Parasitol Res (2005) 97: 259269

DOI 10.1007/s00436-005-1433-4

O R I GI N A L P A P E R

Rosane M. S. Meirelles Andrea Henriques-Pons

Maurilio J. Soares Mario Steindel

Penetration of the salivary glands of Rhodnius domesticus Neiva & Pinto,

1923 (Hemiptera: Reduviidae) by Trypanosoma rangeli Tejera, 1920
(Protozoa: Kinetoplastida)
Received: 14 December 2004 / Accepted: 9 June 2005 / Published online: 5 July 2005

Springer-Verlag 2005

Abstract Penetration of the heteroxenous protozoan

Trypanosoma rangeli into the salivary glands of its
invertebrate host Rhodnius domesticus has been investigated here using dierent approaches. Electron
microscopy showed that epimastigotes coming from the
insect hemocoel cross the basal lamina that surrounds
the salivary glands and penetrate through the gland
cells cytoplasm. After reaching the gland lumen, epimastigote forms remain adhered to the gland cell microvilli
by
their
agella,
while
metacyclic
trypomastigotes are found swimming free in the saliva.
Analysis by ow cytometry, western blotting and
hemolytic activity allowed to demonstrate the presence
in T. rangeli of a hemolytic molecule with antigenic
cross-reactivity with murine perforin, which could be
used by the parasites to reach the salivary gland lumen.
This molecule, which we named as rangelysin, has
120 kDa molecular weight, is able to induce hemolysis
only in acidic pH, and is produced by both trypomastigote and epimastigote forms.

R. M. S. Meirelles M. J. Soares (&)

Laboratorio de Biologia Celular de Microrganismos,
Departamento de Ultra-estrutura e Biologia Celular,
Instituto Oswaldo Cruz/FIOCRUZ, Avenida Brasil 4365,
Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil
E-mail: maurilio@ioc.ocruz.br
Fax: +55-21-22604434
A. Henriques-Pons
Laboratorio de Biologia Celular, Departamento
de Ultra-estrutura e Biologia Celular,
Instituto Oswaldo Cruz/FIOCRUZ, Avenida Brasil 4365,
Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil
M. Steindel
Departamento de Microbiologia e Parasitologia,
Universidade Federal de Santa Catarina,
Caixa Postal 476, 88040-900 Florianopolis,
Santa Catarina, Brazil

Introduction
Trypanosoma rangeli is a heteroxenous trypanosome
rst described in Venezuela by Tejera (1920), which infects mainly triatomine bugs from the Rhodnius genus
(Cuba-Cuba 1998; Guhl and Vallejo 2003). Although T.
rangeli has been assigned to the Stercoraria group of
trypanosomes (those transmitted mainly by the contaminative route), its transmission to the vertebrate host
occurs by the insect bite (DAlessandro and Saravia
1999; Grisard 2002). Mixed infections with T. rangeli
and Trypanosoma cruzi (the causative agent of Chagas
disease) may occur in both vertebrate and invertebrate
hosts (Schaub and Wunderlich 1985; DAlessandro and
Saravia 1999; Grisard et al. 1999). The presence of both
parasites in the mammalian host can be detected by
xenodiagnosis or immunological tests (Garnham 1980;
Acosta et al. 1991; Guhl et al. 2002). In opposition to T.
cruzi, T. rangeli causes a harmless infection in mammals,
including man (Hoare 1972; DAlessandro 1976), but
induces pathological eects in the triatomine bugs, such
as death of nymphs during the molting due to morphological abnormalities (Grewal 1957; Tobie 1965;
Anez 1984).
The life cycle of T. rangeli in the invertebrate host
initiates with the ingestion of blood stream trypomastigote forms from vertebrate hosts. The parasites then
colonize the digestive tube of the insects, adhere to the
intestinal wall and dierentiate into epimastigote forms,
which are able to multiply and transverse the intestinal
epithelium. After reaching the haemolymph, the parasites migrate and infect the salivary glands, but the
evolutive forms responsible for the gland penetration
and the mechanisms involved in this process still remain
poorly known. In the salivary gland lumen the parasites
dierentiate into metacyclic trypomastigote forms,
which are capable of infecting the vertebrate host during
the blood meal (DAlessandro 1976; DAlessandro and
Saravia 1992). In the mammal host, the biological cycle
is also poorly understood, but some evidences from

260

in vitro experiments suggest that the parasites infect and

dierentiate within host monocytes as amastigote-like
forms, which are morphologically dierent from the T.
cruzi amastigotes (Osorio et al. 1995).
Crossing from the intestine lumen to the hemocoel,
and from this compartment to the salivary gland lumen,
are critical steps in the life cycle of T. rangeli in the
invertebrate host. Ultrastructural studies on the penetration of T. rangeli into midgut cells (Hecker et al. 1990;
Oliveira and De Souza 2001) and salivary glands (Ellis
et al. 1980) of Rhodnius prolixus have been carried out,
but the mechanisms used by the trypanosomes to penetrate and transverse these structured epithelia are controversial. Damaged areas of intestinal epithelium in R.
prolixus infected with T. rangeli have been reported
(Watkins 1971). It has been recently suggested that the
parasites transverse the cytoplasm of the midgut cells,
causing cell damage (Oliveira and De Souza 2001).
However, it has been also proposed that T. rangeli cross
the intestinal barrier by an intracellular route without
damaging the cells (Hecker et al. 1990).
Several unicellular parasites have developed various
pore-forming proteins, such as the amoebapores in
Entamoeba histolytica (Lynch et al. 1982; Young et al.
1982; Horstmann et al. 1992), leishporin of Leishmania
(Noronha et al. 1994, 1996), TC-tox in T. cruzi (Andrews 1990; Andrews et al. 1990) and lysterilysin-O in
Lysteria monocytogenes bacteria (Bhakdi and TranunJensen 1988; Bielecki et al. 1990). The aim of our study
was to analyze, by electron microscopy, the interaction
of T. rangeli with the salivary glands of experimentally
infected Rhodnius domesticus, and characterize a possible pore-forming protein that could be used by the
parasites to reach the salivary gland lumen.

obtained as previously described (Koerich et al. 2002).

Briey, epimastigotes were cultivated for 5 days at 27C
in DMEM medium, pH 8.0, supplemented with 5% heat
inactivated fetal bovine serum. Under this condition,
about 80% of the cells transform into trypomastigotes.
Insect infection
Adult R. domesticus of both sexes were inoculated by the
intracelomic route with 103 culture epimastigote forms
(5 ll) of T. rangeli (strain SC-58), by using a Hamilton
syringe. Two days after the inoculation the insects were
allowed to feed on mice sedated with Diazepam (20 mg/
kg), until complete engorgement. One week later a
droplet of hemolymph was collected and examined for
parasite infection. About 80% of the insects presented
parasites in the hemolymph. Fifteen days after parasite
inoculation, insect salivary glands were obtained by
gently pulling o the head of cold-chilled insects into a
droplet of phosphate buered saline, pH 7.4 (PBS).
Scanning electron microscopy (SEM)
Culture epimastigotes and trypomastigotes, as well as
isolated salivary glands infected with T. rangeli, were
xed with 2.5% glutaraldehyde in 0.1 M cacodylate
buer, pH 7.2, washed in buer and post-xed for
30 min with 1% osmium tetroxide in 0.1 M cacodylate
buer, pH 7.2. Thereafter, the samples were dehydrated
in acetone, critical point dried and mounted on SEM
stubs. The samples were coated with a 20-nm thick gold
layer and examined in a Zeiss DSM940 scanning electron microscope. Digital images were acquired and
stored in an IBM-PC compatible computer.

Materials and methods

Chemicals
Polyclonal rabbit anti-mouse perforin IgG was obtained
form Dr. Pedro M. Persechini (Institute of Biophysics,
UFRJ, Rio de Janeiro, RJ, Brazil). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG was
purchased from Caltag (San Francisco, CA, USA).
Molecular weight markers and the silver stain kit were
purchased from BIORAD (San Jose, CA, USA). All
other reagents were purchased from Sigma Chemical
Co. (Saint Louis, MO, USA).
Parasites
Epimastigote forms of T. rangeli, strains SC-58 and
Choachi, were grown with weekly passages at 28C in
LIT (Liver Infusion-Tryptose) medium supplemented
with 20% fetal bovine serum. Five-day-old culture epimastigote forms (at the log phase of growth) were used
for the experiments. Culture trypomastigotes were

Transmission electron microscopy (TEM)

Culture epimastigote and trypomastigote forms, as well
as isolated salivary glands, were xed for 2 h at room
temperature with 2.5% glutaraldehyde in 0.1 M cacodylate buer, pH 7.2. The samples were then washed
twice in buer and post-xed for 1 h with 1% osmium
tetroxide/0.8% potassium ferricyanide/5 mM calcium
chloride in 0.1 M cacodylate buer, pH 7.2 (Forbes
et al. 1977 ). Thereafter, the samples were dehydrated in
graded acetone, incubated overnight in an epoxy resin
(PolyBed 812)/acetone solution (1:1), embedded in pure
resin and then polymerized for 48 h at 60C. Ultra-thin
sections were stained with uranyl acetate and lead citrate
and observed in a Zeiss EM10C transmission electron
microscope, operated at 60 kV.
Hemolytic assays
Culture epimastigote and trypomastigote forms of T.
rangeli (strains Choachi and SC-58) were washed in PBS

261

containing 0.1 mM EDTA and then submitted to ve

cycles of freezing-thawing. The cell extracts were centrifuged for 5 min at 100,000 g and the supernatants
collected and assayed for hemolytic activity at pH 6.5 or
7.5. Hemolytic assays were performed in 96 round-bottomed microplates, by using mouse erythrocytes (4 106
cells/well) and parasite extracts diluted in PBS/0.1 mM
EDTA. To avoid molecular polymerization and inactivation of a possible hemolysin, 10 mM CaCl2 was added
to the incubation mixture just before incubation. After
30 min at 37C, the microplates were centrifuged for
5 min at 200 g and 100 ll of the supernatants was collected for spectrophotometric reading at 414 nm. The
lytic activity was evaluated by quantifying the hemoglobin release, using the following formula:

Experimental lysis %

3 min and submitted to 10% SDS-PAGE (Laemmli

1970). One gel was stained with a silver stain kit, while
the other was transferred to a nitrocellulose membrane.
The membrane was rinsed in washing buer pH 7.4
(0.25% Nonidet NP40/0.25% Tween 20/400 mM NaCl)
and incubated in block buer (4% nonfat dried milk in
washing buer). After washing for three times, the
membrane was incubated with either polyclonal rabbit
anti-mouse perforin IgG, pre-immune serum or antibody buer alone (0.1% nonfat dried milk/10 mM Trizma base/150 mM NaCl/2 mM EDTA, pH 7.4) with
gentle shaking. The membrane was extensively washed
with antibody buer and incubated for 2 h under gentle
shaking in the presence of alkaline phosphatase-conjugated goat anti-rabbit IgG. The membrane was exten-

Absorbance experimental lysis
Abs. spontaneous lysis

 100:
Abs. 100 % lysis
Abs. spontaneous lysis

Spontaneous lysis was obtained as described above,

but in the absence of parasite extract. For 100% lysis,
water was used instead of PBS/EDTA.

sively washed in antibody buer and the protein samples

were revealed using nitroblue tetrazolium (NBT) and
5-bromo-4-chloro-3-indolyl phosphate (BCIP).

Flow cytometry analysis

Results

Trypomastigote and epimastigote forms of T. rangeli

(strains Choachi and SC-58) were xed for 20 min at
4C in 1% paraformaldehyde, centrifuged for 5 min at
10,000 g and then washed three times in PBS. The cells
were then incubated for 20 min in PBS containing 2%
normal goat serum, washed with PBS and incubated for
20 min with permeabilizing buer (PBS/1% bovine
serum albumin/0.1% saponin). In all the following steps,
the samples were maintained in permeabilizing buer.
The samples were then incubated overnight in a buer
containing either polyclonal rabbit anti-mouse perforin
IgG, pre-immune serum or only permeabilizing buer as
control. The samples were then washed for 10 min and
incubated with FITC-conjugated goat anti-rabbit IgG.
After a nal wash the cells were ressuspendend in PBS
and analyzed in a FACScalibur ow cytometer (Becton
and Dickinson, USA).

Ultrastructure of culture epimastigote and

trypomastigote forms of T. rangeli
Culture forms of T. rangeli were analyzed by scanning
and TEM previous to the inoculation into the insect
hemocoel. Epimastigotes of the two strains (SC-58 and
Choachi) appear under short or long forms. The agellum emerges close to the central portion of the cell body,
forming a long undulating membrane (Fig. 1). The
compact kinetoplast presents the typical rod-shaped
structure (Fig. 3). Trypomastigotes appear usually as
short forms with an undulating membrane, with the
agellum emerging at the posterior end and running
toward the anterior tip of the cell (Fig. 2). The kinetoplast in culture trypomastigote forms appears as a
loose basket-like structure (Fig. 4).

Western blotting

Penetration of the salivary glands of R. domesticus

by T. rangeli

Culture epimastigotes (5107 parasites per sample) were

washed three times with PBS/0.1 mM EDTA, pelleted
by centrifugation at 1200 g for 5 min and kept at
70C
until use. The parasites were ressupended in 100 ll of
PBS/0.1 mM EDTA and submitted to ve cycles of
freezing-thawing. The extracts were then centrifuged for
10 min at 1000 g and 20 ll were transferred to 80 ll of
sample buer (0.125 M TrisHCl, pH 6.8/4% SDS/20%
glycerol/10% b-mercaptoethanol). Molecular weight
markers and experimental samples were then boiled for

Fifteen days after the experimental infection of the insects, a high number of parasitesmostly long epimastigoteswere observed adhered to the outer surface
(basal lamina) of the salivary glands of R. domesticus,
either as single individuals or forming large clusters
(Fig. 5). Several parasites, mostly long epimastigotes,
were observed with part of their body inserted into the
basal lamina, usually with the agellum foremost
(Figs. 6, 7). Punctually damaged areas could be observed as holes in the basal lamina, with no visible

262

Figs. 1-4 Fine structure of culture forms of Trypanosoma rangeli

(strain SC-58)
Fig. 1 Scanning electron microscopy showing long epimastigote
forms
Fig. 2 Scanning electron microscopy of a short trypomastigote
form

Fig. 3 Transmission electron microscopy of an epimastigote form

showing the Golgi complex (GC) located near the central nucleus
(N), and the rod-shaped kinetoplast (arrowhead)
Fig. 4 Transmission electron microscopy of a trypomastigote form
showing the kinetoplast with the basket-like structure (arrow). N
nucleus, M mitochondrion

parasites associated to them (Figs. 6, 7). After penetrating the outer layer of the basal lamina, the parasites
could be found in the space between the basal lamina
and the salivary gland epithelium (Fig. 8). Epimastigotes
in the basal lamina then invade the salivary gland cells
by a still unknown mechanism. After reaching the gland
cell cytoplasm, the parasites transverse the gland cell
inside tight (Fig. 9) or loose (Fig. 10) membrane bound

vacuoles, although in a single case we observed a parasite apparently free in the host cell cytoplasm (Fig. 9,
inset). In both cases the parasites were not under the
typical amastigote form, but were either elongated
(Fig. 9) or folded (Fig. 10) parasites with a agellum.
After reaching the gland lumen, the parasites appear
under the epimastigote form and remain attached by the
agellum to the microvilli of the salivary gland cells

263

Figs. 5-8 Scanning (Figs. 5-7) and transmission (Fig. 8) electron

microscopy of Rhodnius domesticus salivary glands infected with T.
rangeli (strain SC-58)
Fig. 5 Low magnication view of the outer surface of the salivary
gland showing the high number of adhered parasites
Fig. 6 Both trypomastigote (t) and epimastigote (e) forms can be
found adhered to the outer surface of the salivary gland
Fig. 7 High magnication showing long epimastigote forms
penetrating the basal lamina of the salivary gland (arrows). Note
punctual damages (arrowheads), with no parasites associated

Fig. 8 Transmission electron microscopy showing parasites located

between the basal lamina (BL) and the salivary gland cell. A rodshaped kinetoplast, typical of epimastigote forms, can be seen
(arrow). A agellum is observed inside a basal lamina layer
(arrowhead)

(Figs. 11, 12). Location of the kinetoplast close to

the nucleus indicates that the epimastigotes start to
dierentiate into trypomastigotes while still attached
to the gland cell microvilli (Fig. 11). No membrane
specialization could be observed at the attachment

region, either at the agellar or the gland cell membranes

(Fig. 12). Large vesicles were observed in the cytoplasm
of the adhered epimastigotes, which could not be seen in
culture epimastigotes (data not shown). Short metacyclic
trypomastigotes are found swimming free in the saliva

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Figs. 9-12 Transmission
electron microscopy of T.
rangeli in the salivary glands of
R. domesticus
Fig. 9 A parasite inside a tight
membrane bound vacuole in the
salivary gland cell cytoplasm.
The arrow indicates the
kinetoplast. Inset: detail of a
free parasite
Fig. 10 An intra-vacuolar
parasite at the salivary gland
cell cytoplasm. The arrow
shows the vacuole membrane.
F agellum
Fig. 11 An intermediary
epimastigote form, with the
kinetoplast (arrow) located
parallel to the nucleus (N),
adhered to the gland cell
microvilli (Mi) by the agellum
(F). L gland lumen
Fig. 12 Epimastigote form
adhered to the gland cell
microvilli by the agellum
(arrow). L gland lumen. Inset:
detail showing the absence of
membrane specialization at the
attachment region (arrowhead)

close to the gland cell microvilli (Fig. 13) and at the

salivary gland lumen (Fig. 14). Notably, these metacyclic trypomastigotes present a rod-shaped kinetoplast
(Fig. 14).

volume of parasite extract sucient to lysate 50% of the

mice erythrocytes) corresponded to 19 ll in epimastigotes, against 33 ll in trypomastigotes (Fig. 15a). These
data prompted us to evaluate the production of this
cytolytic molecule by ow cytometry analysis.

Hemolytic assays
Flow cytometry
As our ultrastructural data suggested an active penetration of T. rangeli into the insect salivary glands, we
have raised the hypothesis that the parasites could be
using cytolytic or pore-forming proteins to transverse
this epithelial barrier. Hemolytic assays with serial
dilutions of epimastigote and trypomastigote extracts
showed a prominent and dose-dependent hemolytic
activity in extracts from both forms at an acidic pH
(pH 6.5, Fig. 15a), but not at pH 7.5 (Fig. 15b). Epimastigotes appear to produce a higher amount of this
hemolytic molecule, since one hemolytic unit (HU, the

Flow cytometry analysis was performed by using a

polyclonal rabbit anti-mouse perforin IgG, expecting a
possible high homology and cross-reactivity between the
lytic molecule and perforin. Perforin is a 70-kDa, Ca2+ dependent, pore-forming protein produced by cytotoxic
lymphocytes and stored within cytoplasmic granules in
these cells (Liu et al. 1995). We found positive labeling in
extracts from both epimastigote and trypomastigote
forms, indicating the presence of an antigenic perforinlike molecule in T. rangeli (Fig. 16a and b). Incubation

265
Fig. 13 Scanning electron
microscopy of trypomastigote
forms free in the saliva, at the
salivary gland lumen
Fig. 14 Transmission electron
microscopy of a trypomastigote
form free in the saliva, at the
salivary gland lumen (L). Note
the rod-shaped kinetoplast. F
agellum, N nucleus

266

Fig. 15 Hemolytic assay with serial dilutions of T. rangeli

epimastigote (lled diamond) and trypomastigote (lled square)
extracts, at pH 6.5 (a) and pH 7.5 (b)

with pre-immune serum showed a lower positive labeling

pattern in epimastigotes (Fig. 16b), thus indicating
cross-reactivity of parasite antigens with the natural
background repertoire of the rabbit serum. No labeling
was found in trypomastigote forms incubated with the
pre-immune serum (Fig. 16c).
Western blotting assays
Polyclonal anti-perforin IgG reacted with a protein of
approximately 120 kDa in epimastigote forms, as well as
with a protein with less than 85 kDa (Fig. 17, lane 1).
Cross-reaction with the latter molecule could be also
detected after incubation with the pre-immune serum
(Fig. 17, lane 2) and thus this molecule is possibly
responsible for the intermediate labeling intensity found
in the FACS analysis (Fig. 16b). Control performed by
incubation with the secondary IgG alone resulted in no
detectable labeling (Fig. 17, lane 3). Silver staining of the
electrophoretic run demonstrated that the 120 kDa
molecule recognized by the anti-perforin antibody is not
a majoritary protein (Fig. 17, lane 4).

Discussion
T. rangeli is a hemoagellate protozoan transmitted to a
variety of wild and domestic animals during the triato-

mine blood meal (DAlessandro 1976). In the invertebrate vector, T. rangeli has a characteristic life cycle,
with a peculiar ability to invade the haemolymph and
then the salivary glands, where a large number of
infective metacyclic trypomastigote forms are generated.
Furthermore, it has been observed a preferential combination between certain parasite strains and Rhodnius
species, indicating the intimate reliance of the parasite
host interaction (Cuba-Cuba 1998). In order to obtain
high salivary gland infection rates we infected triatomines from Santa Catarina, Brazil (R. domesticus) with
parasites isolated from this same geographic region
(strain SC-58). A previous study had demonstrated that
in this case high infection rates can be obtained (Steindel
et al. 1991).
In most trypanosomatids, the parasites adhere to the
digestive tract of their respective insect hosts and form a
homogeneous cellular layer as a critical step in their life
cycle (Tieszen et al. 1986, 1989; Molyneux et al. 1987). In
vitro studies on the interaction of T. rangeli with dissected fragments of the posterior midgut of R. prolixus
showed few parasites attached to sparse epithelial cells,
suggesting that a recognition step is necessary for posterior invasion (Oliveira and De Souza 2001). This
behavior has also been observed in other insect-protozoan models, such as Plasmodium gallinaceum infecting
Aedes aegypti, where the parasites were found associated
preferentially with a well-dened cell type (Shahabuddin
and Pimenta 1998). Analysis of the interaction between
T. rangeli and the epithelium of the salivary glands of R.
domesticus suggests that in this case no such specic
recognition process occurs: the parasites appeared to be
individually penetrating the basal lamina all over the
salivary gland surface. A previous study with FITC-labeled lectins showed that the salivary gland surface
presents dierent carbohydrate residues that may serve
as receptors to which the long epimastigote forms attach
prior to the invasion (Basseri et al. 2002).
A former ultrastructural study on the mechanisms of
T. rangeli penetration into R. prolixus salivary glands
showed that the parasites are able to penetrate the basal
lamina and cross the gland cell cytoplasm (Ellis et al.
1980). Our morphological data showed mainly epimastigote forms transversing the basal lamina through small
holes to reach the glandular epithelium, while trypomastigotes are found adhered to the outer gland surface.
Our data suggest that epimastigote forms can produce a
lytic molecule, such as a pore forming protein (PFP), to
allow the passage of the parasites through the epithelial
barrier. Indeed, hemolytic assays demonstrated a lytic
molecule exclusively active in the acidic environment.
This hemolytic molecule, which we named as rangelysin,
seems to be present in higher amounts in epimastigote
forms, although FACS analysis showed that it is also
present in culture trypomastigotes. It is possible that
only epimastigote forms secrete this lytic molecule in
sucient amounts to damage the basal lamina, thus
allowing the gland cell penetration.

267

Fig. 16 Flow citometry assay with epimastigote (a) and trypomastigote (c) forms of T. rangeli (strain SC-58). In b and d is shown
labeling with the polyclonal rabbit anti-mouse perforin IgG (shaded

curve), the pre-immune serum (solid line) and the FITC-conjugated

goat anti rabbit IgG (dashed line). All analyses were performed in
the region (R1) indicated

Channel formation by PFPs is a well-dened

mechanism of membrane damage used in biological
systems ranging from bacteria to vertebrates (Lynch
et al. 1982; Bhakdi and Tranun-Jensen 1988; Tweten
1995; Ludwig 1996; Suss-Toby et al. 1996; Nickel et al.
1999). Several pathogenic protozoa produce cytolytic
proteins that disrupt target cell membranes by forming
channels in the lipid bilayer (Jiang et al. 1990; Ley et al.
1990; Carruthers 1999). These PFPs are thought to play
a signicant role in the pathogenesis of many protozoan infections, such as in amebiasis (Andrews and
Portnoy 1994; Fiori et al. 1996; Noronha et al. 1996;
Desai and Rosenberg 1997; Horta 1997). Pore-forming
proteins usually bind to the plasma membrane of a
target cell and expose hydrophobic domains, thus
allowing ions and small molecules to pass freely across
the lipid bilayer. Our data indicate that T. rangeli
presents and probably uses a 120 kDa PFP to penetrate
the salivary gland cells of the insect vectors, however
without rupture of the host cells.
We have observed intracellular parasites enclosed in
vacuoles in the gland cell cytoplasm. However, these
forms present a agellum, and thus do not represent

typical intracellular amastigote stages of the parasites.

Ellis et al. (1980) suggested that the parasites cross the
salivary gland cells towards the gland lumen enclosed
within vacuoles until they come near the apical gland cell
portion, where most parasites appear to lose their vacuoles before leaving the gland cells. Indeed, we found
one parasite free in the host cell cytoplasm. It is possible
that T. rangeli, with the help of rangelysin, evades the
parasitophorous vacuole in a way similar to that used by
T. cruzi with the help of TC-tox (Andrews 1990; Andrews et al. 1990).
When the parasites nally reach the gland lumen they
remain adhered to the gland cell microvilli as epimastigotes. As only trypomastigotes were found free in the
saliva, it seems that the metacyclogenesis starts with
adhered epimastigotes and intermediary forms. Ultrastructural analysis showed morphological dierences in
the kinetoplast morphology between trypomastigotes
from culture and from infected salivary glands. Salivary
gland trypomastigotes present a condensed kinetoplast,
and thus this morphological characteristic allows differentiating between metacyclic and culture trypomastigote forms of T. rangeli.

268

Fig. 17 Lanes 13 Western blotting assay with extracts of

epimastigote forms of T. rangeli, strain SC-58. Polyclonal rabbit
anti-mouse perforin IgG was used to detect a 120 kDa protein with
cross-reactivity with perforin in T. rangeli (arrowhead, lane 1).
Cross-reaction with a <85 kDa protein could be detected after
incubation with the pre-immune serum (lane 2). Control performed
by incubation with the secondary IgG alone (alkaline phosphataseconjugated goat anti-rabbit IgG) resulted in no detectable labeling
(lane 3). Silver staining of the electrophoretic run shows that the
120 kDa molecule recognized by the anti-perforin antibody is not a
majoritary protein (lane 4)

Acknowledgements The authors thank Dr. Pedro M. Persechini

(Institute of Biophysics, UFRJ, Rio de Janeiro, Brazil) for kindly
supplying the anti-perforin antibody and Mr. Jose Lopes de Faria
for the photographic work. This work was supported by CNPq,
FAPERJ, PAPES-III/FIOCRUZ and FIOCRUZ. All experiments
were performed according to the Brazilian laws.

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