activation of distinct signal transduction pathways in trypanosoma cruzi isolates with differential...

Activation of distinct signal transduction pathways in Trypanosoma cruziisolates with differential capacity to invade host cells

Ivan Neiraa, Alice T. Ferreirab, Nobuko Yoshidaa,*

aDepartamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, R. Botucatu,

862-6o andar, 04023-062 Sao Paulo, SP, BrazilbDepartamento de Biofısica, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, R. Botucatu, 862-6o andar, 04023-062 Sao Paulo, SP, Brazil

Received 2 November 2001; received in revised form 20 December 2001; accepted 20 December 2001

Abstract

Mammalian cell invasion by Trypanosoma cruzi requires the activation of signal transduction pathways that result in a Ca21 response both

in the parasite and the host cell. By using drugs that interfere with the signalling processes, we investigated if the difference in the ability of T.

cruzi isolates to invade host cells was associated with the activation of distinct signalling routes in the parasites. Experiments were performed

with metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, using the highly invasive isolate CL

and the poorly infective isolate G, which belong to distinct phylogenetic lineages. Treatment of parasites with adenylyl cyclase activator

forskolin increased the infectivity of the G but not of the CL isolate towards HeLa cells. On the other hand, a specific protein tyrosine kinase

inhibitor genistein reduced by ,75% the penetration of CL but not of G isolate into HeLa cells. In the CL but not in the G isolate, protein

tyrosine kinase mediated the phosphorylation of a 175 kDa protein in a manner inducible by a HeLa cell extract. Upon treatment with the

phospholipase C inhibitor U73122, or with drugs such as caffeine, which affects Ca21 release from inositol-1,4,5-triphosphate-sensitive

stores, or thapsigargin, an inhibitor of intracellular Ca21 transport ATPases, the infectivity of the CL but not of the G isolate diminished

significantly ðP , 0:005Þ. In both isolates, a combination of ionomycin plus NH4Cl or nigericin released Ca21 from acidic vacuoles

containing a Ca21/H1 exchange system. This treatment reduced the infectivity of metacyclic forms of the G but not of the CL isolate.

Taken together, these data suggest that, for host cell invasion, distinct signalling pathways are activated in metacyclic trypomastigotes of the

two isolates, leading to Ca21 release from different intracellular compartments. q 2002 Australian Society for Parasitology Inc. Published by

Elsevier Science Ltd. All rights reserved.

Keywords: Trypanosoma cruzi; Metacyclic trypomastigotes; Cell invasion; Signal transduction; Ca21 mobilisation; Acidocalcisomes

1. Introduction

As is the case of many pathogenic microorganisms, the

virulence of Trypanosoma cruzi, the protozoan parasite that

causes Chagas’ disease in humans, is associated with its

ability to invade host cells. A number of studies have

shown that invasion of mammalian cells by T. cruzi requires

the activation of signal transduction pathways leading to an

increase in cytosolic Ca21 concentration, both in the parasite

and the host cell (Moreno et al., 1994; Tardieux et al., 1994;

Ruiz et al., 1998; Yoshida et al., 2000).

The efficiency in entering non-phagocytic mammalian

cells may vary widely between T. cruzi strains and isolates

belonging to different phylogenetic lineages. Trypanosoma

cruzi strains have been placed into two major subgroups or

phylogenetic lineages by ribosomal typing based on the

variable region D7 of the large subunit ribosomal RNA,

mini-exon gene sequence, random amplified polymorphic

DNA and multilocus enzyme electrophoresis (Souto et al.,

1996; Briones et al., 1999; Brisse et al., 2000). The T. cruzi

isolates CL and G, which have been shown to differ in their

ability to invade host cells (Ruiz et al., 1998), were included

in subgroups 1 and 2, respectively (Briones et al., 1999). In

metacyclic trypomastigotes, the developmental forms that

initiate infection in the mammalian host, the variability

between these isolates has been associated with differential

expression of surface glycoproteins, which bind to target

cells in a receptor-mediated manner and exhibit differential

Ca21 signalling activities (Ruiz et al., 1998). One interesting

possibility is that the interaction of these molecules with

their receptors activates different signalling pathways in

metacyclic forms of T. cruzi isolates displaying differential

cell-invasion capacity. Experiments with the highly inva-

sive CL isolate have suggested that engagement of gp82,

International Journal for Parasitology 32 (2002) 405–414

0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(02)00004-8

www.parasitology-online.com

* Corresponding author. Fax: 155-11-571-1095.

E-mail address: [email protected] (N. Yoshida).

a metacyclic stage-specific surface molecule implicated in

cell invasion (Ramirez et al., 1993), transduces the external

signal to the parasite interior upon exposure to the target

cell, triggering a cascade that involves activation of protein

tyrosine kinase (Favoreto et al., 1998) and phospholipase C,

with generation of inositol 1,4,5-triphosphate that mediates

Ca21 mobilisation from inositol 1,4,5-triphosphate-sensitive

intracellular compartments (Yoshida et al., 2000). Whether

poorly invasive T. cruzi isolates engage alternative routes to

enter host cells remains to be investigated.

A question that also awaits clarification is whether,

depending on parasite isolate and/or developmental stage,

distinct intracellular stores may serve as source of Ca21

required for host cell invasion. Studies with amastigotes,

epimastigotes and cell-derived trypomastigotes have been

unable to detect Ca21 release from inositol 1,4,5-tripho-

sphate-sensitive stores (Moreno et al., 1992; Docampo et

al., 1993). Another study, which also did not include meta-

cyclic trypomastigotes, has reported that most of the Ca21

stored in different T. cruzi developmental forms is present in

acidic compartments containing a Ca21/H1 exchange

system (Docampo et al., 1995).

In the present study, we used metacyclic trypomastigotes

of T. cruzi isolates G and CL, differing widely in their ability

to enter mammalian cells, to investigate the possibility that

different signal transduction pathways are activated in these

parasites during cell invasion and that the Ca21 required for

that process is released from distinct intracellular Ca21

stores.

2. Materials and methods

2.1. T. cruzi, mammalian cells and cell-invasion assay

We used in this study two T. cruzi isolates: G, isolated in

the Amazon from a marsupial of the family Didelphidae

(Yoshida, 1983), to which belong the oldest marsupials

known in South America (Simpson, 1980), and CL, isolated

in southern Brazil from Triatoma infestans in dwellings

where people were infected with T. cruzi (Brener and

Chiari, 1963). Parasites were maintained by cyclic passage

in mice and in axenic cultures in liver infusion tryptose

medium (Camargo, 1964). Grace’s medium was also used

to obtain cultures enriched in metacyclic forms, which were

purified by passage through DEAE-cellulose column, as

described (Teixeira and Yoshida, 1986). HeLa cells, the

human carcinoma-derived epithelial cells, were grown at

378C in Dulbecco’s minimum essential medium (DMEM)

supplemented with 10% foetal calf serum (FCS), streptomy-

cin (100 mg/ml) and penicillin (100 U/ml) in a humidified

5% CO2 atmosphere. Host cell invasion assays were carried

out as previously described (Yoshida et al., 1989), by seed-

ing metacyclic forms onto each well of 24-well plates

containing 13-mm diameter round glass coverslip coated

with 1.5 £ 105 HeLa cells, at parasite:cell ratio of 10:1 for

the CL isolate and 50:1 for the G isolate. After 30 min

incubation, the duplicate coverslips were washed in phos-

phate buffered saline (PBS) and stained with Giemsa.

2.2. Detection of tyrosine phosphorylated T. cruzi proteins

In standard experiments, 5 £ 107 parasites were incubated

for 20 min at 378C in the absence or presence of HeLa cell

extract, equivalent to 160 mg/ml protein, in a total volume

of 200 ml. After washing in PBS, the parasites were

disrupted at 48C in a lysis solution containing phosphatase

and protease inhibitors (50 mM Tris–HCl pH 7.4, 150 mM

NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM NaVO4, 1 mM

NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoace-

tamide, 25 mg/ml leupeptin, 25 mg/ml antipain). Lysates

were dissolved in loading buffer and subjected to electro-

phoresis in a 10% sodium dodecyl sulfate (SDS)-polyacry-

lamide gel, under reducing conditions, and the proteins were

transferred to nitrocellulose membrane. Following blockage

with PBS containing 5% defatted milk, the nitrocellulose

membrane was incubated for 1 h at room temperature with

anti-phosphotyrosine antibodies (mouse monoclonal anti-

body (mAb) from Sigma) diluted in PBS-milk. After several

washes in PBS containing 0.05% Tween 20, the membrane

was incubated with anti-mouse IgG conjugated to peroxi-

dase. The final reaction was revealed by chemiluminescence

using the ELC Western blotting detection reagent and

Hyperfilm-MP from Amersham.

2.3. Preparation of HeLa cell extract

The cell extract used in phosphorylation experiments was

prepared as follows. After washings in PBS, HeLa cells

were detached by scraping, suspended in PBS containing

protease inhibitors (1 mM phenylmethylsulfonyl fluoride,

1 mM iodoacetamide, 25 mg/ml leupeptin, 25 mg/ml anti-

pain) and then sonicated on ice in an ultrasonic processor

XL (two pulses of 30 s each). After ascertaining the disrup-

tion of cells under phase contrast microscope, the sonicated

preparation was centrifuged at 12,000 £ g for 5 min, the

supernatant was collected and immediately used for experi-

ments, or stored at 2808C until use.

2.4. Determination of T. cruzi intracellular Ca21

concentration

To measure cytosolic free Ca21, [Ca21]i in metacyclic

trypomastigotes, we proceeded as follows. Parasites were

washed in buffer A, pH 7.2, containing 116 mM NaCl,

5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM N-(2-hydroxyethyl)-

piperazine-N 0-(2-ethanesulfonic acid) (HEPES). After

adjusting the concentration to 108 cells/ml, the parasites

were incubated with 5 mM fura 2/AM (Sigma) for 3 h at

room temperature and non-incorporated fura 2 was washed

out. Fluorescence was read in a fluorophotometer SPEX

AR-CM system (Spex Industries) with dual wavelength

excitation (340 and 380 nm) and emission at 510 nm.

I. Neira et al. / International Journal for Parasitology 32 (2002) 405–414406

Increase in T. cruzi [Ca21]i, resulting from addition of drugs

to 2.5 ml of parasite suspension, was calculated as described

by Grynkiewicz et al. (1985). For each preparation we deter-

mined Rmax and Rmin, which correspond to the fluorescence

ratio at 340 and 380 nm in the presence of saturating Ca21

after treatment with 50 mM digitonin, and in the absence of

Ca21, upon addition of 10 mM EGTA, respectively.

2.5. Treatment of parasites with different drugs

Treatment of metacyclic trypomastigotes with 250 mM

genistein or 10 mM forskolin was performed by incubation

of the parasites at 378C for 30 min in the presence of the

drug, followed by washing in PBS. Other treatments

included incubation with: 1 mM thapsigargin for 30 min at

378C; 1 mM U73122 for 4 min at 378C; 100 mM neomycin

for 4 h at 378C; 10 mM caffeine for 30 min at 378C; iono-

mycin (1 mM) plus NH4Cl (20 mM) or nigericin (1 mM) for

10 min at 378C. For treatment with BAPTA-AM, the tetra-

acetoxymethyl ester of bis-(o-aminophenoxy)-ethane-

N,N,N 0,N 0 tetraacetic acid, we followed the procedure

described for tissue culture trypomastigotes (Yakubu et

al., 1994) by incubating the parasites for 2 h at room

temperature with 25 mM of the drug.

2.6. Statistics

Student’s t test was used to determine significance.

3. Results

3.1. Metacyclic trypomastigotes of T. cruzi isolates G and

CL differ widely in infectivity in vitro and in vivo

Cell-invasion assays have systematically shown a marked

difference in the ability of metacyclic forms of CL and G

isolates to enter mammalian cells. Fig. 1A shows the result

of assays in which the parasites were incubated for 3 h with

HeLa cells, at parasite:cell ratio of 10:1. The number of CL

isolate metacyclic trypomastigotes that invaded HeLa cells

was about eight times higher than that of the G isolate. Such

a difference in infectivity was also observed with Vero cells

and mouse 3T3 fibroblasts (data not shown), being consis-

tent with the finding that metacyclic trypomastigotes of

these T. cruzi isolates produce distinct courses of infection

in mice, high parasitaemia resulting from inoculation with

CL isolate whereas infection with G isolate is always subpa-

tent (Yoshida, 1983). Due to the low infectivity of the G

isolate, for short time (30 min) invasion assays, such as

those aimed at examining the signalling events, we used a

parasite:cell ratio of 50:1. Even at this high multiplicity of

infection, fewer metacyclic forms of the G isolate entered

HeLa cells as compared with the CL isolate, at parasite:cell

ratio of 10:1 (Fig. 1B). In parallel with Giemsa staining, in a

few cell-invasion assays the internalised parasites were also

visualised by sequential reaction with antibodies directed to

a host cell lysosomal membrane glycoprotein and to human

IgG conjugated to fluorescein. This procedure allows an

accurate monitoring of intracellular localisation of parasites,

which enter target cells internalised in a membrane vacuole

formed with the contribution of lysosomes and are therefore

detectable by staining lysosomal markers (Tardieux et al.,

1992). We obtained similar results by Giemsa staining and

detection of lysosome markers, indicating that the simple

Giemsa staining is reliable if one counts only the parasites

judged to be unequivocally located inside the cells.

3.2. Host cell invasion by metacyclic forms of T. cruzi G and

CL isolates is differentially affected by treatment of

parasites with protein tyrosine kinase inhibitor

We examined the effect of genistein, a specific inhibitor

of protein tyrosine kinase which scarcely inhibits the

enzyme activities of serine- and threonine-specific protein

kinases such as cAMP-dependent protein kinase, phosphor-

ylase kinase and the Ca21/phospholipid-dependent enzyme

protein kinase C (Akiyama et al., 1987). Metacyclic trypo-

mastigotes and HeLa cells were incubated separately in the

absence or presence of the drug at 250 mM. After washing

in PBS, control and genistein-treated parasites were incu-

bated with treated or untreated HeLa cells, for 30 min at

378C, in DMEM medium containing 10% FCS. Following

washings in PBS, HeLa cells were stained with Giemsa and

the number of intracellular parasites was counted in at least

I. Neira et al. / International Journal for Parasitology 32 (2002) 405–414 407

Fig. 1. Differential infectivity of metacyclic trypomastigotes of Trypano-

soma cruzi CL and G isolates. (A) Metacyclic forms were incubated with

HeLa cells at 378C for 3 h at parasite:cell ratio of 10:1. (B) Parasites were

incubated with HeLa cells at 378C for 30 min at the indicated multiplicity of

infection. In both cases, HeLa cells were washed in PBS and the number of

intracellular parasites was counted in a total of at least 500 Giemsa-stained

cells. Values are the means ^ SD of 10 experiments (A) or six experiments

(B).

500 cells. As shown in Table 1, genistein significantly ðP ,

0:0001Þ inhibited cell invasion (,75%) of CL isolate meta-

cyclic forms, whereas the infectivity of the G isolate

towards HeLa cells was not affected. Pretreatment of

HeLa cells with 250 mM genistein for 30 min did not impair

the invasion of either CL or G isolate, confirming reports

that host cell protein tyrosine kinase is not implicated in T.

cruzi entry into non-phagocytic mammalian cells (Rodri-

guez et al., 1995; Favoreto et al., 1998).

3.3. Protein tyrosine kinase-mediated p175 phosphorylation

is inducible by HeLa cell extract in CL isolate but not in G

isolate metacyclic trypomastigotes

In CL isolate metacyclic forms, protein tyrosine kinase

mediates tyrosine phosphorylation of a 175 kDa protein

(p175) in a manner inducible by soluble extract of HeLa

cells but not of K562 cells which are resistant to T. cruzi

infection (Favoreto et al., 1998), consistent with protein

tyrosine kinase-dependent parasite internalisation (Table

1). To determine the G isolate protein tyrosine kinase activ-

ity, we assayed the presence of HeLa cell-inducible p175,

using the CL isolate as positive control. Metacyclic trypo-

mastigotes of both T. cruzi isolates were incubated in the

presence or absence of HeLa cell extract for 20 min at 378C.

After washing in PBS, the parasites were disrupted by

adding 1% Triton X-100 solution containing phosphatase

inhibitors. The whole lysates were then analysed by

Western blotting using anti-phosphotyrosine antibodies. In

contrast to CL isolate p175, which had its phosphorylation

levels greatly augmented by parasite exposure to HeLa cell

extract, G isolate p175 was highly tryrosine phosphorylated

regardless of parasite contact with HeLa cell soluble compo-

nents (Fig. 2). Genistein inhibited protein tyrosine kinase

activity of both isolates, as deduced from diminished p175

phosphorylation levels after treatment with the drug (Fig. 2).

The differences observed in the intensity of p175 bands in

different samples are not due to an eventual unequal loading

for we ascertained that equal amounts of parasite extracts

were loaded, either by staining the SDS-PAGE gel with

Coomassie blue or the nitrocellulose membrane with

Ponceau-S. p175 appears to be associated with the parasite

cytoskeleton for it remains in the pellet of Triton X-100-

solubilised parasite extract (data not shown).

3.4. Differential effect of adenylyl cyclase activation on

infectivity of CL and G isolate metacyclic trypomastigotes

Cyclic AMP, synthesised by adenylyl cyclase from ATP,

is a ubiquitous second messenger that in T. cruzi has been

shown to play a role in the control of growth and differen-

tiation (Oliveira et al., 1984; Gonzalez-Perdomo et al.,

1988). Here, we tested the effect of forskolin, an activator

of adenylyl cyclase (Seamon and Daly, 1981; Seamon et al.,

1981; Van Sande et al., 1981), on HeLa cell invasion by T.

cruzi. Metacyclic trypomastigotes and HeLa cells were trea-

ted separately with 10 mM forskolin. After washing in PBS,

the untreated and forskolin-treated parasites were incubated


Fig. 2. Differential tyrosine phosphorylation of p175 in metacyclic trypo-

mastigotes of Trypanosoma cruzi G and CL isolates. Metacyclic forms,

either untreated or treated with 250 mM genistein, were incubated for

20 min at 378C, in absence or presence of HeLa cell extract (HCE), washed

in PBS and disrupted in 1% Triton X-100 lysis solution. The total lysates

were subjected to SDS-PAGE and analysed by immunoblotting using anti-

phosphotryrosine antibodies. Note the HCE-induced increase in genistein-

susceptible p175 phosphorylation levels in CL but not in G isolate parasites.

Table 1

Effect of inhibition of Trypanosoma cruzi protein tyrosine kinase on host cell invasiona

Trypanosoma cruzi isolate Treatment with genistein No. of intracellular

parasites/100 cellsb

% Invasion

Parasite HeLa cell

CL 2 2 24.4 ^ 3.6 100.00

1 2 6.2 ^ 1.5 25.41

2 1 23.4 ^ 4.1 95.90

G 2 2 15.5 ^ 4.5 100.00

1 2 18.2 ^ 5.4 117.42

2 1 19.1 ^ 5.5 123.23

a Metacyclic trypomastigotes, treated (1) or not (2) with genistein, were incubated with HeLa cells pretreated (1) or not (2) with genistein, at 378C for

30 min. After washes in PBS, the number of intracellular parasites was counted in at least 500 Giemsa-stained cells.b The values are the means ^ SD of six experiments performed in duplicate. For CL isolate, the difference between untreated controls and genistein-treated

parasites was significant ðP , 0:0001Þ by Student’s t test.

with control or forskolin-treated HeLa cells, at 378C for

30 min. As shown in Table 2, forskolin-treated metacyclic

forms of the G strain entered cells in higher numbers than

untreated controls whereas the CL isolate infectivity was

unaltered by forskolin. Treatment of HeLa cells with forsko-

lin did not affect infection by CL or G isolate metacyclic

trypomastigotes, even at 100 mM, in contrast with the report

by Rodriguez et al. (1999). These authors treated NRK cells

with 100 mM forskolin and observed an increase in Ca21-

dependent exocytosis of lysosomes, which is essential for

parasite internalisation, as well as in lysosome-mediated

cell invasion by tissue culture-derived trypomastigotes.

The involvement of cAMP in the internalisation of the G

isolate metacyclic forms was further investigated in experi-

ments in which the parasites were preincubated with dibu-

tyryl cAMP, an inhibitor of the enzyme phosphodiesterase

that breaks down cAMP. Pretreatment of parasites with

10 mM dibutyryl cAMP at room temperature for 15 min

resulted in twofold increase in parasite entry into HeLa

cells (data not shown).

3.5. Treatment of parasites with drugs that interfere with

intracellular Ca21 mobilisation differentially affects host

cell invasion by CL and G isolate metacyclic

trypomastigotes

The Ca21 response induced in trypomastigotes upon

interaction with host cells is essential for parasite internali-

sation (Yakubu et al., 1994; Dorta et al., 1995). Accord-

ingly, the parasite invasion is reduced by decreasing the

cytoplasmic free calcium. Metacyclic trypomastigotes (CL

isolate) had their rate of HeLa cell invasion diminished by

,75% when they were pretreated with 25 mM calcium

chelator BAPTA-AM. In the CL isolate, the Ca21 signalling

is affected by drugs that interfere with inositol 1,4,5-tripho-

sphate generation or with inositol 1,4,5-triphosphate-depen-

dent Ca21 release from intracellular stores (Yoshida et al.,

2000). Here, we performed a comparative analysis by test-

ing the effect of several of these drugs on CL and G isolate

infectivity.

To determine the effect of U73122, a specific inhibitor of

phospholipase C (Bleasdale et al., 1990), which mediates

inositol 1,4,5-triphosphate production (Berridge, 1993), the

parasites were incubated in absence or in the presence of

1 mM U73122 and then assayed for HeLa cell invasion.

U73122-treated CL isolate entered HeLa cells in signifi-

cantly lower numbers ðP , 0:0001Þ than untreated controls

whereas the G isolate infectivity was not affected by the

drug (Table 3). In addition to U73122, the effect of neomy-

cin, another phospholipase C inhibitor (Ma and Michel,

1998), was examined. Parasites were incubated with

100 mM neomycin for 4 h at 378C, washed in PBS and


Table 2

Effect of activation of Trypanosoma cruzi adenylyl cyclase on host cell invasiona

Trypanosoma cruzi isolate Treatment with forskolin No. of intracellular


% Invasion

Parasite HeLa cell

CL 2 2 26.4 ^ 4.2 100.00

1 2 23.8 ^ 4.0 91.54

2 1 23.3 ^ 4.2 89.62

G 2 2 17.3 ^ 2.4 100.00

1 2 34.7 ^ 4.7 200.58

2 1 19.5 ^ 2.1 112.72

a Metacyclic trypomastigotes, treated (1) or not (2) with forskolin, were incubated with HeLa cells pretreated (1) or not (2) with forskolin, at 378C for

30 min. After washes in PBS, the number of intracellular parasites was counted in at least 500 Giemsa-stained cells.b The values are the means ^ SD of four experiments performed in duplicate. For G isolate, the difference between untreated controls and forskolin-treated

parasites was significant ðP , 0:001Þ by Student’s t test.

Table 3

Effect of inhibition of Trypanosoma cruzi Ca21 mobilisation on HeLa cell

invasiona

Trypanosoma

cruzi isolate

Inhibitor Cell invading


% Invasion

CL U73122 2 26.9 ^ 3.8 100.00

1 9.1 ^ 3.2 33.83

Thapsigargin 2 27.3 ^ 5.4 100.00

1 6.6 ^ 4.9 24.17

Caffeine 2 29.8 ^ 2.5 100.00

1 10.4 ^ 5.0 34.90

G U73122 2 15.1 ^ 3.4 100.00

1 15.0 ^ 4.5 99.33

Thapsigargin 2 15.1 ^ 1.8 100.00

1 13.4 ^ 2.2 88.74

Caffeine 2 13.7 ^ 1.5 100.00

1 14.0 ^ 1.2 102.19

a Metacyclic trypomastigotes, pretreated (1) or not (2) with the indi-

cated inhibitor, were incubated with HeLa cells for 30 min at 378C and the

number of intracellular parasites was counted in at least 500 Giemsa stained

cells.b The values are the means ^ SD of at least four experiments performed

in duplicate. For the CL isolate, the difference between untreated controls

and the parasites treated either with U73122 ðP , 0:0001Þ, thapsigargin

ðP , 0:005Þ or caffeine ðP , 0:0001Þ was significant by Student’s t test.

then used for invasion assay. Although to a lesser degree

than U73122, neomycin also inhibited (,47%) the CL but

not the G isolate infectivity.

The effect of thapsigargin, a sesquiterpene lactone that

depletes intracellular Ca21 stores in many mammalian cell

types by specific inhibition of endoplasmic reticulum Ca21-

ATPase (Thastrup et al., 1990), was also examined. Para-

sites were treated with 1 mM thapsigargin, at 378C for

30 min before seeding onto HeLa cells. Control parasites

were incubated under the same conditions in the presence

of 0.1% DMSO, equivalent to the DMSO concentration in

the thapsigargin-treated samples. As shown in Table 3, thap-

sigargin significantly inhibited the penetration of CL but not

of G isolate metacyclic forms into HeLa cells. Similar

results were obtained when the parasites were pretreated

at 378C for 30 min with 10 mM caffeine, which inhibits

inositol 1,4,5-triphosphate-induced Ca21 release in different

cell types (Brown et al., 1992; Hirose et al., 1993). In both

assays, incubation of the parasites with the drug was carried

out in buffer A, pH 7.2.

3.6. Host cell invasion by CL and G isolate metacyclic forms

is differentially affected by treatment of parasites with drugs

that release Ca21 from acidocalcisomes

Acidocalcisomes, which are acidic vacuoles containing

Ca21/H1 exchange system and rich in Ca21, have been

detected in epimastigotes, amastigotes and cell-derived

trypomastigotes (Docampo et al., 1995). To examine the

participation of acidocalcisomes in the Ca21 response

required for host cell invasion, we first ascertained the

presence of these organelles in metacyclic trypomastigotes

and then assayed the parasites pretreated with drugs that

release Ca21 from acidocalcisomes for HeLa cell invasion.

The presence of acidocalcisomes was assayed as follows.

Fura 2-loaded metacyclic trypomastigotes in buffer A, with-

out Ca21, were exposed to Ca21 ionophore ionomycin

(1 mM) and subsequently to NH4Cl (20 mM) or nigericin

(1 mM), both of which increase the pH of acidic compart-

ments. Confirming the observations with other T. cruzi

developmental stages (Docampo et al., 1995), Ca21 was

released by ionomycin in relatively small amounts. In the

presence of NH4Cl or nigericin Ca21 release was greatly

potentiated both in CL and G isolate metacyclic forms

(Fig. 3A, B). Similar results were obtained in assays

performed in the presence of the Ca21 chelator EGTA in

the medium.

For invasion assay, which in this case was performed in

PBS containing 1% bovine serum albumin (BSA) instead of

DMEM plus 10% FCS, metacyclic trypomastigotes were

pretreated with ionomycin (1 mM) plus NH4Cl (20 mM) or

nigericin (1 mM) for 10 min at 378C and were then seeded

onto HeLa. As shown in Table 4, the infectivity of the G

isolate was significantly ðP , 0:005Þ reduced by either

treatment whereas that of the CL isolate was unchanged.

Ionomycin by itself had no effect. In addition, we tested

the effect of the proton ionophore carbonyl cyanide p-

trifluoromethoxyphenylhydrazone, by incubating the para-

sites with 1 mM of the drug for 10 min at 378C before cell-

invasion assay. The ability of G isolate to enter HeLa cells,

but not of CL isolate metacyclic forms, was also signifi-

cantly diminished by carbonyl cyanide p-trifluoromethoxy-

phenylhydrazone (Table 4). The inhibitory effect of the drug

was not further augmented when combined with ionomycin.

4. Discussion

Our results indicate that, in order to invade host cells,

metacyclic trypomastigotes of T. cruzi G and CL isolates,

which differ widely in their infectivity, engage distinct

signal transduction pathways. As surface molecules are

differentially expressed in metacyclic forms of these

isolates, different molecules could associate with distinct

signalling routes upon interaction with target cells. Meta-

cyclic forms of the CL isolate, which are highly invasive,

appear to attach to host cells preferentially through gp82

(Ramirez et al., 1993). The cell binding site of this meta-

cyclic stage-specific glycoprotein is a peptide sequence,

located in the central domain of the molecule and possibly


Table 4

Effect of drugs that release Ca21 from Trypanosoma cruzi acidocalcisomes on host cell invasiona

Trypanosoma cruzi isolate Drug Cell invading parasites/100 cellsb % Invasion

CL – 19.8 ^ 2.4 100.00

Ionomycin 1 NH4Cl 19.0 ^ 3.7 95.96

Ionomycin 1 nigericin 13.2 ^ 2.7 67.67

FCCP 19.9 ^ 3.5 100.51

G – 18.3 ^ 2.2 100.00

Ionomycin 1 NH4Cl 7.1 ^ 2.4 38.30

Ionomycin 1 nigericin 5.6 ^ 1.6 30.60

FCCP 9.9 ^ 0.04 54.64

a Metacyclic trypomastigotes, pretreated (1) or not (2) with the indicated drug, were incubated with HeLa cells for 30 min at 378C and the number of

intracellular parasites was counted in at least 500 Giemsa stained cells.b The values are the means ^ SD of three experiments performed in duplicate. For the G isolate, the difference between untreated controls and the parasites

with ionomycin 1 NH4Cl, ionmycin 1 nigericin or FCCP, was significant ðP , 0:005Þ by Student’s t test.

formed by two sequences separated by a hydrophobic

stretch (Manque et al., 2000). On the other hand, host cell

binding of poorly invasive metacyclic forms of the G isolate

is predominantly mediated by gp35/50 (Ruiz et al., 1998).

These mucin-like glycoproteins interact with target cells via

their carbohydrate moiety (Yoshida et al., 1967, 1989).

In the CL isolate, gp82 has been proposed to be the key

mediator in the activation of parasite Ca21 response

required for host cell invasion (Ruiz et al., 1998). Following

gp82–target cell interaction, the upstream events in the para-

site signalling cascade, which include protein tyrosine

kinase activation and p175 tyrosine phosphorylation (Favor-

eto et al., 1998), would ultimately lead to intracellular Ca21

mobilisation (Yoshida et al., 2000). In accord with this

view, treatment of CL isolate metacyclic forms with protein

tyrosine kinase inhibitor genistein impaired Ca21 mobilisa-

tion (Yoshida et al., 2000) and reduced the parasite penetra-

tion into host cells (Table 1). Much less is known about the

signal transduction pathway activated in G isolate metacyc-

lic forms. Although genistein-inhibitable protein tyrosine

kinase was detected in the G isolate, it apparently does

not play a role in cell invasion. One interesting possibility

is that different isoforms of p175 are expressed in CL and G

isolates and are phosphorylated at different tyrosine resi-

dues. In the CL isolate, the inducible tyrosine phosphoryla-

tion of p175 could lead to its association with the

downstream components of the signal transduction cascade,

whereas the phosphorylated tyrosine residues of G isolate

p175 would be unable to establish such an association. On

the other hand, as the Ca21 response is triggered in the G

isolate by a mAb directed to the carbohydrate portion of

gp35/50 (Ruiz et al., 1998), this mucin-like molecule is

thought to be involved in signalling. This is compatible

with the hypothesis that, for host cell entry, the metacyclic

forms of the G strain preferentially engage gp35/50, which

is presumably associated with an alternative protein tyrosine

kinase-independent route. The fact that internalisation of the

G isolate is not affected by treatment of parasites with genis-

tein (Table 1), in addition to the observation that adenylyl

cyclase activator forskolin increases G isolate but not CL

isolate infectivity (Table 2), is consistent with our assump-

tion that alternative signalling pathways are activated in

these T. cruzi isolates during target cell invasion. While

these data shed some light on aspects of T. cruzi signalling

(Table 5), they also raise a number of questions for future

investigations. For instance, does the CL isolate surface

gp82 associate with a transmembrane protein with protein

tyrosine kinase activity or does it establish a connection

through a linker protein? What is the role played in the

CL isolate by p175 and in the G isolate by cAMP generated

by adenylyl cyclase? We envisage the possibility that p175

may be an isoform of phospholipase C, whose activation

follows phosphorylation of key tyrosine residues, as is the

case of phospholipase C-g1 (145–150 kDa) of mammalian

cells (Watson and Arkinstall, 1994). In the same manner as

the phospholipase C-gamma in rat embryo fibroblasts

(McBride et al., 1991), p175 appears to be associated with

the cytoskeleton for it remains in the pellet of Triton X-100-

solubilised parasite extract. The observations suggesting

that in the CL isolate the activation of protein tyrosine

kinase is associated with phospholipase C activation, plus

the finding that p175 is the only protein whose tyrosine

phosphorylation levels are enhanced upon parasite–host

cell interaction, are not incompatible with that hypothesis.

Results of the present study suggest that, despite the

presence of acidocalcisomes, CL isolate metacyclic trypo-

mastigotes do not rely on these acidic compartments for

Ca21 mobilisation required for parasite internalisation.

Treatment of parasites with Ca21 ionophore ionomycin

which, combined either with NH4Cl or nigericin, released

Ca21 from acidocalcisomes (Fig. 3) did not affect parasite

infectivity (Table 4). Observations from a previous study

(Yoshida et al., 2000), and herein confirmed (Table 3), indi-

cate rather that Ca21 is released from thapsigargin-sensitive

intracellular stores in inositol 1,4,5-triphosphate-dependent

manner. Penetration of CL isolate metacyclic forms into

host cell decreased when inositol 1,4,5-triphosphate genera-

tion was inhibited by treatment of parasites with the phos-

pholipase C inhibtors U73122 or neomycin, or when inositol

1,4,5-triphosphate-induced Ca21 release was affected by

drugs such as caffeine and heparin (Yoshida et al., 2000;

Table 3). Neither of these drugs affected the ability of G

isolate metacyclic trypomastigotes to enter host cells (Table

3). In the G isolate, the Ca21 necessary for cell invasion


Table 5

Diversity between metacyclic trypomastigotes of Trypanosoma cruzi CL and G isolates in the process of host cell invasion

Parasite component or event Involvement in cell invasion

CL isolate G isolate

Invasion-promoting surface glycoprotein gp82 gp35/50

gp90 as negative modulator No Yes

Protein tyrosine kinase Yes No

Protein tyrosine kinase substrate p175 ? No

Adenylyl cyclase/cAMP No Yes

Phospholipase C Yes No

Inositol 1,4,5-triphosphate-mediated Ca21 release Yes No

Source of intracellular Ca21 Inositol 1,4,5-triphosphate-sensitive store Acidocalcisome

possibly originates from acidocalcisomes, for treatment of

parasites with ionomycin, in combination with NH4Cl or

nigericin, significantly inhibited host cell entry (Table 4).

Our speculative view is that in the CL isolate, which gener-

ates inositol 1,4,5-triphosphate upon contact with host cells,

Ca21 is released from thapsigargin-sensitive compartments

expressing inositol 1,4,5-triphosphate-dependent receptors.

In the G isolate, which engages an alternative signalling

pathway leading to the enhancement of cAMP, Ca21

would be released from acidocalcisomes through a hypothe-

tical channel, opened for instance by the action of a cMP-

dependent protein kinase. Phosphorylation of the calcium

release channel of the cardiac sarcoplasmic reticulum by

cMP-dependent protein kinase has been reported to result

in the opening of that channel (Marx et al., 2000). The Ca21

release by drugs such as thapsigargin and the combination

of ionomycin plus NH4Cl, observed in both the CL and the

G isolates, is not associated with the process of cell inva-

sion. It simply indicates that distinct calcium stores, either

thapsigargin-sensitive or containing Ca21/H1 exchange

system, are present. Therefore, although comparable

amounts of Ca21 can be released by thapsigargin and by

ionomycin plus NH4Cl (data not shown), as during meta-

cyclic trypomastigote–target cell interaction different

second messengers are produced depending on the T.

cruzi isolate; Ca21 is released by different mechanisms

from distinct parasite organelles.

In addition to the differences found in this study between

CL and G isolates, several others have been reported (Table

5). A stage-specific surface glycoprotein gp90, which has

been shown to negatively modulate the parasite infectivity

(Malaga and Yoshida, 2001), is expressed in G but not in CL

isolate metacyclic trypomastigotes (Teixeira and Yoshida,

1986). Structural differences of related molecules have also

been identified. G isolate gp35/50 contains b-galactofura-

nose among the galactose residues of O-linked oligosac-

charide chains (Acosta-Serrano et al., 1995, 2001). This

b-galactofuranose is part of the epitope recognised by an

mAb capable of inhibiting host cell entry of G isolate meta-

cyclic trypomastigotes (Yoshida et al., 1989). On the other


Fig. 3. Drug-induced Ca21 release from Trypanosoma cruzi acidocalcisomes. Fura 2-loaded metacyclic trypomastigotes, in buffer A, were exposed to: (A)

Ca21 ionophore ionomycin (1 mM) and subsequently to NH4Cl (20 mM) or (B) ionomycin (1 mM) and nigericin (1 mM), at the indicated times, and Ca21 was

measured. Results representative of three experiments are presented.

hand, CL isolate expresses a variant form of gp35/50, which

is not recognised by the aforementioned mAb (Mortara et

al., 1992). In this molecule most of the galactose residues

are present as b-galactopyranose. Another difference, that

may be relevant for infectivity in vivo, is the susceptibility

to complement-mediated lysis by diverse anti-T. cruzi

immune sera, CL isolate metacyclic forms being resistant

to immune sera that lyse 100% of the G isolate (Yoshida,

1983). A relevant question in this context is that related to

the phylogenetic divergence between CL and G isolates.

They belong to distinct T. cruzi subgroups 1 and 2 (Briones

et al., 1999). The CL isolate derived from the insect vector

fed on humans is a member of the subgroup 1 that is mainly

represented in the domestic cycle whereas the G isolate

from a marsupial is indigenous in South America and

belongs to the subgroup 2 that predominates in the sylvatic

cycle. At the time when a massive migration started through

the land connection between North and South Americas

(Simpson, 1980), North American placental mammals

spread throughout South America, carrying subgroup 1

parasites, while few South American marsupials migrated

towards North, carrying subgroup 2 parasites. The isolation

time of these two groups would be of at least 70 millions

years, during which they would have accumulated enough

divergence (Briones et al., 1999). Several different markers

suggest that the divergence is ancient and the results here

described might be a consequence of independent evolution

of Cl and G isolates, representing subgroups 1 and 2 (Kawa-

shita et al., 2001).

Acknowledgements

This work was supported by Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (FAPESP), Brazil. I.N.

is recipient of PhD fellowship from Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq). We

thank Dr Marcelo Briones for valuable suggestions and Dr

Sergio Schenkman for critical reading of the manuscript.

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