50 kDa mucin-like component are involved (Ramirez
et al., 1993; Ruiz et al., 1993, 1998; Yoshida et al., 1989). It
is thus conceivable that the engagement of different surface
molecules by metacyclic and tissue-culture trypomastigote,
particularly the mucin-like glycoproteins (Acosta-Serrano
W.K. Andreoli, R.A. Mortara / International Journal for Parasitology 33 (2003) 185–197
et al., 2001), could have consequences on signalling
pathways (Burleigh and Andrews, 1998; Yoshida et al.,
2000) and the receptors mobilised and consequently, on
parasitophorous vacuole formation and parasite escape.
Differences in surface antigen composition could provide
the parasite better resources to survive in the parasitophorous vacuole acid environment. It has been shown that
metacyclic trypomastigotes are more resistant than tissueculture trypomastigotes to severe conditions and are capable
of avoiding gastric secretions and invade the gastric
mucosal epithelium (Hoft, 1996). This ability may be
related to the highly glycosilated mucin-like glycoproteins
expressed by metacyclic trypomastigotes that differ from
those found on tissue-culture trypomastigotes (Acosta-Serrano et al., 2001). Thus, metacyclic trypomastigote mucins
could provide additional means for survival in low pH
conditions such as the parasitophorous vacuole microenvironment, for longer periods than tissue-culture
trypomastigotes.
The observations made here that the two trypomastigote
stages will form parasitophorous vacuoles with different
intracellular fates and maturation characteristics, suggest
that the different surface molecules found on each T. cruzi
form could also interact with the molecules responsible for
the endocytic fusion machinery. Recent experiments
performed by Desjardins and colleagues showed the
influence of lipophosphoglycan on Leishmania donovani
promastigotes phagosome maturation (Dermine et al., 2000;
Scianimanico et al., 1999). Lipophosphoglycan from L.
donovani promastigotes can prevent fusion of the parasitophorous vacuole to late compartment and degradation
within lysosomes (Dermine et al., 2000). Lipophosphoglycan appears to act by controlling the acquisition of Rab7 and
LAMP-1 molecules until promastigotes transform into
amastigotes. Once transformation occurs, L. donovani
loses lipophosphoglycan and the phagosome begins to
acquire hydrolases to which amastigotes are resistant
(Scianimanico et al., 1999). The characterisation of
trypomastigote parasitophorous vacuoles by immunofluorescence labelling for EEA1, Rab7, and LAMP-1 revealed
that metacyclic trypomastigote parasitophorous vacuole
maturation occurs in a more organised and orderly fashion
when compared with tissue-culture trypomastigote parasitophorous vacuoles (Fig. 4A, B). This was also observed
when we examined the parasitophorous vacuole distribution
in relation to the C. burnetii vacuole. Whereas EEA1
labelling was typically observed around recently invading
parasites (Fig. 6A –C), late endocytic markers labelled
parasitophorous vacuoles closer to the C. burnetii vacuoles
(Fig. 7A – D).
Another interesting aspect that arose from the present
studies is the pH dependence on the extent of fusion between
metacyclic parasitophorous vacuole and C. burnetii vacuole. Clearly parasite parasitophorous vacuoles fused preferentially with the population of C. burnetti vacuoles
displaying the lower pHs (Fig. 9) and raising vacuolar pH
195
with bafilomycin A1, concanamycin A or chloroquine
inhibited this process (Fig. 8). At least two possible
explanations, not mutually exclusive, can be put forward
at this point. It is possible that the more acidic C. burnetii
vacuoles could be more fusogenic but it seem also
reasonable to imagine that some signal of the fusing
machinery could be involved in the parasitophorous vacuole
membrane recognition of the more acidic rickettsia
vacuoles.
We are currently examining the kinetics of parasitophorous vacuole formation and escape of different T. cruzi
infective forms in cultured cells in order to expand our
knowledge in this area, aiming at a better understanding of
the interactions that occur during the intracellular trafficking
and maturation of the parasite parasitophorous vacuole.
Acknowledgements
We are deeply grateful to Michel Rabinovitch for
introducing us to the Coxiella system, for providing the
initial support to this project, and for all the ideas. We thank
Sergio Schenkman for his help with cells and suggestions.
We are also indebted to Dr Nobuko Yoshida for her
suggestions on the manuscript and Elettra Greene for
carefully reviewing the text. W.K.A. is recipient of a
Doctorate Fellowship from CAPES. The financial support
from FAPESP and CNPq through grants and fellowships to
R.A.M. are also acknowledged.
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