ORIGINAL PAPER

Rosane M. S. Meirelles Æ Andrea Henriques-Pons

Maurilio J. Soares Æ 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 protozoanTrypanosoma rangeli into the salivary glands of itsinvertebrate host Rhodnius domesticus has been inves-tigated here using different approaches. Electronmicroscopy showed that epimastigotes coming from theinsect hemocoel cross the basal lamina that surroundsthe salivary glands and penetrate through the glandcells cytoplasm. After reaching the gland lumen, epi-mastigote forms remain adhered to the gland cell mi-crovilli by their flagella, while metacyclictrypomastigotes are found swimming free in the saliva.Analysis by flow cytometry, western blotting andhemolytic activity allowed to demonstrate the presencein T. rangeli of a hemolytic molecule with antigeniccross-reactivity with murine perforin, which could beused by the parasites to reach the salivary gland lumen.This molecule, which we named as rangelysin, has120 kDa molecular weight, is able to induce hemolysisonly in acidic pH, and is produced by both trypo-mastigote and epimastigote forms.

Introduction

Trypanosoma rangeli is a heteroxenous trypanosomefirst described in Venezuela by Tejera (1920), which in-fects mainly triatomine bugs from the Rhodnius genus(Cuba-Cuba 1998; Guhl and Vallejo 2003). Although T.rangeli has been assigned to the Stercoraria group oftrypanosomes (those transmitted mainly by the con-taminative route), its transmission to the vertebrate hostoccurs by the insect bite (D’Alessandro and Saravia1999; Grisard 2002). Mixed infections with T. rangeliand Trypanosoma cruzi (the causative agent of Chagas’disease) may occur in both vertebrate and invertebratehosts (Schaub and Wunderlich 1985; D’Alessandro andSaravia 1999; Grisard et al. 1999). The presence of bothparasites in the mammalian host can be detected byxenodiagnosis 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; D’Alessandro 1976), butinduces pathological effects in the triatomine bugs, suchas death of nymphs during the molting due to mor-phological abnormalities (Grewal 1957; Tobie 1965;Anez 1984).

The life cycle of T. rangeli in the invertebrate hostinitiates with the ingestion of blood stream trypomasti-gote forms from vertebrate hosts. The parasites thencolonize the digestive tube of the insects, adhere to theintestinal wall and differentiate into epimastigote forms,which are able to multiply and transverse the intestinalepithelium. After reaching the haemolymph, the para-sites migrate and infect the salivary glands, but theevolutive forms responsible for the gland penetrationand the mechanisms involved in this process still remainpoorly known. In the salivary gland lumen the parasitesdifferentiate into metacyclic trypomastigote forms,which are capable of infecting the vertebrate host duringthe blood meal (D’Alessandro 1976; D’Alessandro andSaravia 1992). In the mammal host, the biological cycleis also poorly understood, but some evidences from

R. M. S. Meirelles Æ 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, BrazilE-mail: maurilio@ioc.fiocruz.brFax: +55-21-22604434

A. Henriques-PonsLaboratorio de Biologia Celular, Departamentode Ultra-estrutura e Biologia Celular,Instituto Oswaldo Cruz/FIOCRUZ, Avenida Brasil 4365,Manguinhos, 21040-900 Rio de Janeiro, RJ, Brazil

M. SteindelDepartamento de Microbiologia e Parasitologia,Universidade Federal de Santa Catarina,Caixa Postal 476, 88040-900 Florianopolis,Santa Catarina, Brazil

Parasitol Res (2005) 97: 259–269DOI 10.1007/s00436-005-1433-4

in vitro experiments suggest that the parasites infect anddifferentiate within host monocytes as amastigote-likeforms, which are morphologically different 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 theinvertebrate host. Ultrastructural studies on the pene-tration of T. rangeli into midgut cells (Hecker et al. 1990;Oliveira and De Souza 2001) and salivary glands (Elliset al. 1980) of Rhodnius prolixus have been carried out,but the mechanisms used by the trypanosomes to pen-etrate and transverse these structured epithelia are con-troversial. Damaged areas of intestinal epithelium in R.prolixus infected with T. rangeli have been reported(Watkins 1971). It has been recently suggested that theparasites transverse the cytoplasm of the midgut cells,causing cell damage (Oliveira and De Souza 2001).However, it has been also proposed that T. rangeli crossthe intestinal barrier by an intracellular route withoutdamaging the cells (Hecker et al. 1990).

Several unicellular parasites have developed variouspore-forming proteins, such as the amoebapores inEntamoeba 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 (An-drews 1990; Andrews et al. 1990) and lysterilysin-O inLysteria monocytogenes bacteria (Bhakdi and Tranun-Jensen 1988; Bielecki et al. 1990). The aim of our studywas to analyze, by electron microscopy, the interactionof T. rangeli with the salivary glands of experimentallyinfected Rhodnius domesticus, and characterize a possi-ble pore-forming protein that could be used by theparasites to reach the salivary gland lumen.

Materials and methods

Chemicals

Polyclonal rabbit anti-mouse perforin IgG was obtainedform Dr. Pedro M. Persechini (Institute of Biophysics,UFRJ, Rio de Janeiro, RJ, Brazil). Fluorescein isothi-ocyanate (FITC)-conjugated goat anti-rabbit IgG waspurchased from Caltag (San Francisco, CA, USA).Molecular weight markers and the silver stain kit werepurchased from BIORAD (San Jose, CA, USA). Allother reagents were purchased from Sigma ChemicalCo. (Saint Louis, MO, USA).

Parasites

Epimastigote forms of T. rangeli, strains SC-58 andChoachi, were grown with weekly passages at 28�C inLIT (Liver Infusion-Tryptose) medium supplementedwith 20% fetal bovine serum. Five-day-old culture epi-mastigote forms (at the log phase of growth) were usedfor the experiments. Culture trypomastigotes were

obtained as previously described (Koerich et al. 2002).Briefly, epimastigotes were cultivated for 5 days at 27�Cin DMEMmedium, pH 8.0, supplemented with 5% heatinactivated 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 theintracelomic route with 103 culture epimastigote forms(5 ll) of T. rangeli (strain SC-58), by using a Hamiltonsyringe. Two days after the inoculation the insects wereallowed to feed on mice sedated with Diazepam (20 mg/kg), until complete engorgement. One week later adroplet of hemolymph was collected and examined forparasite infection. About 80% of the insects presentedparasites in the hemolymph. Fifteen days after parasiteinoculation, insect salivary glands were obtained bygently pulling off the head of cold-chilled insects into adroplet of phosphate buffered saline, pH 7.4 (PBS).

Scanning electron microscopy (SEM)

Culture epimastigotes and trypomastigotes, as well asisolated salivary glands infected with T. rangeli, werefixed with 2.5% glutaraldehyde in 0.1 M cacodylatebuffer, pH 7.2, washed in buffer and post-fixed for30 min with 1% osmium tetroxide in 0.1 M cacodylatebuffer, pH 7.2. Thereafter, the samples were dehydratedin acetone, critical point dried and mounted on SEMstubs. The samples were coated with a 20-nm thick goldlayer and examined in a Zeiss DSM940 scanning elec-tron microscope. Digital images were acquired andstored in an IBM-PC compatible computer.

Transmission electron microscopy (TEM)

Culture epimastigote and trypomastigote forms, as wellas isolated salivary glands, were fixed for 2 h at roomtemperature with 2.5% glutaraldehyde in 0.1 M caco-dylate buffer, pH 7.2. The samples were then washedtwice in buffer and post-fixed for 1 h with 1% osmiumtetroxide/0.8% potassium ferricyanide/5 mM calciumchloride in 0.1 M cacodylate buffer, pH 7.2 (Forbeset al. 1977 ). Thereafter, the samples were dehydrated ingraded acetone, incubated overnight in an epoxy resin(PolyBed 812)/acetone solution (1:1), embedded in pureresin and then polymerized for 48 h at 60�C. Ultra-thinsections were stained with uranyl acetate and lead citrateand observed in a Zeiss EM10C transmission electronmicroscope, operated at 60 kV.

Hemolytic assays

Culture epimastigote and trypomastigote forms of T.rangeli (strains Choachi and SC-58) were washed in PBS

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containing 0.1 mM EDTA and then submitted to fivecycles of freezing-thawing. The cell extracts were cen-trifuged for 5 min at 100,000 g and the supernatantscollected and assayed for hemolytic activity at pH 6.5 or7.5. Hemolytic assays were performed in 96 round-bot-tomed microplates, by using mouse erythrocytes (4 · 106

cells/well) and parasite extracts diluted in PBS/0.1 mMEDTA. To avoid molecular polymerization and inacti-vation of a possible hemolysin, 10 mM CaCl2 was addedto the incubation mixture just before incubation. After30 min at 37�C, the microplates were centrifuged for5 min at 200 g and 100 ll of the supernatants was col-lected for spectrophotometric reading at 414 nm. Thelytic activity was evaluated by quantifying the hemo-globin release, using the following formula:

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

Flow cytometry analysis

Trypomastigote and epimastigote forms of T. rangeli(strains Choachi and SC-58) were fixed for 20 min at4�C in 1% paraformaldehyde, centrifuged for 5 min at10,000 g and then washed three times in PBS. The cellswere then incubated for 20 min in PBS containing 2%normal goat serum, washed with PBS and incubated for20 min with permeabilizing buffer (PBS/1% bovineserum albumin/0.1% saponin). In all the following steps,the samples were maintained in permeabilizing buffer.The samples were then incubated overnight in a buffercontaining either polyclonal rabbit anti-mouse perforinIgG, pre-immune serum or only permeabilizing buffer ascontrol. The samples were then washed for 10 min andincubated with FITC-conjugated goat anti-rabbit IgG.After a final wash the cells were ressuspendend in PBSand analyzed in a FACScalibur flow cytometer (Bectonand Dickinson, USA).

Western blotting

Culture epimastigotes (5·107 parasites per sample) werewashed three times with PBS/0.1 mM EDTA, pelletedby centrifugation at 1200 g for 5 min and kept at �70�Cuntil use. The parasites were ressupended in 100 ll ofPBS/0.1 mM EDTA and submitted to five cycles offreezing-thawing. The extracts were then centrifuged for10 min at 1000 g and 20 ll were transferred to 80 ll ofsample buffer (0.125 M Tris–HCl, pH 6.8/4% SDS/20%glycerol/10% b-mercaptoethanol). Molecular weightmarkers and experimental samples were then boiled for

3 min and submitted to 10% SDS-PAGE (Laemmli1970). One gel was stained with a silver stain kit, whilethe other was transferred to a nitrocellulose membrane.The membrane was rinsed in washing buffer pH 7.4(0.25% Nonidet NP40/0.25% Tween 20/400 mM NaCl)and incubated in block buffer (4% nonfat dried milk inwashing buffer). After washing for three times, themembrane was incubated with either polyclonal rabbitanti-mouse perforin IgG, pre-immune serum or anti-body buffer alone (0.1% nonfat dried milk/10 mM Tri-zma base/150 mM NaCl/2 mM EDTA, pH 7.4) withgentle shaking. The membrane was extensively washedwith antibody buffer and incubated for 2 h under gentleshaking in the presence of alkaline phosphatase-conju-gated goat anti-rabbit IgG. The membrane was exten-

sively washed in antibody buffer and the protein sampleswere revealed using nitroblue tetrazolium (NBT) and5-bromo-4-chloro-3-indolyl phosphate (BCIP).

Results

Ultrastructure of culture epimastigote andtrypomastigote forms of T. rangeli

Culture forms of T. rangeli were analyzed by scanningand TEM previous to the inoculation into the insecthemocoel. Epimastigotes of the two strains (SC-58 andChoachi) appear under short or long forms. The flagel-lum emerges close to the central portion of the cell body,forming a long undulating membrane (Fig. 1). Thecompact kinetoplast presents the typical rod-shapedstructure (Fig. 3). Trypomastigotes appear usually asshort forms with an undulating membrane, with theflagellum emerging at the posterior end and runningtoward the anterior tip of the cell (Fig. 2). The kine-toplast in culture trypomastigote forms appears as aloose ‘‘basket-like’’ structure (Fig. 4).

Penetration of the salivary glands of R. domesticusby T. rangeli

Fifteen days after the experimental infection of the in-sects, a high number of parasites—mostly long epim-astigotes—were 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 thebasal lamina, usually with the flagellum foremost(Figs. 6, 7). Punctually damaged areas could be ob-served as holes in the basal lamina, with no visible

Experimental lysis ð%Þ ¼ Absorbance experimental lysis�Abs. spontaneous lysis

Abs. 100 % lysis�Abs. spontaneous lysis� 100:

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parasites associated to them (Figs. 6, 7). After pene-trating the outer layer of the basal lamina, the parasitescould be found in the space between the basal laminaand the salivary gland epithelium (Fig. 8). Epimastigotesin the basal lamina then invade the salivary gland cellsby a still unknown mechanism. After reaching the glandcell cytoplasm, the parasites transverse the gland cellinside tight (Fig. 9) or loose (Fig. 10) membrane bound

vacuoles, although in a single case we observed a para-site apparently free in the host cell cytoplasm (Fig. 9,inset). In both cases the parasites were not under thetypical amastigote form, but were either elongated(Fig. 9) or folded (Fig. 10) parasites with a flagellum.

After reaching the gland lumen, the parasites appearunder the epimastigote form and remain attached by theflagellum to the microvilli of the salivary gland cells

Figs. 1-4 Fine structure of culture forms of Trypanosoma rangeli(strain SC-58)Fig. 1 Scanning electron microscopy showing long epimastigoteformsFig. 2 Scanning electron microscopy of a short trypomastigoteform

Fig. 3 Transmission electron microscopy of an epimastigote formshowing the Golgi complex (GC) located near the central nucleus(N), and the rod-shaped kinetoplast (arrowhead)Fig. 4 Transmission electron microscopy of a trypomastigote formshowing the kinetoplast with the ‘‘basket-like’’ structure (arrow). Nnucleus, M mitochondrion

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(Figs. 11, 12). Location of the kinetoplast close tothe nucleus indicates that the epimastigotes start todifferentiate into trypomastigotes while still attachedto the gland cell microvilli (Fig. 11). No membranespecialization could be observed at the attachment

region, either at the flagellar or the gland cell membranes(Fig. 12). Large vesicles were observed in the cytoplasmof the adhered epimastigotes, which could not be seen inculture epimastigotes (data not shown). Short metacyclictrypomastigotes are found swimming free in the saliva

Figs. 5-8 Scanning (Figs. 5-7) and transmission (Fig. 8) electronmicroscopy of Rhodnius domesticus salivary glands infected with T.rangeli (strain SC-58)Fig. 5 Low magnification view of the outer surface of the salivarygland showing the high number of adhered parasitesFig. 6 Both trypomastigote (t) and epimastigote (e) forms can befound adhered to the outer surface of the salivary glandFig. 7 High magnification showing long epimastigote formspenetrating the basal lamina of the salivary gland (arrows). Notepunctual damages (arrowheads), with no parasites associated

Fig. 8 Transmission electron microscopy showing parasites locatedbetween the basal lamina (BL) and the salivary gland cell. A rod-shaped kinetoplast, typical of epimastigote forms, can be seen(arrow). A flagellum is observed inside a basal lamina layer(arrowhead)

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close to the gland cell microvilli (Fig. 13) and at thesalivary gland lumen (Fig. 14). Notably, these metacy-clic trypomastigotes present a rod-shaped kinetoplast(Fig. 14).

Hemolytic assays

As our ultrastructural data suggested an active pene-tration of T. rangeli into the insect salivary glands, wehave raised the hypothesis that the parasites could beusing cytolytic or pore-forming proteins to transversethis epithelial barrier. Hemolytic assays with serialdilutions of epimastigote and trypomastigote extractsshowed a prominent and dose-dependent hemolyticactivity in extracts from both forms at an acidic pH(pH 6.5, Fig. 15a), but not at pH 7.5 (Fig. 15b). Epim-astigotes appear to produce a higher amount of thishemolytic molecule, since one hemolytic unit (HU, the

volume of parasite extract sufficient to lysate 50% of themice erythrocytes) corresponded to 19 ll in epimastig-otes, against 33 ll in trypomastigotes (Fig. 15a). Thesedata prompted us to evaluate the production of thiscytolytic molecule by flow cytometry analysis.

Flow cytometry

Flow cytometry analysis was performed by using apolyclonal rabbit anti-mouse perforin IgG, expecting apossible high homology and cross-reactivity between thelytic molecule and perforin. Perforin is a 70-kDa, Ca2+ -dependent, pore-forming protein produced by cytotoxiclymphocytes and stored within cytoplasmic granules inthese cells (Liu et al. 1995). We found positive labeling inextracts from both epimastigote and trypomastigoteforms, indicating the presence of an antigenic perforin-like molecule in T. rangeli (Fig. 16a and b). Incubation

Figs. 9-12 Transmissionelectron microscopy of T.rangeli in the salivary glands ofR. domesticusFig. 9 A parasite inside a tightmembrane bound vacuole in thesalivary gland cell cytoplasm.The arrow indicates thekinetoplast. Inset: detail of afree parasiteFig. 10 An intra-vacuolarparasite at the salivary glandcell cytoplasm. The arrowshows the vacuole membrane.F flagellumFig. 11 An intermediaryepimastigote form, with thekinetoplast (arrow) locatedparallel to the nucleus (N),adhered to the gland cellmicrovilli (Mi) by the flagellum(F). L gland lumenFig. 12 Epimastigote formadhered to the gland cellmicrovilli by the flagellum(arrow). L gland lumen. Inset:detail showing the absence ofmembrane specialization at theattachment region (arrowhead)

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Fig. 13 Scanning electronmicroscopy of trypomastigoteforms free in the saliva, at thesalivary gland lumenFig. 14 Transmission electronmicroscopy of a trypomastigoteform free in the saliva, at thesalivary gland lumen (L). Notethe rod-shaped kinetoplast. Fflagellum, N nucleus

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with pre-immune serum showed a lower positive labelingpattern in epimastigotes (Fig. 16b), thus indicatingcross-reactivity of parasite antigens with the naturalbackground repertoire of the rabbit serum. No labelingwas found in trypomastigote forms incubated with thepre-immune serum (Fig. 16c).

Western blotting assays

Polyclonal anti-perforin IgG reacted with a protein ofapproximately 120 kDa in epimastigote forms, as well aswith a protein with less than 85 kDa (Fig. 17, lane 1).Cross-reaction with the latter molecule could be alsodetected after incubation with the pre-immune serum(Fig. 17, lane 2) and thus this molecule is possiblyresponsible for the intermediate labeling intensity foundin the FACS analysis (Fig. 16b). Control performed byincubation with the secondary IgG alone resulted in nodetectable labeling (Fig. 17, lane 3). Silver staining of theelectrophoretic run demonstrated that the 120 kDamolecule recognized by the anti-perforin antibody is nota majoritary protein (Fig. 17, lane 4).

Discussion

T. rangeli is a hemoflagellate protozoan transmitted to avariety of wild and domestic animals during the triato-

mine blood meal (D’Alessandro 1976). In the inverte-brate vector, T. rangeli has a characteristic life cycle,with a peculiar ability to invade the haemolymph andthen the salivary glands, where a large number ofinfective metacyclic trypomastigote forms are generated.Furthermore, it has been observed a preferential com-bination between certain parasite strains and Rhodniusspecies, indicating the intimate reliance of the parasite–host interaction (Cuba-Cuba 1998). In order to obtainhigh salivary gland infection rates we infected triato-mines from Santa Catarina, Brazil (R. domesticus) withparasites isolated from this same geographic region(strain SC-58). A previous study had demonstrated thatin this case high infection rates can be obtained (Steindelet al. 1991).

In most trypanosomatids, the parasites adhere to thedigestive tract of their respective insect hosts and form ahomogeneous cellular layer as a critical step in their lifecycle (Tieszen et al. 1986, 1989; Molyneux et al. 1987). Invitro studies on the interaction of T. rangeli with dis-sected fragments of the posterior midgut of R. prolixusshowed few parasites attached to sparse epithelial cells,suggesting that a recognition step is necessary for pos-terior invasion (Oliveira and De Souza 2001). Thisbehavior has also been observed in other insect-proto-zoan models, such as Plasmodium gallinaceum infectingAedes aegypti, where the parasites were found associatedpreferentially with a well-defined cell type (Shahabuddinand Pimenta 1998). Analysis of the interaction betweenT. rangeli and the epithelium of the salivary glands of R.domesticus suggests that in this case no such specificrecognition process occurs: the parasites appeared to beindividually penetrating the basal lamina all over thesalivary gland surface. A previous study with FITC-la-beled lectins showed that the salivary gland surfacepresents different carbohydrate residues that may serveas receptors to which the long epimastigote forms attachprior to the invasion (Basseri et al. 2002).

A former ultrastructural study on the mechanisms ofT. rangeli penetration into R. prolixus salivary glandsshowed that the parasites are able to penetrate the basallamina and cross the gland cell cytoplasm (Ellis et al.1980). Our morphological data showed mainly epimas-tigote forms transversing the basal lamina through smallholes to reach the glandular epithelium, while trypom-astigotes are found adhered to the outer gland surface.Our data suggest that epimastigote forms can produce alytic molecule, such as a pore forming protein (PFP), toallow the passage of the parasites through the epithelialbarrier. Indeed, hemolytic assays demonstrated a lyticmolecule exclusively active in the acidic environment.This hemolytic molecule, which we named as rangelysin,seems to be present in higher amounts in epimastigoteforms, although FACS analysis showed that it is alsopresent in culture trypomastigotes. It is possible thatonly epimastigote forms secrete this lytic molecule insufficient amounts to damage the basal lamina, thusallowing the gland cell penetration.

Fig. 15 Hemolytic assay with serial dilutions of T. rangeliepimastigote (filled diamond) and trypomastigote (filled square)extracts, at pH 6.5 (a) and pH 7.5 (b)

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Channel formation by PFPs is a well-definedmechanism of membrane damage used in biologicalsystems ranging from bacteria to vertebrates (Lynchet al. 1982; Bhakdi and Tranun-Jensen 1988; Tweten1995; Ludwig 1996; Suss-Toby et al. 1996; Nickel et al.1999). Several pathogenic protozoa produce cytolyticproteins that disrupt target cell membranes by formingchannels in the lipid bilayer (Jiang et al. 1990; Ley et al.1990; Carruthers 1999). These PFPs are thought to playa significant role in the pathogenesis of many proto-zoan infections, such as in amebiasis (Andrews andPortnoy 1994; Fiori et al. 1996; Noronha et al. 1996;Desai and Rosenberg 1997; Horta 1997). Pore-formingproteins usually bind to the plasma membrane of atarget cell and expose hydrophobic domains, thusallowing ions and small molecules to pass freely acrossthe lipid bilayer. Our data indicate that T. rangelipresents and probably uses a 120 kDa PFP to penetratethe salivary gland cells of the insect vectors, howeverwithout rupture of the host cells.

We have observed intracellular parasites enclosed invacuoles in the gland cell cytoplasm. However, theseforms present a flagellum, and thus do not represent

typical intracellular amastigote stages of the parasites.Ellis et al. (1980) suggested that the parasites cross thesalivary gland cells towards the gland lumen enclosedwithin vacuoles until they come near the apical gland cellportion, where most parasites appear to lose their vac-uoles before leaving the gland cells. Indeed, we foundone parasite free in the host cell cytoplasm. It is possiblethat T. rangeli, with the help of rangelysin, evades theparasitophorous vacuole in a way similar to that used byT. cruzi with the help of TC-tox (Andrews 1990; An-drews et al. 1990).

When the parasites finally reach the gland lumen theyremain adhered to the gland cell microvilli as epim-astigotes. As only trypomastigotes were found free in thesaliva, it seems that the metacyclogenesis starts withadhered epimastigotes and intermediary forms. Ultra-structural analysis showed morphological differences inthe kinetoplast morphology between trypomastigotesfrom culture and from infected salivary glands. Salivarygland trypomastigotes present a condensed kinetoplast,and thus this morphological characteristic allows dif-ferentiating between metacyclic and culture trypomas-tigote forms of T. rangeli.

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

curve), the pre-immune serum (solid line) and the FITC-conjugatedgoat anti rabbit IgG (dashed line). All analyses were performed inthe region (R1) indicated

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Acknowledgements The authors thank Dr. Pedro M. Persechini(Institute of Biophysics, UFRJ, Rio de Janeiro, Brazil) for kindlysupplying the anti-perforin antibody and Mr. Jose Lopes de Fariafor the photographic work. This work was supported by CNPq,FAPERJ, PAPES-III/FIOCRUZ and FIOCRUZ. All experimentswere performed according to the Brazilian laws.

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Fig. 17 Lanes 1–3 Western blotting assay with extracts ofepimastigote forms of T. rangeli, strain SC-58. Polyclonal rabbitanti-mouse perforin IgG was used to detect a 120 kDa protein withcross-reactivity with perforin in T. rangeli (arrowhead, lane 1).Cross-reaction with a <85 kDa protein could be detected afterincubation with the pre-immune serum (lane 2). Control performedby incubation with the secondary IgG alone (alkaline phosphatase-conjugated goat anti-rabbit IgG) resulted in no detectable labeling(lane 3). Silver staining of the electrophoretic run shows that the120 kDa molecule recognized by the anti-perforin antibody is not amajoritary protein (lane 4)

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