EXPERIMENTAL PARASITOLOGY 73, 1-14 (191)
Trypanosoma cruzi: Amastigotes and Trypomastigotes Interact Different Structures on the Surface of HeLa Cells
with
RENATO A. MORTARA Department
of Microbiology,
Immunology, and Parasitology, Escola Paulista 862 6” andar, 04023, Scio Pa&o, SP, Brazil
de Medicina,
Rua Botucatu,
MORTARA,RENATO A. 1991. Trypanosoma cruzi: Amastigotes and trypomastigotes interact with different structures on the surface of HeLa cells. Experimental Parasitology 73, 1-14. It is generally accepted that Trypanosoma cruzi trypomastigotes represent the infective forms of the etiological agent of Chagas’ disease. However, the invasive capacity of amastigotes and their ability to sustain a complete infective cycle in mammalian cultured cells and hosts has been recently demonstrated. In order to compare the process of cell invasion by these different infective forms, I examined the interactions of trypomastigotes and amastigotes with HeLa cells using a new and simple method that improves parasitecell interactions and significantly reduces incubation periods. T. cruzi forms were centrifuged onto HeLa cells grown on coverslips and parasite-cell interactions were examined by fluorescence and scanning electron microscopy. As expected, it was observed that all parasite forms attach and eventually enter the cells. However, whereas trypomastigotes preferentially invade HeLa cells at the edges, as has recently been demonstrated for other cell types, the initial steps of amastigote-HeLa cell interaction involve binding and entangling of the parasite to surface microvilli. Thus, different T. cruzi infective forms interact with different cell surface structures that could express different receptors at the HeLa cell membrane. 0 1991Academic Press, ICC INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Amastigote; Metacyclic trypomastigote; HeLa cell; Parasite-mammalian cell interactions; Actin; Cytoskeleton; Microvillus; RPM1 1640 containing 10% fetal bovine serum (R-10); Cytochalasin D (CD); Scanning electron microscopy &EM).
and hosts (Pan 1978; Umezawa et al. 1985; Ley et al. 1988). In an elegant piece of work, Schenkman et al. (1988) demonstrated that T. cruzi trypomastigotes invade mammalian cells in a polarized fashion and preferentially penetrate fibroblasts at the cell periphery. By contrast, the mode of interaction of amastigotes with mammalian cells has not yet been carefully examined. In spite of extensive studies on this subject, the molecular nature of the membrane elements involved in the initial steps of parasite attachment is only beginning to emerge (Quaissi et al. 1984; Kierszenbaum and Stiles 1985; Zingales and Colli 1985). Since actin microtilaments are the major contractile elements of the mammalian cell cytoskeleton associated with the plasma membrane (Alberts et al. 1983), their involvement in the uptake by phagocytic and
In order to establish a complete life cycle within the mammalian host, Trypan~~rna cruzi, the ethiological agent of Chagas’ disease, must invade cells, divide intracellularly as amastigotes, and, finally, transform into trypomastigotes that may eventually be ingested by triatomine vectors. It has been known for a while that trypomastigotes, derived from blood of infected mammalian hosts or mammalian cultured cells, and metacyclic trypomastigotes, obtained from axenic cultures or urine from infected vectors, represent the classic infective forms of the parasite (Brener 1973; De Souza 1984). More recent work, however, has also demonstrated that amastigotes, derived from infected cells or organs, can also infect and multiply within mammalian cells 1
0014-4894/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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RENATOA.MORTARA
nonphagocytic cells of the various parasite previously infected cells. Supematants were centristages has been examined by several au- fuged at 200s for 2 min to eliminate most cellular debris and parasites were subsequently collected at thors. The evidences are somewhat con- 25OOgfor 15 min. During the isolation procedure, tryflicting and mostly based on the analysis of pomastigotes spontaneously differentiated into the effect of cytochalasin B on the host cell amastigotes (Andrews et al. 1987) and mixtures conbut, nevertheless, favor a possible partici- taining from 5 to 50% trypomastigotes were usually pation of actin filaments in a classical en- obtained in this way. After purification, metacyclic trypomastigotes (7 x 10’ parasites ml-‘) and trypodocytic interiorization, particularly of try- mastigote-amastigote mixtures (2-4 X 10’ parasites pomastigotes (Alexander 1975; Nogueira ml-‘) were resuspended in R-10 medium and incuand Cohn 1976; Kipnis et al. 1979; Hen- bated at 37°C for 30 min before the centrifugation procedure. Parasite/cell ratios usually ranged between 5: 1 riquez et al. 1981; Meirelles et al. 1982). In a series of studies on the interaction and1511. The centrifugation of parasites onto HeLa cells was between enteropathogenic Escherichia coli carried out essentially as described earlier (Silva et (I/. and HeLa cells, it was demonstrated that 1989).Briefly, HeLa cells were grown for at least 24 hr localized adherence of the bacteria caused onto glass coverslips in 2-cm-diameter petri dishes aggregation of actin filaments due to bacte- containing 1.5 ml R-IO medium. The medium was asrial clumping of surface microvilli (Silva et pirated and after addition of the parasite suspensions dishes were immediately centrifuged in a swing-out al. 1989). To study the interactions between the rotor at 2000~ for 15 min at 30°C. After centrifugation, T. cruzi and a mammalian cultured cell line, unattached parasites were removed by washing the I have adapted the centrifugation method coverslips 10 times with phosphate-buffered saline used in that work (Silva et al. 1989) which (PBS). Associated parasites produced normal infecimproves parasite-cell interactions and re- tions as determined by the observation of amastigotecells after 2448 hr and by the presence of duces the time of manipulations. Followed bearing trypomastigotes in culture supernatants after 4-5 days. by a combination of fluorescence and scan- One set of coverslips was fixed with methanol and ning electron microscopy, the mode of as- stained with the Giemsa reagent. Duplicate sets of insociation between different T. cruzi forms fected and control coverslips were immediately fixed and HeLa cells was examined. It was ob- in 3.7% formaldehyde/PBS for 30 min at room temperature for immunofluorescence observations or proserved that trypomastigotes tend to invade cessed for scanning electron microscopy (SEM). HeLa cells at the periphery, as expected The formaldehyde-fixed coverslips were washed from previous studies (Schenkman et al. three times with PBS and after a 15-min incubation in 1988). By contrast, amastigotes, which can 50 mM NH&l/PBS to quench excess aldehyde groups, induce the formation of actin aggregates, the coverslips were treated with PBS containing 0.25% 0.05% NaN, (PGN), and 0.1% saponin to imdetected at the optical microscopy level, gelatin, prove permeabilization and prevent nonspecific antibind to microvilli forming small aggregates body binding during the subsequent incubations. In at the cell surface before entering the HeLa the series of experiments carried out to determine parasite interiorization, coverslips were fixed in 2% glutcells. MATERIALSAND
METHODS
The T. cruzi G strain used throughout this study was isolated from an infected opossum by Mena-Barreto in the Amazon (Yoshida 1983). Epimastigotes were grown in LIT medium (Camargo 1964)and metacyclic trypomastigotes were isolated from late stationary LIT cultures as previously described (Mortara ef al. 1988). HeLa cells were grown at 37°C in RPM1 1640medium supplemented with 10% fetal bovine serum and 40 ug ml-’ gentamycin (R-10 medium) in a 5% CO, humid atmosphere. HeLa cell-derived trypomastigotes and amastigotes were isolated from culture supernatants of
araldehyde (Sigma, EM grade) in PBS for 1 hr at room temperature. After three washings in PBS, the coverslips were incubated with 0.138 M ethanolamine, pH 8.3, for 30 min at room temperature to block excess aldehyde groups, washed with PBS, and soaked in PGN. Permeabilization of glutaraldehyde-fixed samples was achieved by soaking the coverslips in PGN containing 1% Nonidet-P40 (NP-40) before antibody incubation as described below. After 15-30 min, excess liquid was blotted with filter paper and the coverslips were inverted for 45 min onto 25-pl drops of 2C2 (Andrews et al. 1987)or 3F5 antiT. cruzi monoclonal antibodies (3F5 reacts with a surface glycoconjugate expressed on the cell surface of metacyclic trypomastigotes and epimastigotes-R. A. Mor-
T. cruzi INFECTIVE FORMS tara and S. da Silva, manuscript in preparation)-both ascitic fluids diluted 1:100 in PGN-in a humid chamber. Subsequently, samples were washed five times with PBS and treated in the same way with a solution containing the appropriate FITC conjugate (Dako Corp.) and phalloidin coupled to rhodamine (Faulstich ef al. 1983), a kind gift of H. Faulstich. T. cruzi actin was not labeled with the concentrations of phalloidinrhodamine used in this study and required to 30- to 40-fold increase to be visualized (Mortara 1989; R. A. Mortara, unpublished observations). After washing the conjugate/phalloidin mixture as above, coverslips were mounted in 90% glyceroli0.2M glycine, pH 9.2. Photographs were taken using black and white Ilford HP5 400 ASA films on a Leitz Laborlux D microscope equipped for epifluorescence with selective FITC and rhodamine filters using 40 and 100x phase-contrast objectives (Silva et al. 1989). For experiments with cytochalasin D (CD, obtained from Sigma Chemical Co.), appropriate amounts (OS5 (~1)of a 5 mg ml-’ stock solution in dimethylsulfoxide were added to the HeLa cell supematants and samples incubated for 15 min (final CD concentration ranged between 1 and 5 pg ml-‘). After this period, coverslips were washed three times with R-10 medium before parasite centrifugations. The parasite suspensions were then added and the dishes subjected to the centrifugation procedure. For SEM cells grown on glass coverslips were washed 10 times in PBS after parasite centrifugation, fixed with 2.5% glutaraldehyde (Sigma, EM Grade) in 0.1 M phosphate buffer, pH 7.2, postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2, dehydrated in an ethanol series, and critical-point dried with CO,. The coverslips were mounted on appropriate stubs, coated with gold, and examined on a JEOL JSM 25 SII scanning electron microscope operated at 25 kV.
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cells and images suggesting parasite invasion were frequently observed under phasecontrast microscopy and immunofluorescence (Figs. IA and IB). Following the attachment of metacyclic trypomastigotes, rearrangements of actin microfilaments from HeLa cells were not frequently detected, as judged by the analysis of phalloidin-rhodamine distribution under conventional fluorescence microscopy (Fig. IC). Further observation of attached metacyclic trypomastigotes showed that after 24 hr all parasites had entered the cells leading to normal infections of more than 60% of the cells, as judged by the presence of intracellular amastigote forms (result not shown). The observation of corresponding samples at the scanning electron microscope showed that the dorsal surface of the HeLa cells used in this study (see also Silva et al. 1989) was covered with microvilli (Fig. 2). A substantial proportion (94%, II = 254) of the metacyclic trypomastigotes observed were end-on associated with their posterior (kinetoplast) portion toward the cells and had part of their cell body already inside the HeLa cells, suggesting that they were engaged in the process of invasion (e.g., Fig. 2B). The trypomastigotes were usually (89% of parasites scored, IZ = 224) observed at cellular edges (Fig. 2A) and intercellular contacts (Fig. 2B) not more than 1 km away from the cell borders and did not RESULTS appear to associate with surface microvilli The interaction of different T. cruzi de- (Figs. 2B-2D). Interestingly, in some cases velopmental forms with HeLa cells was ex- more than one metacyclic trypomastigote amined in this study. To improve the asso- was found quite close to others(s) paraciation with HeLa cells and reduce the ma- site(s) penetrating a cell. Under identical nipulation time, parasites were centrifuged experimental conditions, epimastigotes did onto HeLa cells grown on coverslips and not attach to HeLa cells (see Table I). When mixtures of tissue culture-derived after washing unattached parasites, samamastigotes and trypomastigotes were cenples were processed for immunofluorestrifuged onto HeLa cells, actin aggregation cence and scanning electron microscopy. On average, these experiments were per- underneath the sites of amastigote adhesion formed within 18 min from the addition of was observed (Figs. 3A-3D). At the optical the parasites to the cells until sample fixa- microscopy level it was observed that, using the centrifugation protocol, amastigotes tion. Under such conditions, metacyclic trypomastigotes readily attached to HeLa usually displayed a greater tendency to as-
RENATO
A. MORTARA
sociate with HeLa cells than tissue culturederived trypomastigotes (see Table I). Using glutaraldehyde-fixed and unpermeabilized samples, it could be determined that amastigotes were already inside the cell after the centrifugation step since parasites visible by phase contrast were not labeled with the monoclonal antibody (Figs. 3A3D). By contrast, when samples were fixed in glutaraldehyde and subsequently permeabilized with 1% NP-40, all amastigotes associated with the HeLa cells were labeled with monoclonal antibody 2C2 (Figs. 3D3F). Actin aggregation was evident at the sites of amastigote attachment and the amastigotes without the corresponding microfilament aggregate were usually those that did not label with the antibody and were considered to be already within the cell (Figs. 3A-3D). Quantitation of these samples indicated that after 15 min of centrifugation, 41% of the attached amastigotes were already inside the HeLa cells (Table II). Since all changes in actin distribution after amastigote attachment to HeLa cells were monitored at the fluorescence microscope by double-labeling samples with phalloidin-rhodamine and monoclonal antibody 2C2, controls were performed to ensure that 2C2 (raised against the major surface glycoprotein of Y strain amastigotes-Andrews et al. 1987, 1988) gave a positive reaction with 100% of the amastigotes of the G strain and that phalloidinrhodamine, at the concentration used in this study, did not label parasite actin (Fig. 4). Attachment and entry of amastigotes also led to successful infections and cells displaying several perinuclearly arranged FIG. 1. HeLa cell actin distribution following T. cruzi metacyclic trypomastigote centrifugation. (A) Phase-contrast micrograph of cells with associated parasites (arrows); (B) immunofluorescence image of metacyclics stained with monoclonal antibody 3F5 with suggestive images (arrows) of cell invasion; (C) same fields labeled with phalloidin-rhodamine, with typical actin filament bundles. Magnification: x685. Bar = 10 (*m.
FIG. 2. Scanning electron microscopy of HeLa cells after centrifugation of T. cruzi metacyclic trypomastigotes. (A-D) Images of parasites invading HeLa cells at the edges. (C and D) Metacyclic trypomastigotes penetrating a cell close to each other. Magnifications: (A) X 1000; (B and C) x4500; (D) x3000. Bars = 5 km.
amastigotes were already detectable 24 hr postinfection (Table II and Fig. 5). Examination by SEM of samples fixed immediately after the centrifugation step revealed that amastigotes adhered to the dorsal surface of HeLa cells (Fig. 6A). At higher magnifications, the adherence sites consistently displayed microvilli attached to the parasite surface forming a small clump (Figs. 6B-6F). Quantitative studies carried out at high magnifications (X 10,000) indicated that 95% of the attached amastigotes were actually entangled with surface microvilli whereas the number of contaminating trypomastigotes associated with the cells was comparatively small. Up to 10 amastigotes could associate with a single HeLa cell but the majority of infected cells contained between one and three parasites on their dorsal surface (Fig. 7). The role of microtilaments on the attachment and interiorization of T. cruzi into
mammalian cells has been probed with cytochalasin B. Cytochalasin D was used in this study because it has a higher affinity for actin filaments and its effects on mammalian cells is also more specific than those of cytochalasin B (Cooper 1987). Using the glutaraldehyde fixation approach it was possible to distinguish between attached and internalized amastigotes in cytochalasin D-treated HeLa cells. Cytochalasin D treatment as performed here (5 p.g ml - ’ for 15 min) caused dramatic changes in cell morphology (see below) and despite a significant reduction in the proportion of intracellular parasites, it did not block the entry of amastigotes (Table II). Figure 8 shows one of such samples in which attached (labeled) and internalized (unlabeled) amastigotes can be distinguished. The distribution of actin in CD-treated cells was also affected and microfilament aggregates were visible throughout the cytoplasm (Fig. 8).
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TABLE I Treatment of HeLa Cells with Cytochalasin D: Effect on Ttypanosoma cruzi Association*
T. cruzi form
Metacyclic trypomastigote@ Amastigotesl trypomastigotes (70:30 ratio)
Cytochalasin D (wg ml-‘)
2 5 Epimastigotes
3.18 k 0.36 2.65 -r- 0.32 2.48 2 0.40
0 2 5
0
0
No. of parasites associated per cell
Ad T A T A T
3.01 2 0.24 2 3.48 4 0.33 ? 2.93 t 0.34 2 0
0.86 0.03 0.79 0.13 0.93 0.11
0 Parasites scored on at least 400 cells on duplicate cover slips after Giemsa staining. b Figures represent means 5 standard error of two independent experiments. c Figures represent means k standard error of three independent experiments. d A, amastigotes; T, trypomastigotes.
Another quantitative evaluation of the effect of treating HeLa cells with cytochalasin D on T. cruzi association was obtained with samples stained with Giemsa reagent. Quantitation was expressed in terms of parasites associated per cell, since this staining procedure does not allow the clear distinction between interiorized and attached forms (Table I). Metacyclic trypomastigote association with HeLa cells was hardly affected by CD treatment and also no significant differences were observed in the attachment of amastigotes, both to control and to CD-treated cells (Table I). Similarly, tissue culture-derived trypomastigotes associated with CD-treated HeLa cells in levels comparable to that of the control samples (Table I). Incubation of cytochalasin D-treated samples for 24 hr (in medium without the drug) resulted in infections comparable to that of the controls, both for amastigotes (Table II) and metacyclic trypomastigotes (not shown). DISCUSSION
Phalloidin staining also revealed that at some sites of amastigote attachment, cellular actin appeared to surround the entire parasite, suggesting that a membrane process is preventing the binding of monoclonal 2C2 to the parasites (vertical arrows in Figs. 8A and SC). Cells treated with cytochalasin D displayed profound morphological changes. They tended to round up and their attachments to the substratum appeared to retract; moreover, their surface presented few microvilli and became covered with blebs (Fig. 9A). Quantitation of amastigote binding to CD-treated HeLa cells at the SEM level (Figs. 9C and 9D) was not possible since the abundance of surface blebs masked the attached parasites (see, for instance, Fig. 90). On the other hand, metacyclic trypomastigotes are frequently seen invading CD-treated HeLa cells, preserving their preference for the cell periphery (Figs. 9E and 9F).
Based on previous observations that enteropathogenic E. coli cause actin aggregation after localized adhesion of the bacteria to HeLa cells (Silva et al. 1989) and considering the evidences of a possible involvement of actin filaments in the interiorization process of T. cruzi in mammalian cells (Nogueira and Cohn 1976; Kipnis et al. 1979; Henriquez et al. 1981; Meirelles et al. 1982), the interaction of T. cruzi with HeLa cells was studied in order to examine possible effects of different developmental forms of the parasite on the host cell microfilament system. For such studies a centrifugation procedure previously described in our laboratory (Silva et al. 1989) was employed to maximize parasite-HeLa cell interactions and to reduce manipulation time. Results with metacyclic trypomastigotes confirmed previous observations (Schenkman et al. 1988) that these forms tend to invade cells at the periphery (Fig. 2). By contrast, amastigotes attach preferentially
T.
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FIG. 3. Centrifugation of T. cruzi amastigotes onto HeLa cells induces aggregation of actin filaments and leads to parasite entry. (A-C) Phase-contrast microscopy, 2C2 staining, and actin distribution in glutaraldehyde-fixed samples. (D-F) Equivalent samples stained as before after permeabilization with 1% Nonidet-P40-note that all parasites are labeled with the antibody. Black arrows in (A): intemalized amastigotes; black arrowheads in (A) indicate attached parasites, also labeled with 2C2 (see B); white arrows in (C): actin aggregates corresponding to attached amastigotes. Magnification: x710. Bar = 10 pm.
to the dorsal surface of HeLa cells where they induce actin filament aggregation that corresponds to clumps of surface microvilli and parasites (Figs. 4 and 6). Attached and internalized amastigotes could be distinguished using differential antibody accessibility after glutaraldehyde fixation (Fig. 4 and Table II). The finding that 41% of the
amastigotes were localized within the cells after 15 min required for the centrifugation step supports the notion that this procedure promotes infection by amastigote forms. Under the experimental conditions described here, trypomastigotes derived from infected cells displayed a lower degree of association with HeLa than amastigotes. It
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TABLE II Entry of Trypanosoma cruzi Amastigotes” into HeLa Cellsb Effect of Cytochalasin D Percentage of Cytochalasin DC parasitesd inside No. amastigotes’ inside 100 cells the cells (kg mlm’) 0 5 0 5
41.3 k 11.2 2 97.1 2 95.4 k
6.8 0.9 l.lf 2.P
155 t 422 255 ? 247 2
13 10 35 42
a These parasite preparations contained more than 95% amastigotes. b Parasites were centrifuged onto HeLa cells and after glutaraldehyde fixation, samples were stained with monoclonal antibody 2C2. c Cells were incubated for 15 min with 5 ug ml-’ CD. d Percentage refers to the ratio of parasites not labeled/total parasites on at least 200 cells on duplicate cover slips. Figures represent means rt standard error of two independent experiments. p Intracellular amastigotes scored on 200 cells on duplicate coverslips. Figures represent means 2 standard error of two independent experiments. f Figures obtained following incubation in drug-free medium for 24 hr, after centrifugation.
should be pointed out, however, that these associations do not reflect the relative infectivities under conventional conditions where trypomastigotes clearly display a much greater capacity to infect mammalian cells (e.g., Schenkman et al. 1988). Surface projections like microvilli and filopodia are probing organelles (Vasiliev 1981) thought to be involved in initial steps of parasitexell interactions (McGee et al. 1988). The interaction of amastigotes with microvilli on the dorsal surface of HeLa cells forming small clumps (Fig. 6) confirms that these membrane projections act as docking elements to the spheroid amastigotes. These interactions could involve specific parasite and microvillus membrane components. This apparently specific association with microvilli could imply that these surface protrusions concentrate specific membrane receptor molecules for amastigotes, as seems to occur in other systems (e.g., Koch and Smith, 1978). Inter-
FIG. 4. Control of T. cruzi amastigote staining with monoclonal antibody 2C2 and phalloidin-rhodamine. (A) Phase-contrast micrograph of parasites; (B) 2C2 monoclonal antibody staining, and (C) phalloidinrhodamine staining of actin using the same concentrations of the reagent as in Figs. 1, 3, and 8.
estingly, when HeLa cells are treated with cytochalasin D at concentrations that dramatically altered their surface morphology (Fig. 9) and completely blocked localized adherence of enteropathogenic E. coli (Silva et al. 1989), amastigote (and trypomastigote) adhesion was still observed in levels comparable to those of the controls (Table I). Since cytochalasin D-treated cells express fewer microvilli while retaining their capacity to physically attach to amastigotes, it could be tentatively assumed that the putative receptor molecule(s) remains functionally expressed at the surface and that the microvillar structure is not required for parasite adhesion.
T. cruzi INFECTIVE FORMS
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ments with CD greatly inhibited amastigote entry after the centrifugation procedure (Table II). At this point it would be interesting to consider the role of host cell actin microtilaments on T. cruzi attachment and invasion. Previous studies based mainly on the inhibitory effect of cytochalasin B were taken as indications that microfilaments participate in the uptake of T. cruzi by cells (Alexander 1975; Nogueira and Cohn 1976; Henriquez et al. 1981; Meirelles et al. 1982). In these studies it was shown that severe disruption of the cortical microtilament system with cytochalasin D did not cause a significant effect on the degree of association of different T. cruzi infective forms with HeLa cells (Table I) and subsequent incubations in medium without the drug resulted in normal infections (Table II). It is noteworthy that metacyclic trypomastigotes retain their preference to invade the cells at the periphery, even in CDtreated cells (see Figs. 2 and 9). In a recent study, Schenkman et al. (1991) observed that CD, at the same concentration used here, did not affect the entry of Y-strain trypomastigotes into HeLa cells. These findings support the notion that (1) attachment is required for invasion and (2) attached parasites will eventually enter (even CD-treated) cells. This is intriguing in view of the accepted notion that a classical reFIG. 5. Amastigote attachment and invasion causes ceptor-mediated endocytosis mechanism is normal infection. Amastigotes were centrifuged onto believed to operate in the uptake of the parHeLa cells, washed, and incubated for 24 hr before asite (Kierszenbaum and Stiles, 1985; fixation and 2C2 staining. (A) Phase-contrast and (B) Schenkman et al. 1988; Zingales and Colli 2C2 labeling of amastigotes. Magnification: X260. Bar 1985). In such a mechanism, motility of = 10 pm. membrane receptors tethered to the cytoskeleton leading to the uptake of surface This is again in contrast to the effect ob- ligands is thought to require a functional served with the adhesion of enteropatho- cortical meshwork of actin microfilaments, genie E. cofi that is almost abolished when since such processes are promptly inhibited HeLa cells are exposed to conditions that by cytochalasins (e.g., Cooper 1985). The reduce the expression of microvilli (Silva ef observations that the infective forms of T. al. 1989). Although cytochalasin D did not cruzi keep their capacity to associate with significantly affect parasite attachment (Ta- and invade (albeit to a lower degree in the case of the amastigotes) cells bearing disble I), the disruption of cellular microfila-
FIG. 6. T. cruzi amastigote centrifuged onto HeLa cells entangle to microvilli. (A-H) Scanning electron micrographs showing amastigotes attached to the dorsal surface of HeLa cells. Images in (B-H) show details of clumps formed with microvilh bound to amastigotes. Magnifications: (A) X 1500; (B) x4500; (C and D) x7000; (E) x4500; (F) x7000; (G and H) X10,000. Bars in A, B, C, and H = 5 pm.
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FIG. 7. Frequency of T. cruzi amastigote attach-
ment per HeLa cells after parasite centrifugation. Black bars: determined by SEM; hatched bars: determined after Giemsa staining.
rupted microfilaments and altered surface morphology is consistent with the notion that the interaction of parasite ligands with appropriate functional receptor molecules at specific surface sites, as envisaged by King (1988), could be sufficient to trigger a parasite-directed endocytosis (McGee ef al. 1988) efficient enough to overcome the damage caused to the host cell cytoskeleton by cytochalasin D. The highly motile trypomastigotes would be more efficient than amastigotes in such an internalization mechanism involving a positive participation of T. cruzi, which could be envisaged as an active entry process as previously suggested by Kipnis et al. (1979). Finally, the preference of trypomastigotes for the cell periphery and the association of amastigotes with surface microvilli
FIG. 8. Cytochalasin D inhibits but does not block amastigote entry into HeLa cells. Cells were fixed with glutaraldehyde after treatment with 5 ug ml - t CD for 15 min and parasite centrifugation. (A) Phasecontrast micrograph, (B) 2C2 staining, and (C) actin distribution. Black arrows in (A), internalized amastigotes; arrowheads in (A), attached amastigotes (see B); white arrows in (C), actin aggregates at amastigote attachment (horizontal) and internalization (vertical) sites. Vertical arrows at top right in (A) and (C) indicate internalized amastigote tightly covered with cellular actin. Magnification x800. Bar = 10 urn.
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FIG. 9. T. cruzi forms attach to cytochalasin D-treated HeLa cells. Scanning electron microscopy of different T. cruzi infective forms centrifuged onto HeLa cells after incubation for 15 min with 5 p.g ml-’ CD. (A) General aspect of treated cells. (B-D) Cells after amastigote:trypoamstigote mixture (70:30 ratio) centrifugation (arrow in D points to an attached amastigote); (E and F) cells after metacyclic trypomastigote centrifugation; note trypomastigotes penetrating the cells at the edges. Magnifications: (A) x1000; (B) x2000; (C-E) x4500; (F) x4000. Bars in A, B, D, and F = 5 pm.
shows that different T. cruzi developmental forms use or mobilize distinct structures and/or receptors to attach and invade HeLa cells. ACKNOWLEDGMENTS I thank Sebastiio Cruz, Helena C. Barros, and Wanderley de Souza for their help with the scanning microscopy. I am grateful to my colleagues Nobuko
Yoshida and Jose Franc0 da Silveira for their comments and suggestions on the manuscript. I am particularly grateful to Sergio Schenkman for helpful discussions and communication of his results prior to publication. This work was supported by grants from the Fundacao de Amparo a Pesquisa do Estado de Slo Paulo-FAPESP and Conselho National de desenvolvimento Cientitico e Tecnologico-CNPq, and UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Rockefeller Foundation.
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REFERENCES
ALBERTS, B., BRAY, D., LEWIS, J., RAFF, M., ROBERTS,K., AND WATSON,J. D. 1983. The cytoskeleton. In “The Molecular Biology of the Cell,” pp. 549-609. Garland, New York/London. ALEXANDER, J. 1975. Effect of the antiphagocytic agent cytochalasin B on macrophage invasion by Leishmaniu mexicana promastigotes and Trypanosoma cruzi epimastigotes. Journal of Protozoology 22, 237-240. ANDREWS, N. W., HONG, K. S., ROBBINS, E. S., AND NUSSENZWEIG,V. 1987. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Experimental Parasitology
64, 474-484.
ANDREWS, N. W., ROBBINS,E. S., LEY, V., HONG, K. S., AND NUSSENZWEIG,V. 1988. Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi. The Journal Medicine 167, 300-3 14.
of Experimental
BRENER,Z. 1973. Biology of Trypanosoma nual Reviews of Microbiology
cruzi. An27, 347-554.
CAMARGO,E. P. 1964. Growth and differentiation in Trypanosoma cruzi: Origin of metacyclic trypomastigotes in liquid media. Revista do Instituto de Medicina Tropical de Sdo Paul0 6, 93-100. COOPER,J. A. 1987. Effects of cytochalasins and phalloidin on actin. Journal of Cell Biology 105, 14731478. DE SOUZA, W. 1984. Cell Biology of Trypanosoma cruzi. international Reviews of Cytology 86, 197283. FAULSTICH, H., TRISCHMAN, H., AND MAYER, D. 1983. Preparation of tetramethyl rhodaminylphalloidin and uptake of the toxin into short-term cultured hepatocytes. Experimental Cell Research 144, 63-72.
HENRIQUEZ, D., PIUS, R., AND PIUS, M. M. 1981. The effect of surface membrane modifications of tibroblastic cells on the entry process of Trypanosoma cruzi trypomastigotes. Molecular and Biochemical Parasitology
2, 35%366.
KIERSZENBAUM,R., AND STILES, B. 1985. Evidence supporting the existence of a host cell surface receptor for Ttypanosoma cruzi. Journal of Protozoology 32, 364-366. KING, C. A. 1988. Cell motility of sporozoan protozoa. Parasitology Today 4( 1I), 3 15-3 19. KIPNIS, T. L., CALICH, V. L. G., AND DIAS DA SILVA, W. 1979. Active entry of bloodstream forms of Trypanosoma cruzi into macrophages. Parasitology 78, 89-98.
FORMS
13
KOCH, G. L. E., AND SMITH, M. J. 1978. An association between actin and the major histocompatibility antigen H-2. Nature (London) 273, 274-278. LEY, V., ANDREWS, N. W., ROBBINS, E. S., AND NUSSENZWEIG,V. 1988. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. Journal of Experimental Medicine 168, 649659. MCGEE, Z., GORBY,G. L., WYRICK, P. B., HODINKA, R., AND HOFFMAN, L. H. 1988. Parasite-directed endocytosis. Reviews on Infectious Diseases 10, S311-S316. MEIRELLES,M. N. L., ARA~JOJORGE,T. C., AND DE SOUZA, W. 1982. Interaction of Trypanosoma cruzi with macrophages in vitro: Dissociation of the attachment and internalization phases by low temperature and cytochalasin B. Zeitschrift fir Parasitenkunde 68, 7-14.
MORTARA, R. A., ARAGUTH, M. F., AND YOSHIDA, N. 1988.Reactivity of stage-specific monoclonal antibody lG7 with metacyclic trypomastigotes of Trypanosoma cruzi strains: Lytic property and antigen polymorphism. Parasite Immunology 10, 369-378. MORTARA,R. A. 1989. Studies on trypanosomatid actin. I. lmmunochemical and biochemical identification. Journal of Protozoology 36, 8-13. NOGUEIRA, N., AND COHN, Z. 1976. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cells. Journal of Experimental Medicine 143, 1402-1420. NOISIN, E. L., AND VILLALTA, F. 1989. Fibronectin increases Trypanosoma cruzi amastigote binding to and uptake by murine macrophages and human monocytes. Infection and Immunity 57, 1030-1034. OUAISSI,M. A., AFCHAIN, A., CAPRON,A., AND GRIMAUD, J. A. 1984. Fibronectin receptors on Trypanosoma cruzi trypomastigotes and their biological role. Nature (London) 308, 380-382. PAN, S. C.-T. 1978. Trypanosoma cruzi: In virro interactions between cultured amastigotes and human skin-muscle cells. Experimental Parasitology 45, 274-286. SCHENKMAN, S., ANDREWS, N. W., NUSSENZWEIG, V., AND ROBBINS, E. S. 1988. Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner. Cell 55, 157-165. SCHENKMAN,S., ROBINS,E. S., AND NUSSENZWEIG, V. 1991. The attachment and penetration of Trypanosoma cruzi into mammalian cells require parasite energy and can be independent of the target cell cytoskeleton. Infection and Immunity, 59, 645-654. SILVA, M. L. M., MORTARA, R. A., BARROS,H. C., DE SOUZA, W., AND TRABULSI, L. R. 1989. Aggre-
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gation of membrane associated actin filaments following localized adherence of enteropathogenic Escherichia coli to HeLa cells. Journa/ of Cell Science 93, 4394l6. UMEZAWA, E. S., MILDER, R. V., AND ABRAHAMcruzi amastigotes: SON, 1. A. 1985. Trypanosoma Development in vitro and infectivity in vivo of the forms isolated from spleen and liver. Acta Tropica
cells. In “International Cell Biology” (H. G. Schweiger, Ed.). Springer-Verlag, Berlin. YOSHIDA, N. 1983. Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi. Infection and Immunity
40, 836-839.
B., AND COLLI, W. 1985. Trypanosoma cruzi: Interaction with host cells. Current Topics in Microbiology and Immunology 117, 129-152.
ZINGALES,
42, 25-32.
J. M. 1981. The role of pseudopodial reactions in the attachment of normal and transformed
VASILIEV,
Received 24 July 1990; accepted with revision 22 January 1991
Trypanosoma cruzi: Amastigotes and Trypomastigotes Interact Different Structures on the Surface of HeLa Cells
with
RENATO A. MORTARA Department
of Microbiology,
Immunology, and Parasitology, Escola Paulista 862 6” andar, 04023, Scio Pa&o, SP, Brazil
de Medicina,
Rua Botucatu,
MORTARA,RENATO A. 1991. Trypanosoma cruzi: Amastigotes and trypomastigotes interact with different structures on the surface of HeLa cells. Experimental Parasitology 73, 1-14. It is generally accepted that Trypanosoma cruzi trypomastigotes represent the infective forms of the etiological agent of Chagas’ disease. However, the invasive capacity of amastigotes and their ability to sustain a complete infective cycle in mammalian cultured cells and hosts has been recently demonstrated. In order to compare the process of cell invasion by these different infective forms, I examined the interactions of trypomastigotes and amastigotes with HeLa cells using a new and simple method that improves parasitecell interactions and significantly reduces incubation periods. T. cruzi forms were centrifuged onto HeLa cells grown on coverslips and parasite-cell interactions were examined by fluorescence and scanning electron microscopy. As expected, it was observed that all parasite forms attach and eventually enter the cells. However, whereas trypomastigotes preferentially invade HeLa cells at the edges, as has recently been demonstrated for other cell types, the initial steps of amastigote-HeLa cell interaction involve binding and entangling of the parasite to surface microvilli. Thus, different T. cruzi infective forms interact with different cell surface structures that could express different receptors at the HeLa cell membrane. 0 1991Academic Press, ICC INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma cruzi; Amastigote; Metacyclic trypomastigote; HeLa cell; Parasite-mammalian cell interactions; Actin; Cytoskeleton; Microvillus; RPM1 1640 containing 10% fetal bovine serum (R-10); Cytochalasin D (CD); Scanning electron microscopy &EM).
and hosts (Pan 1978; Umezawa et al. 1985; Ley et al. 1988). In an elegant piece of work, Schenkman et al. (1988) demonstrated that T. cruzi trypomastigotes invade mammalian cells in a polarized fashion and preferentially penetrate fibroblasts at the cell periphery. By contrast, the mode of interaction of amastigotes with mammalian cells has not yet been carefully examined. In spite of extensive studies on this subject, the molecular nature of the membrane elements involved in the initial steps of parasite attachment is only beginning to emerge (Quaissi et al. 1984; Kierszenbaum and Stiles 1985; Zingales and Colli 1985). Since actin microtilaments are the major contractile elements of the mammalian cell cytoskeleton associated with the plasma membrane (Alberts et al. 1983), their involvement in the uptake by phagocytic and
In order to establish a complete life cycle within the mammalian host, Trypan~~rna cruzi, the ethiological agent of Chagas’ disease, must invade cells, divide intracellularly as amastigotes, and, finally, transform into trypomastigotes that may eventually be ingested by triatomine vectors. It has been known for a while that trypomastigotes, derived from blood of infected mammalian hosts or mammalian cultured cells, and metacyclic trypomastigotes, obtained from axenic cultures or urine from infected vectors, represent the classic infective forms of the parasite (Brener 1973; De Souza 1984). More recent work, however, has also demonstrated that amastigotes, derived from infected cells or organs, can also infect and multiply within mammalian cells 1
0014-4894/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
RENATOA.MORTARA
nonphagocytic cells of the various parasite previously infected cells. Supematants were centristages has been examined by several au- fuged at 200s for 2 min to eliminate most cellular debris and parasites were subsequently collected at thors. The evidences are somewhat con- 25OOgfor 15 min. During the isolation procedure, tryflicting and mostly based on the analysis of pomastigotes spontaneously differentiated into the effect of cytochalasin B on the host cell amastigotes (Andrews et al. 1987) and mixtures conbut, nevertheless, favor a possible partici- taining from 5 to 50% trypomastigotes were usually pation of actin filaments in a classical en- obtained in this way. After purification, metacyclic trypomastigotes (7 x 10’ parasites ml-‘) and trypodocytic interiorization, particularly of try- mastigote-amastigote mixtures (2-4 X 10’ parasites pomastigotes (Alexander 1975; Nogueira ml-‘) were resuspended in R-10 medium and incuand Cohn 1976; Kipnis et al. 1979; Hen- bated at 37°C for 30 min before the centrifugation procedure. Parasite/cell ratios usually ranged between 5: 1 riquez et al. 1981; Meirelles et al. 1982). In a series of studies on the interaction and1511. The centrifugation of parasites onto HeLa cells was between enteropathogenic Escherichia coli carried out essentially as described earlier (Silva et (I/. and HeLa cells, it was demonstrated that 1989).Briefly, HeLa cells were grown for at least 24 hr localized adherence of the bacteria caused onto glass coverslips in 2-cm-diameter petri dishes aggregation of actin filaments due to bacte- containing 1.5 ml R-IO medium. The medium was asrial clumping of surface microvilli (Silva et pirated and after addition of the parasite suspensions dishes were immediately centrifuged in a swing-out al. 1989). To study the interactions between the rotor at 2000~ for 15 min at 30°C. After centrifugation, T. cruzi and a mammalian cultured cell line, unattached parasites were removed by washing the I have adapted the centrifugation method coverslips 10 times with phosphate-buffered saline used in that work (Silva et al. 1989) which (PBS). Associated parasites produced normal infecimproves parasite-cell interactions and re- tions as determined by the observation of amastigotecells after 2448 hr and by the presence of duces the time of manipulations. Followed bearing trypomastigotes in culture supernatants after 4-5 days. by a combination of fluorescence and scan- One set of coverslips was fixed with methanol and ning electron microscopy, the mode of as- stained with the Giemsa reagent. Duplicate sets of insociation between different T. cruzi forms fected and control coverslips were immediately fixed and HeLa cells was examined. It was ob- in 3.7% formaldehyde/PBS for 30 min at room temperature for immunofluorescence observations or proserved that trypomastigotes tend to invade cessed for scanning electron microscopy (SEM). HeLa cells at the periphery, as expected The formaldehyde-fixed coverslips were washed from previous studies (Schenkman et al. three times with PBS and after a 15-min incubation in 1988). By contrast, amastigotes, which can 50 mM NH&l/PBS to quench excess aldehyde groups, induce the formation of actin aggregates, the coverslips were treated with PBS containing 0.25% 0.05% NaN, (PGN), and 0.1% saponin to imdetected at the optical microscopy level, gelatin, prove permeabilization and prevent nonspecific antibind to microvilli forming small aggregates body binding during the subsequent incubations. In at the cell surface before entering the HeLa the series of experiments carried out to determine parasite interiorization, coverslips were fixed in 2% glutcells. MATERIALSAND
METHODS
The T. cruzi G strain used throughout this study was isolated from an infected opossum by Mena-Barreto in the Amazon (Yoshida 1983). Epimastigotes were grown in LIT medium (Camargo 1964)and metacyclic trypomastigotes were isolated from late stationary LIT cultures as previously described (Mortara ef al. 1988). HeLa cells were grown at 37°C in RPM1 1640medium supplemented with 10% fetal bovine serum and 40 ug ml-’ gentamycin (R-10 medium) in a 5% CO, humid atmosphere. HeLa cell-derived trypomastigotes and amastigotes were isolated from culture supernatants of
araldehyde (Sigma, EM grade) in PBS for 1 hr at room temperature. After three washings in PBS, the coverslips were incubated with 0.138 M ethanolamine, pH 8.3, for 30 min at room temperature to block excess aldehyde groups, washed with PBS, and soaked in PGN. Permeabilization of glutaraldehyde-fixed samples was achieved by soaking the coverslips in PGN containing 1% Nonidet-P40 (NP-40) before antibody incubation as described below. After 15-30 min, excess liquid was blotted with filter paper and the coverslips were inverted for 45 min onto 25-pl drops of 2C2 (Andrews et al. 1987)or 3F5 antiT. cruzi monoclonal antibodies (3F5 reacts with a surface glycoconjugate expressed on the cell surface of metacyclic trypomastigotes and epimastigotes-R. A. Mor-
T. cruzi INFECTIVE FORMS tara and S. da Silva, manuscript in preparation)-both ascitic fluids diluted 1:100 in PGN-in a humid chamber. Subsequently, samples were washed five times with PBS and treated in the same way with a solution containing the appropriate FITC conjugate (Dako Corp.) and phalloidin coupled to rhodamine (Faulstich ef al. 1983), a kind gift of H. Faulstich. T. cruzi actin was not labeled with the concentrations of phalloidinrhodamine used in this study and required to 30- to 40-fold increase to be visualized (Mortara 1989; R. A. Mortara, unpublished observations). After washing the conjugate/phalloidin mixture as above, coverslips were mounted in 90% glyceroli0.2M glycine, pH 9.2. Photographs were taken using black and white Ilford HP5 400 ASA films on a Leitz Laborlux D microscope equipped for epifluorescence with selective FITC and rhodamine filters using 40 and 100x phase-contrast objectives (Silva et al. 1989). For experiments with cytochalasin D (CD, obtained from Sigma Chemical Co.), appropriate amounts (OS5 (~1)of a 5 mg ml-’ stock solution in dimethylsulfoxide were added to the HeLa cell supematants and samples incubated for 15 min (final CD concentration ranged between 1 and 5 pg ml-‘). After this period, coverslips were washed three times with R-10 medium before parasite centrifugations. The parasite suspensions were then added and the dishes subjected to the centrifugation procedure. For SEM cells grown on glass coverslips were washed 10 times in PBS after parasite centrifugation, fixed with 2.5% glutaraldehyde (Sigma, EM Grade) in 0.1 M phosphate buffer, pH 7.2, postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2, dehydrated in an ethanol series, and critical-point dried with CO,. The coverslips were mounted on appropriate stubs, coated with gold, and examined on a JEOL JSM 25 SII scanning electron microscope operated at 25 kV.
3
cells and images suggesting parasite invasion were frequently observed under phasecontrast microscopy and immunofluorescence (Figs. IA and IB). Following the attachment of metacyclic trypomastigotes, rearrangements of actin microfilaments from HeLa cells were not frequently detected, as judged by the analysis of phalloidin-rhodamine distribution under conventional fluorescence microscopy (Fig. IC). Further observation of attached metacyclic trypomastigotes showed that after 24 hr all parasites had entered the cells leading to normal infections of more than 60% of the cells, as judged by the presence of intracellular amastigote forms (result not shown). The observation of corresponding samples at the scanning electron microscope showed that the dorsal surface of the HeLa cells used in this study (see also Silva et al. 1989) was covered with microvilli (Fig. 2). A substantial proportion (94%, II = 254) of the metacyclic trypomastigotes observed were end-on associated with their posterior (kinetoplast) portion toward the cells and had part of their cell body already inside the HeLa cells, suggesting that they were engaged in the process of invasion (e.g., Fig. 2B). The trypomastigotes were usually (89% of parasites scored, IZ = 224) observed at cellular edges (Fig. 2A) and intercellular contacts (Fig. 2B) not more than 1 km away from the cell borders and did not RESULTS appear to associate with surface microvilli The interaction of different T. cruzi de- (Figs. 2B-2D). Interestingly, in some cases velopmental forms with HeLa cells was ex- more than one metacyclic trypomastigote amined in this study. To improve the asso- was found quite close to others(s) paraciation with HeLa cells and reduce the ma- site(s) penetrating a cell. Under identical nipulation time, parasites were centrifuged experimental conditions, epimastigotes did onto HeLa cells grown on coverslips and not attach to HeLa cells (see Table I). When mixtures of tissue culture-derived after washing unattached parasites, samamastigotes and trypomastigotes were cenples were processed for immunofluorestrifuged onto HeLa cells, actin aggregation cence and scanning electron microscopy. On average, these experiments were per- underneath the sites of amastigote adhesion formed within 18 min from the addition of was observed (Figs. 3A-3D). At the optical the parasites to the cells until sample fixa- microscopy level it was observed that, using the centrifugation protocol, amastigotes tion. Under such conditions, metacyclic trypomastigotes readily attached to HeLa usually displayed a greater tendency to as-
RENATO
A. MORTARA
sociate with HeLa cells than tissue culturederived trypomastigotes (see Table I). Using glutaraldehyde-fixed and unpermeabilized samples, it could be determined that amastigotes were already inside the cell after the centrifugation step since parasites visible by phase contrast were not labeled with the monoclonal antibody (Figs. 3A3D). By contrast, when samples were fixed in glutaraldehyde and subsequently permeabilized with 1% NP-40, all amastigotes associated with the HeLa cells were labeled with monoclonal antibody 2C2 (Figs. 3D3F). Actin aggregation was evident at the sites of amastigote attachment and the amastigotes without the corresponding microfilament aggregate were usually those that did not label with the antibody and were considered to be already within the cell (Figs. 3A-3D). Quantitation of these samples indicated that after 15 min of centrifugation, 41% of the attached amastigotes were already inside the HeLa cells (Table II). Since all changes in actin distribution after amastigote attachment to HeLa cells were monitored at the fluorescence microscope by double-labeling samples with phalloidin-rhodamine and monoclonal antibody 2C2, controls were performed to ensure that 2C2 (raised against the major surface glycoprotein of Y strain amastigotes-Andrews et al. 1987, 1988) gave a positive reaction with 100% of the amastigotes of the G strain and that phalloidinrhodamine, at the concentration used in this study, did not label parasite actin (Fig. 4). Attachment and entry of amastigotes also led to successful infections and cells displaying several perinuclearly arranged FIG. 1. HeLa cell actin distribution following T. cruzi metacyclic trypomastigote centrifugation. (A) Phase-contrast micrograph of cells with associated parasites (arrows); (B) immunofluorescence image of metacyclics stained with monoclonal antibody 3F5 with suggestive images (arrows) of cell invasion; (C) same fields labeled with phalloidin-rhodamine, with typical actin filament bundles. Magnification: x685. Bar = 10 (*m.
FIG. 2. Scanning electron microscopy of HeLa cells after centrifugation of T. cruzi metacyclic trypomastigotes. (A-D) Images of parasites invading HeLa cells at the edges. (C and D) Metacyclic trypomastigotes penetrating a cell close to each other. Magnifications: (A) X 1000; (B and C) x4500; (D) x3000. Bars = 5 km.
amastigotes were already detectable 24 hr postinfection (Table II and Fig. 5). Examination by SEM of samples fixed immediately after the centrifugation step revealed that amastigotes adhered to the dorsal surface of HeLa cells (Fig. 6A). At higher magnifications, the adherence sites consistently displayed microvilli attached to the parasite surface forming a small clump (Figs. 6B-6F). Quantitative studies carried out at high magnifications (X 10,000) indicated that 95% of the attached amastigotes were actually entangled with surface microvilli whereas the number of contaminating trypomastigotes associated with the cells was comparatively small. Up to 10 amastigotes could associate with a single HeLa cell but the majority of infected cells contained between one and three parasites on their dorsal surface (Fig. 7). The role of microtilaments on the attachment and interiorization of T. cruzi into
mammalian cells has been probed with cytochalasin B. Cytochalasin D was used in this study because it has a higher affinity for actin filaments and its effects on mammalian cells is also more specific than those of cytochalasin B (Cooper 1987). Using the glutaraldehyde fixation approach it was possible to distinguish between attached and internalized amastigotes in cytochalasin D-treated HeLa cells. Cytochalasin D treatment as performed here (5 p.g ml - ’ for 15 min) caused dramatic changes in cell morphology (see below) and despite a significant reduction in the proportion of intracellular parasites, it did not block the entry of amastigotes (Table II). Figure 8 shows one of such samples in which attached (labeled) and internalized (unlabeled) amastigotes can be distinguished. The distribution of actin in CD-treated cells was also affected and microfilament aggregates were visible throughout the cytoplasm (Fig. 8).
RENATO A. MORTARA
6
TABLE I Treatment of HeLa Cells with Cytochalasin D: Effect on Ttypanosoma cruzi Association*
T. cruzi form
Metacyclic trypomastigote@ Amastigotesl trypomastigotes (70:30 ratio)
Cytochalasin D (wg ml-‘)
2 5 Epimastigotes
3.18 k 0.36 2.65 -r- 0.32 2.48 2 0.40
0 2 5
0
0
No. of parasites associated per cell
Ad T A T A T
3.01 2 0.24 2 3.48 4 0.33 ? 2.93 t 0.34 2 0
0.86 0.03 0.79 0.13 0.93 0.11
0 Parasites scored on at least 400 cells on duplicate cover slips after Giemsa staining. b Figures represent means 5 standard error of two independent experiments. c Figures represent means k standard error of three independent experiments. d A, amastigotes; T, trypomastigotes.
Another quantitative evaluation of the effect of treating HeLa cells with cytochalasin D on T. cruzi association was obtained with samples stained with Giemsa reagent. Quantitation was expressed in terms of parasites associated per cell, since this staining procedure does not allow the clear distinction between interiorized and attached forms (Table I). Metacyclic trypomastigote association with HeLa cells was hardly affected by CD treatment and also no significant differences were observed in the attachment of amastigotes, both to control and to CD-treated cells (Table I). Similarly, tissue culture-derived trypomastigotes associated with CD-treated HeLa cells in levels comparable to that of the control samples (Table I). Incubation of cytochalasin D-treated samples for 24 hr (in medium without the drug) resulted in infections comparable to that of the controls, both for amastigotes (Table II) and metacyclic trypomastigotes (not shown). DISCUSSION
Phalloidin staining also revealed that at some sites of amastigote attachment, cellular actin appeared to surround the entire parasite, suggesting that a membrane process is preventing the binding of monoclonal 2C2 to the parasites (vertical arrows in Figs. 8A and SC). Cells treated with cytochalasin D displayed profound morphological changes. They tended to round up and their attachments to the substratum appeared to retract; moreover, their surface presented few microvilli and became covered with blebs (Fig. 9A). Quantitation of amastigote binding to CD-treated HeLa cells at the SEM level (Figs. 9C and 9D) was not possible since the abundance of surface blebs masked the attached parasites (see, for instance, Fig. 90). On the other hand, metacyclic trypomastigotes are frequently seen invading CD-treated HeLa cells, preserving their preference for the cell periphery (Figs. 9E and 9F).
Based on previous observations that enteropathogenic E. coli cause actin aggregation after localized adhesion of the bacteria to HeLa cells (Silva et al. 1989) and considering the evidences of a possible involvement of actin filaments in the interiorization process of T. cruzi in mammalian cells (Nogueira and Cohn 1976; Kipnis et al. 1979; Henriquez et al. 1981; Meirelles et al. 1982), the interaction of T. cruzi with HeLa cells was studied in order to examine possible effects of different developmental forms of the parasite on the host cell microfilament system. For such studies a centrifugation procedure previously described in our laboratory (Silva et al. 1989) was employed to maximize parasite-HeLa cell interactions and to reduce manipulation time. Results with metacyclic trypomastigotes confirmed previous observations (Schenkman et al. 1988) that these forms tend to invade cells at the periphery (Fig. 2). By contrast, amastigotes attach preferentially
T.
Cruzi
INFECTIVE
FORMS
7
FIG. 3. Centrifugation of T. cruzi amastigotes onto HeLa cells induces aggregation of actin filaments and leads to parasite entry. (A-C) Phase-contrast microscopy, 2C2 staining, and actin distribution in glutaraldehyde-fixed samples. (D-F) Equivalent samples stained as before after permeabilization with 1% Nonidet-P40-note that all parasites are labeled with the antibody. Black arrows in (A): intemalized amastigotes; black arrowheads in (A) indicate attached parasites, also labeled with 2C2 (see B); white arrows in (C): actin aggregates corresponding to attached amastigotes. Magnification: x710. Bar = 10 pm.
to the dorsal surface of HeLa cells where they induce actin filament aggregation that corresponds to clumps of surface microvilli and parasites (Figs. 4 and 6). Attached and internalized amastigotes could be distinguished using differential antibody accessibility after glutaraldehyde fixation (Fig. 4 and Table II). The finding that 41% of the
amastigotes were localized within the cells after 15 min required for the centrifugation step supports the notion that this procedure promotes infection by amastigote forms. Under the experimental conditions described here, trypomastigotes derived from infected cells displayed a lower degree of association with HeLa than amastigotes. It
RENATO A. MORTARA
8
TABLE II Entry of Trypanosoma cruzi Amastigotes” into HeLa Cellsb Effect of Cytochalasin D Percentage of Cytochalasin DC parasitesd inside No. amastigotes’ inside 100 cells the cells (kg mlm’) 0 5 0 5
41.3 k 11.2 2 97.1 2 95.4 k
6.8 0.9 l.lf 2.P
155 t 422 255 ? 247 2
13 10 35 42
a These parasite preparations contained more than 95% amastigotes. b Parasites were centrifuged onto HeLa cells and after glutaraldehyde fixation, samples were stained with monoclonal antibody 2C2. c Cells were incubated for 15 min with 5 ug ml-’ CD. d Percentage refers to the ratio of parasites not labeled/total parasites on at least 200 cells on duplicate cover slips. Figures represent means rt standard error of two independent experiments. p Intracellular amastigotes scored on 200 cells on duplicate coverslips. Figures represent means 2 standard error of two independent experiments. f Figures obtained following incubation in drug-free medium for 24 hr, after centrifugation.
should be pointed out, however, that these associations do not reflect the relative infectivities under conventional conditions where trypomastigotes clearly display a much greater capacity to infect mammalian cells (e.g., Schenkman et al. 1988). Surface projections like microvilli and filopodia are probing organelles (Vasiliev 1981) thought to be involved in initial steps of parasitexell interactions (McGee et al. 1988). The interaction of amastigotes with microvilli on the dorsal surface of HeLa cells forming small clumps (Fig. 6) confirms that these membrane projections act as docking elements to the spheroid amastigotes. These interactions could involve specific parasite and microvillus membrane components. This apparently specific association with microvilli could imply that these surface protrusions concentrate specific membrane receptor molecules for amastigotes, as seems to occur in other systems (e.g., Koch and Smith, 1978). Inter-
FIG. 4. Control of T. cruzi amastigote staining with monoclonal antibody 2C2 and phalloidin-rhodamine. (A) Phase-contrast micrograph of parasites; (B) 2C2 monoclonal antibody staining, and (C) phalloidinrhodamine staining of actin using the same concentrations of the reagent as in Figs. 1, 3, and 8.
estingly, when HeLa cells are treated with cytochalasin D at concentrations that dramatically altered their surface morphology (Fig. 9) and completely blocked localized adherence of enteropathogenic E. coli (Silva et al. 1989), amastigote (and trypomastigote) adhesion was still observed in levels comparable to those of the controls (Table I). Since cytochalasin D-treated cells express fewer microvilli while retaining their capacity to physically attach to amastigotes, it could be tentatively assumed that the putative receptor molecule(s) remains functionally expressed at the surface and that the microvillar structure is not required for parasite adhesion.
T. cruzi INFECTIVE FORMS
9
ments with CD greatly inhibited amastigote entry after the centrifugation procedure (Table II). At this point it would be interesting to consider the role of host cell actin microtilaments on T. cruzi attachment and invasion. Previous studies based mainly on the inhibitory effect of cytochalasin B were taken as indications that microfilaments participate in the uptake of T. cruzi by cells (Alexander 1975; Nogueira and Cohn 1976; Henriquez et al. 1981; Meirelles et al. 1982). In these studies it was shown that severe disruption of the cortical microtilament system with cytochalasin D did not cause a significant effect on the degree of association of different T. cruzi infective forms with HeLa cells (Table I) and subsequent incubations in medium without the drug resulted in normal infections (Table II). It is noteworthy that metacyclic trypomastigotes retain their preference to invade the cells at the periphery, even in CDtreated cells (see Figs. 2 and 9). In a recent study, Schenkman et al. (1991) observed that CD, at the same concentration used here, did not affect the entry of Y-strain trypomastigotes into HeLa cells. These findings support the notion that (1) attachment is required for invasion and (2) attached parasites will eventually enter (even CD-treated) cells. This is intriguing in view of the accepted notion that a classical reFIG. 5. Amastigote attachment and invasion causes ceptor-mediated endocytosis mechanism is normal infection. Amastigotes were centrifuged onto believed to operate in the uptake of the parHeLa cells, washed, and incubated for 24 hr before asite (Kierszenbaum and Stiles, 1985; fixation and 2C2 staining. (A) Phase-contrast and (B) Schenkman et al. 1988; Zingales and Colli 2C2 labeling of amastigotes. Magnification: X260. Bar 1985). In such a mechanism, motility of = 10 pm. membrane receptors tethered to the cytoskeleton leading to the uptake of surface This is again in contrast to the effect ob- ligands is thought to require a functional served with the adhesion of enteropatho- cortical meshwork of actin microfilaments, genie E. cofi that is almost abolished when since such processes are promptly inhibited HeLa cells are exposed to conditions that by cytochalasins (e.g., Cooper 1985). The reduce the expression of microvilli (Silva ef observations that the infective forms of T. al. 1989). Although cytochalasin D did not cruzi keep their capacity to associate with significantly affect parasite attachment (Ta- and invade (albeit to a lower degree in the case of the amastigotes) cells bearing disble I), the disruption of cellular microfila-
FIG. 6. T. cruzi amastigote centrifuged onto HeLa cells entangle to microvilli. (A-H) Scanning electron micrographs showing amastigotes attached to the dorsal surface of HeLa cells. Images in (B-H) show details of clumps formed with microvilh bound to amastigotes. Magnifications: (A) X 1500; (B) x4500; (C and D) x7000; (E) x4500; (F) x7000; (G and H) X10,000. Bars in A, B, C, and H = 5 pm.
T. cruzi
123456769 AMASTIGOTES
INFECTIVE
PER CELL
FIG. 7. Frequency of T. cruzi amastigote attach-
ment per HeLa cells after parasite centrifugation. Black bars: determined by SEM; hatched bars: determined after Giemsa staining.
rupted microfilaments and altered surface morphology is consistent with the notion that the interaction of parasite ligands with appropriate functional receptor molecules at specific surface sites, as envisaged by King (1988), could be sufficient to trigger a parasite-directed endocytosis (McGee ef al. 1988) efficient enough to overcome the damage caused to the host cell cytoskeleton by cytochalasin D. The highly motile trypomastigotes would be more efficient than amastigotes in such an internalization mechanism involving a positive participation of T. cruzi, which could be envisaged as an active entry process as previously suggested by Kipnis et al. (1979). Finally, the preference of trypomastigotes for the cell periphery and the association of amastigotes with surface microvilli
FIG. 8. Cytochalasin D inhibits but does not block amastigote entry into HeLa cells. Cells were fixed with glutaraldehyde after treatment with 5 ug ml - t CD for 15 min and parasite centrifugation. (A) Phasecontrast micrograph, (B) 2C2 staining, and (C) actin distribution. Black arrows in (A), internalized amastigotes; arrowheads in (A), attached amastigotes (see B); white arrows in (C), actin aggregates at amastigote attachment (horizontal) and internalization (vertical) sites. Vertical arrows at top right in (A) and (C) indicate internalized amastigote tightly covered with cellular actin. Magnification x800. Bar = 10 urn.
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FIG. 9. T. cruzi forms attach to cytochalasin D-treated HeLa cells. Scanning electron microscopy of different T. cruzi infective forms centrifuged onto HeLa cells after incubation for 15 min with 5 p.g ml-’ CD. (A) General aspect of treated cells. (B-D) Cells after amastigote:trypoamstigote mixture (70:30 ratio) centrifugation (arrow in D points to an attached amastigote); (E and F) cells after metacyclic trypomastigote centrifugation; note trypomastigotes penetrating the cells at the edges. Magnifications: (A) x1000; (B) x2000; (C-E) x4500; (F) x4000. Bars in A, B, D, and F = 5 pm.
shows that different T. cruzi developmental forms use or mobilize distinct structures and/or receptors to attach and invade HeLa cells. ACKNOWLEDGMENTS I thank Sebastiio Cruz, Helena C. Barros, and Wanderley de Souza for their help with the scanning microscopy. I am grateful to my colleagues Nobuko
Yoshida and Jose Franc0 da Silveira for their comments and suggestions on the manuscript. I am particularly grateful to Sergio Schenkman for helpful discussions and communication of his results prior to publication. This work was supported by grants from the Fundacao de Amparo a Pesquisa do Estado de Slo Paulo-FAPESP and Conselho National de desenvolvimento Cientitico e Tecnologico-CNPq, and UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases and the Rockefeller Foundation.
T. cruzi
INFECTIVE
REFERENCES
ALBERTS, B., BRAY, D., LEWIS, J., RAFF, M., ROBERTS,K., AND WATSON,J. D. 1983. The cytoskeleton. In “The Molecular Biology of the Cell,” pp. 549-609. Garland, New York/London. ALEXANDER, J. 1975. Effect of the antiphagocytic agent cytochalasin B on macrophage invasion by Leishmaniu mexicana promastigotes and Trypanosoma cruzi epimastigotes. Journal of Protozoology 22, 237-240. ANDREWS, N. W., HONG, K. S., ROBBINS, E. S., AND NUSSENZWEIG,V. 1987. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Experimental Parasitology
64, 474-484.
ANDREWS, N. W., ROBBINS,E. S., LEY, V., HONG, K. S., AND NUSSENZWEIG,V. 1988. Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi. The Journal Medicine 167, 300-3 14.
of Experimental
BRENER,Z. 1973. Biology of Trypanosoma nual Reviews of Microbiology
cruzi. An27, 347-554.
CAMARGO,E. P. 1964. Growth and differentiation in Trypanosoma cruzi: Origin of metacyclic trypomastigotes in liquid media. Revista do Instituto de Medicina Tropical de Sdo Paul0 6, 93-100. COOPER,J. A. 1987. Effects of cytochalasins and phalloidin on actin. Journal of Cell Biology 105, 14731478. DE SOUZA, W. 1984. Cell Biology of Trypanosoma cruzi. international Reviews of Cytology 86, 197283. FAULSTICH, H., TRISCHMAN, H., AND MAYER, D. 1983. Preparation of tetramethyl rhodaminylphalloidin and uptake of the toxin into short-term cultured hepatocytes. Experimental Cell Research 144, 63-72.
HENRIQUEZ, D., PIUS, R., AND PIUS, M. M. 1981. The effect of surface membrane modifications of tibroblastic cells on the entry process of Trypanosoma cruzi trypomastigotes. Molecular and Biochemical Parasitology
2, 35%366.
KIERSZENBAUM,R., AND STILES, B. 1985. Evidence supporting the existence of a host cell surface receptor for Ttypanosoma cruzi. Journal of Protozoology 32, 364-366. KING, C. A. 1988. Cell motility of sporozoan protozoa. Parasitology Today 4( 1I), 3 15-3 19. KIPNIS, T. L., CALICH, V. L. G., AND DIAS DA SILVA, W. 1979. Active entry of bloodstream forms of Trypanosoma cruzi into macrophages. Parasitology 78, 89-98.
FORMS
13
KOCH, G. L. E., AND SMITH, M. J. 1978. An association between actin and the major histocompatibility antigen H-2. Nature (London) 273, 274-278. LEY, V., ANDREWS, N. W., ROBBINS, E. S., AND NUSSENZWEIG,V. 1988. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. Journal of Experimental Medicine 168, 649659. MCGEE, Z., GORBY,G. L., WYRICK, P. B., HODINKA, R., AND HOFFMAN, L. H. 1988. Parasite-directed endocytosis. Reviews on Infectious Diseases 10, S311-S316. MEIRELLES,M. N. L., ARA~JOJORGE,T. C., AND DE SOUZA, W. 1982. Interaction of Trypanosoma cruzi with macrophages in vitro: Dissociation of the attachment and internalization phases by low temperature and cytochalasin B. Zeitschrift fir Parasitenkunde 68, 7-14.
MORTARA, R. A., ARAGUTH, M. F., AND YOSHIDA, N. 1988.Reactivity of stage-specific monoclonal antibody lG7 with metacyclic trypomastigotes of Trypanosoma cruzi strains: Lytic property and antigen polymorphism. Parasite Immunology 10, 369-378. MORTARA,R. A. 1989. Studies on trypanosomatid actin. I. lmmunochemical and biochemical identification. Journal of Protozoology 36, 8-13. NOGUEIRA, N., AND COHN, Z. 1976. Trypanosoma cruzi: Mechanism of entry and intracellular fate in mammalian cells. Journal of Experimental Medicine 143, 1402-1420. NOISIN, E. L., AND VILLALTA, F. 1989. Fibronectin increases Trypanosoma cruzi amastigote binding to and uptake by murine macrophages and human monocytes. Infection and Immunity 57, 1030-1034. OUAISSI,M. A., AFCHAIN, A., CAPRON,A., AND GRIMAUD, J. A. 1984. Fibronectin receptors on Trypanosoma cruzi trypomastigotes and their biological role. Nature (London) 308, 380-382. PAN, S. C.-T. 1978. Trypanosoma cruzi: In virro interactions between cultured amastigotes and human skin-muscle cells. Experimental Parasitology 45, 274-286. SCHENKMAN, S., ANDREWS, N. W., NUSSENZWEIG, V., AND ROBBINS, E. S. 1988. Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner. Cell 55, 157-165. SCHENKMAN,S., ROBINS,E. S., AND NUSSENZWEIG, V. 1991. The attachment and penetration of Trypanosoma cruzi into mammalian cells require parasite energy and can be independent of the target cell cytoskeleton. Infection and Immunity, 59, 645-654. SILVA, M. L. M., MORTARA, R. A., BARROS,H. C., DE SOUZA, W., AND TRABULSI, L. R. 1989. Aggre-
14
RENATO A. MORTARA
gation of membrane associated actin filaments following localized adherence of enteropathogenic Escherichia coli to HeLa cells. Journa/ of Cell Science 93, 4394l6. UMEZAWA, E. S., MILDER, R. V., AND ABRAHAMcruzi amastigotes: SON, 1. A. 1985. Trypanosoma Development in vitro and infectivity in vivo of the forms isolated from spleen and liver. Acta Tropica
cells. In “International Cell Biology” (H. G. Schweiger, Ed.). Springer-Verlag, Berlin. YOSHIDA, N. 1983. Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi. Infection and Immunity
40, 836-839.
B., AND COLLI, W. 1985. Trypanosoma cruzi: Interaction with host cells. Current Topics in Microbiology and Immunology 117, 129-152.
ZINGALES,
42, 25-32.
J. M. 1981. The role of pseudopodial reactions in the attachment of normal and transformed
VASILIEV,
Received 24 July 1990; accepted with revision 22 January 1991
