Trypanosoma cruzi: Amastigotes and ... - ResearchGate

EXPERIMENTAL PARASITOLOGY 73, 1-14 (191) 

Trypanosoma cruzi: Amastigotes and Trypomastigotes Interact with 

Different Structures on the Surface of HeLa Cells 

RENATO A. MORTARA 

Department of Microbiology, Immunology, and Parasitology, Escola Paulista de Medicina, Rua Botucatu, 

862 6” andar, 04023, Scio Pa&o, SP, Brazil 

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 1991 Academic 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). 

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 

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 

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- 

25OOg for 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 trymastigote-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 proriquez 

et al. 1981; Meirelles et al. 1982). cedure. Parasite/cell ratios usually ranged between 5: 1 

In a series of studies on the interaction and 1511. 

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 

the dishes were immediately centrifuged in a swing-out 

al. 1989). To study the interactions between 

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 amastigotebearing 

cells after 2448 hr and by the presence of 

duces the time of manipulations. Followed 

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. 

1988). By contrast, amastigotes, which can 

induce the formation of actin aggregates, 

detected at the optical microscopy level, 

bind to microvilli forming small aggregates 

at the cell surface before entering the HeLa 

cells. 

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 1640 medium 

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 

three times with PBS and after a 15-min incubation in 

50 mM NH&l/PBS to quench excess aldehyde groups, 

the coverslips were treated with PBS containing 0.25% 

gelatin, 0.05% NaN, (PGN), and 0.1% saponin to improve 

permeabilization and prevent nonspecific antibody 

binding during the subsequent incubations. In 

the series of experiments carried out to determine parasite 

interiorization, coverslips were fixed in 2% glutaraldehyde 

(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 metaamastigotes 

were isolated from culture supernatants of cyclic trypomastigotes and epimastigotes-R. A. Mor-

T. cruzi INFECTIVE FORMS 3 

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 (OS- 

5 (~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. 

RESULTS 

The interaction of different T. cruzi developmental 

forms with HeLa cells was examined 

in this study. To improve the association 

with HeLa cells and reduce the manipulation 

time, parasites were centrifuged 

onto HeLa cells grown on coverslips and 

after washing unattached parasites, samples 

were processed for immunofluorescence 

and scanning electron microscopy. 

On average, these experiments were performed 

within 18 min from the addition of 

the parasites to the cells until sample fixation. 

Under such conditions, metacyclic 

trypomastigotes readily attached to HeLa 

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 

appear to associate with surface microvilli 

(Figs. 2B-2D). Interestingly, in some cases 

more than one metacyclic trypomastigote 

was found quite close to others(s) parasite(s) 

penetrating a cell. Under identical 

experimental conditions, epimastigotes did 

not attach to HeLa cells (see Table I). 

When mixtures of tissue culture-derived 

amastigotes and trypomastigotes were centrifuged 

onto HeLa cells, actin aggregation 

underneath the sites of amastigote adhesion 

was observed (Figs. 3A-3D). At the optical 

microscopy level it was observed that, using 

the centrifugation protocol, amastigotes 

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. 3A- 

3D). 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. 3D- 

3F). 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).

6 RENATO A. MORTARA 

TABLE I 

Treatment of HeLa Cells with Cytochalasin D: 

Effect on Ttypanosoma cruzi Association* 

No. of 

parasites 

Cytochalasin D associated 

T. cruzi form (wg ml-‘) per cell 

Metacyclic 

trypomastigote@ 0 3.18 k 0.36 

2 2.65 -r- 0.32 

5 2.48 2 0.40 

Amastigotesl 

trypomastigotes 

(70:30 ratio) 0 Ad 3.01 2 0.86 

T 0.24 2 0.03 

2 A 3.48 4 0.79 

T 0.33 ? 0.13 

5 A 2.93 t 0.93 

T 0.34 2 0.11 

Epimastigotes 0 0 

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. 

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). 

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 

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


TABLE II 

Entry of Trypanosoma cruzi Amastigotes” into HeLa 

Cellsb Effect of Cytochalasin D 

Percentage of 

Cytochalasin DC parasitesd inside No. amastigotes’ 

(kg mlm’) the cells inside 100 cells 

0 41.3 k 6.8 155 t 13 

5 11.2 2 0.9 422 10 

0 97.1 2 l.lf 255 ? 35 

5 95.4 k 2.P 247 2 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.


FIG. 5. Amastigote attachment and invasion causes 

normal infection. Amastigotes were centrifuged onto 

HeLa cells, washed, and incubated for 24 hr before 

fixation and 2C2 staining. (A) Phase-contrast and (B) 

2C2 labeling of amastigotes. Magnification: X260. Bar 

= 10 pm. 

This is again in contrast to the effect observed 

with the adhesion of enteropathogenie 

E. cofi that is almost abolished when 

HeLa cells are exposed to conditions that 

reduce the expression of microvilli (Silva ef 

al. 1989). Although cytochalasin D did not 

significantly affect parasite attachment (Table 

I), the disruption of cellular microfila- 

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 receptor-mediated 

endocytosis mechanism is 

believed to operate in the uptake of the parasite 

(Kierszenbaum and Stiles, 1985; 

Schenkman et al. 1988; Zingales and Colli 

1985). In such a mechanism, motility of 

membrane receptors tethered to the cytoskeleton 

leading to the uptake of surface 

ligands is thought to require a functional 

cortical meshwork of actin microfilaments, 

since such processes are promptly inhibited 

by cytochalasins (e.g., Cooper 1985). The 

observations that the infective forms of T. 

cruzi keep their capacity to associate with 

and invade (albeit to a lower degree in the 

case of the amastigotes) cells bearing dis-

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.


123456769 

AMASTIGOTES PER CELL 

FIG. 7. Frequency of T. cruzi amastigote attachment 

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.

12 RENATO A.MORTARA 

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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Received 24 July 1990; accepted with revision 22 January 

1991

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