J. Cell Sci. 33, 285-299 (1978) 285Printed in Great Britain © Company of Biologists Limited 197S

THE CELL SURFACE OF TRYPANOSOMA CRUZI:

CYTOCHEMISTRY AND FREEZE-FRACTURE

W. DE SOUZA,* A. MARTfNEZ-PALOMOfAND A. GONZALEZ-ROBLESf*Instituto de Biofisica, Universidade Federal do Rio de Janeiro, 20.000, Rio de Janeiro,Brazil, and fDepartamento de Biologia Celular, Centra de Investigacidn del IPN,Apartado Postal 14-740, Mexico, D.F.

SUMMARY

The ultrastructure of epimastigotes of Trypanosoma cruzi, obtained from, accllular cultures,and bloodstream, trypomastigotes, isolated from infected mice, were studied by thin-sectioningand freeze-fracturing techniques. Epimastigotes showed a thin (5 nm) surface coat whenstained with, ruthenium red, while the surface coat of trypomastigotes was more prominent(15 nm thick). Both P and E faces of the plasma membrane of T. cruzi had roughly the samenumber of intramembranous particles (IMP) as seen by freeze-fracture. The plasma membraneof bloodstream trypomastigotes had less IMP than epimastigotes. Several differentiations of theplasma membrane were observed. In epimastigotes a cytostome appears as a particle-poorregion delimited by a pallisade-like row of adjacent IMP. Bloodstream trypomastigotes did nothave a cytostome. Instead, abundant pinocytic vesicles were observed. At the base of theflagellum of epimastigotes a ciliary necklace was found. At this region, the surface coat wasdifferentiated as long, hair-like projections after staining with ruthenium red. The flagellarmembrane had less IMP than the body membrane. Clusters of IMP were present on both facesof the flagellar membrane at the flagellar-body adhesion zone of epimastigotes. Linear arraysof IMP were also seen. In bloodstream trypomastigotes clusters of particles were observed bothon the flagellar and cell body membranes. Our observations demonstrate the presence of con-siderable structural variations of the T. cruzi plasma membrane at the two stages of the lifecycle studied.

INTRODUCTION

One approach to an understanding of the host-parasite interactions present inChagas' disease is the study of the outer membrane of the causative agent, the proto-zoon Trypanosoma cruzi. A comparison between the surface of the invasive trypom-astigote form with that of the non-invasive stage, the epimastigote, could give someinformation on the factors that determine the invasive behaviour of the parasitetowards host cells.

Previous studies on the cell surface of T. cruzi carried out by us have centred on thedetection of specific carbohydrate components by means of lectin-binding studies(Chiari, De Souza, Romanha, Chiari & Brener, 1978), the measurement of total surfacecharge estimated both as binding of cationized ferritin and electrophoretic mobility(De Souza et al. 1977) and the study of the plasma membrane of epimastigote formsby ultrastructural cytochemistry, and electron microscopy of freeze-fracture replicas(Martinez-Palomo, De Souza & Gonzalez-Robles, 1976).

* For all correspondence.19 C E I. 33

286 W. De Souza, A. Martinez-Palomo and A, Gonzdlez-Robles

The introduction of the freeze-fracture technique has increased our understandingof the structure and organization of animal membranes in general, and of varioushuman parasites, in particular, such as Trypanosoma brucei (Smith, Njogu, Cayer &Jarlfors, 1974; Hogan & Patton, 1976), Entamoeba histolytica (Pinto da Silva, Martinez-Palomo & Gonzalez-Robles, 1975; Martinez-Palomo, Pinto da Silva & Chavez, 1976)and Onchocerca volvulus (Martinez-Palomo, 1978).

We have now applied the freeze-fracture technique to study the fine structure ofthe plasma membrane of both invasive blood forms and non-invasive culture forms ofT. cruzi. The parasite plasma membrane was found to present considerable variationsat these 2 stages of the life cycle.

MATERIALS AND METHODS

Microorganisms

Trypanosoma cruzi, strain Y, was cultivated in. LIT (liver infusion-tryptosc) medium(Camargo, 1964) for 4 days at 28 °C. Under these conditions only epimastigote forms wereobtained. Bloodstream trypomastigote forms were obtained from mice infected with the Ystrain. The blood was collected in the presence of sodium citrate and centrifuged at 50 g for10 min at 4 °C. The pellet was discarded. The supernatant fluid, which contained the trypano-somes, was collected and centrifuged at 800 g for 15 min at 4 °C. The pellet obtained waswashed twice in cold 001 M phosphate-buffered 0-15 M saline (PBS), pH 72.

Electron microscopy

Cells were fixed in glutaraldehyde 2 5 % (v/v) in o-i M cacodylate buffer, pH 7 2 , for 1 h atroom temperature. After a rinse in buffer the cells were postfixed in 1 % (v/v) OsO.t in. O'i Mcacodylate buffer, pH 7 2 , for 1 h at room temperature. Then they were dehydrated through agraded ethanol series and embedded in Epon.

Ruthenium red

After fixation in glutaraldehyde the cells were washed twice in cacodylate buffer and postfixedin 1 % OsOi in o-i M cacodylate buffer + o-s mg/ml ruthenium red for 1 h in the dark at roomtemperature (Luft, 1971).

Freeze-fracture

Cells fixed in glutaraldehyde were washed twice in o-i M cacodylate buffer, pH 72, andgradually impregnated during 30 min with glycerol in cacodylate buffer up to 20 % concentra-tion, where they were left for about 60 min at room temperature. They were then mounted onBalzer's support disks and rapidly frozen in the liquid phase of partially solidified Freon 22,

Fig. 1. Epimastigote form of T. cruzi treated with ruthenium red. The surface coatof the flagellum (/) shows a local modification in the form of a hairy coat (arrows), k,kinetoplast. x 75 000.Fig. 2. Cell surface of T. cruzi showing the uniform disposition of subpellicularmicrotubules. x 80 000.Fig. 3. The cytostome (c) found in epimastigotes appears as a funnel-shapeddepression covered by a ruthenium red-positive coat, x 75 000.Fig. 4. Region of adhesion between the cell body (cb) and the flagellum (/) ofepimastigotes. At the cytoplasmic side of the body surface, macular densities are seen(arrowheads), x 85 000.

Cell surface of T. cruzi 2S7

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288 W. De Souza, A. Martinez-Palomo and A. Gonzalez-Robles

Cell surface of T. cruzi 289

cooled by liquid nitrogen, and stored in liquid nitrogen until used. Freeze-fracture was carriedout at — 115 °C in a Balzer's 300 apparatus equipped with a turbomolecular pump. Replicaswere produced by evaporation from a platinum-carbon source. The specimens were shadowedat 267 x io~' N m~2 within 2 s of fracturing and the knife edge positioned under conditionsthat minimize contamination. Replicas were recovered in distilled water, cleaned with Cloroxand distilled water and mounted on 200-mesh grids coated with Formvar.

Thin sections and replicas were studied with an EM9S-2 or an EM 10 Zeiss electron micro-scope. Micrographs are mounted with the shadow direction from bottom to top except whenindicated otherwise. Shadows are white.

RESULTS

Epimastigote forms

As a reference for interpretation of the freeze-fracture replicas, the general aspect ofthe epimastigote form of Trypanosoma cruzi in ultrathin sections, is shown in Figs. 1-3.The periplast is composed of a plasma membrane 8-10 nm thick, covering the cellbody, the flagellum, and lining the flagellar pocket region. About 8 nm below theplasma membrane there is a single layer of microtubules in parallel array, arrangedalong the major axis of the cell (Fig. 2). With standard techniques, no surface coat canbe detected on the plasma membrane (Fig. 2). However, ruthenium red stainingreveals a 5-nm-thick surface coat (Fig. 1).

In the region of the cytostome, a local differentiation of the cell surface and theplasma membrane has been described by us previously (Martinez-Palomo et al. 1976).The cytostome appears as a funnel-shaped depression lined by the cell membrane,close to the region where the Golgi complex is located (Fig. 3). At the junctionsbetween body and flagellum faint macular densities can be seen only on the cyto-plasmic side of the body surface membrane (Fig. 4).

Trypanosomes studied by freeze-fracture replication may cleave through thecytoplasm (Figs. 5, 6), or through the hydrophobic region of the plasma membrane,as in other cells (Pinto da Silva & Branton, 1970), exposing large surfaces of the inneror outer membranes halves (Fig. 7). An overall picture of the cytoplasm of T. cruzias seen in replicas obtained by freeze-fracture is shown in Fig. 5. The nucleus, thekinetoplast, the Golgi complex and the emergence of the flagellum are evident. Infavourable fractures, the nuclear membrane can be seen with its characteristic pores(Fig. 6). At low magnifications, the plasma membrane of epimastigotes shows auniform distribution of intraniembranous particles (IMP), except at the cytostome andthe flagellar regions, where IMP are either sparse or absent (Fig. 7).

Contrary to what has been found in most cells studied with the freeze-fracturetechnique, the difference between the number of IMP on the P face (which represents

Figs. 5-7. General aspect of the epimastigote form of T. cruzi as seen by freeze-fracture. In Figs. 5 and 6 the cells were cleaved through the cytoplasm showing thenucleus (n) with its pores, the kinetoplast (k), Golgi complex (g) and the emergence ofthe flagellum (/). In Fig. 7 a large area of the cell body and the flagellar membrane isshown. Intramembranous particles are sparse or absent in the flagellar membrane (/)and cytostome (c). Figs. 5 and 7, x 25 000; Fig. 6, x 16 000.

290 W. De Souza, A. Martmez-Palomo and A. Gonzdlez-Robles

the outer aspect of the inner membrane half) and IMP on the E face (which corre-sponds to the inner aspect of the outer membrane half) is not large. Estimations of thenumber of IMP per square micrometre of membrane area reveal a particle density ofi83o//tm2 on the P face and i45o//im2 on the E face. IMP on both faces of T. cruziplasma membrane are very heterogeneous, both in size and shape, as shown in Fig. 8.

The flagellar membrane differs in structure when compared with the membrane ofthe cell body. In general, IMP are absent except in a few regions of the flagellarmembrane (Fig. 9) where IMP are arranged in clusters spaced at irregular intervals.Such clusters of IMP are observed both on P and E faces of the flagellar membrane(Figs. 9, 10), but are lacking on P or E faces of the cell body plasma membrane.

A second specialization of the flagellar membrane, associated with the region ofattachment to the cell body, appears as a linear array of closely adjacent IMP locatedin the portion of the flagellum where it emerges from the base (Fig. 11). The lineararrays are found both in E and P faces of the flagellum, but are absent from the plasmamembrane of the cell body.

A third specialization of the flagellum is formed by circular concentrations of IMPat the base of the flagellum, forming 5-6 circular rows (Fig. 12). At this site the surfacecoat of the flagellum shows in thin sections a local modification in the form of a fuzzycollar (Fig. 1).

Bloodstream trypomastigote forms

The ultrastructure of the bloodstream trypomastigote forms of T. cruzi as seen insections has been described previously (Maria, Tafuri & Brener, 1972). The finestructure is basically similar to that found in epimastigotes. However, various differ-ences can be demonstrated: (a) the nuclei are more elongated; and (b) the kinetoplastappears as a spherical body, localized at one tip (called the posterior tip), and showinga different pattern in the array of the DNA-containing fibrils. The cytostome foundin epimastigotes (Martinez-Palomo et al. 1976) was not detected in bloodstream formsof T. cruzi.

In sections of trypomastigotes stained with ruthenium red, a prominent rutheniumred-positive surface coat is found (Fig. 13). At the zone of adhesion between body andflagellum, adjacent membranes run parallel and the converging surface coats seem tooverlap, giving rise to a median dense line (Fig. 13). Observations of freeze-fracturereplicas of plasma membranes reveal the presence of intramembranous particles inboth E and P faces (Fig. 14). As found in epimastigotes, there is little difference in thenumber of IMP on the P face and E face. However, both faces show many fewerparticles than the cell membrane of epimastigote forms. The average number of IMPper/tm2 of cell membrane is 122 on P faces and 126 on E faces. Therefore, the plasmamembrane of bloodstream forms roughly contains 10 times fewer IMP than thesurface membrane of culture forms. The absence of a cytostome in bloodstream formsof T. cruzi was confirmed in freeze-fracture replicas. Instead, abundant pinocytoticvesicles are found on the plasma membrane (Fig. 14).

As in epimastigotes, the flagellar membrane of trypomastigotes is almost devoid ofIMP both on P and E faces, except at the region of contact with the cell body, where

Cell surface of T. cnisi

Fig. 8. P (P) and E (E) faces of the cell membrane of two epimastigotes. The numberof intramembranous particles in the two faces is roughly similar, x 90 000.Fig. 9. Particles arranged in clusters (arrowheads) are seen in the flagellar membraneof epimastigotes. x 150000.

2Q2 W. De Sousa, A. Martinez-Palomo and. A. Gonzdlez-Robles

Fig. 10. Micrography showing the difference in the number of particles between thecell body (cb) and the flagellar (/) membrane, x 85 000.Fig. 11. Specialization of the flagellar membrane of epimastigote in the region ofattachment of the flagellum to the body. Particles in linear array are observed,x 95 000.Fig, 12. Ciliary necklace localized at the base of the flagellum (arrows), x 116 000.

Cell surface of T. cruzi 293

rows of IMP clusters are found. The flagellum is located over a specialized region ofthe cell body membrane which appears as a regular pit (Fig. 15). Linear arrays of IMPclusters are not limited to E and P faces of the flagellar membrane; they are alsopresent on both fracture faces of the cell body membrane at the region of attachmentof the flagellum (Figs. 15, 16). Furthermore, in replicas where the fracture jumps fromthe P face of the flagellar membrane to the E face of the cell body membrane, and viceversa, the rows of IMP on adjacent membranes are found to be in register (Fig. 15).

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Fig. 13. Thin section of bloodstream trypomastigote stained with ruthenium red. Aprominent surface coat is evident. The stain penetrates at the zone of adhesion (arrow)between the body (cb) and the flagellum. ( /) . x 51 000.

DISCUSSION

The fine structure of trypanosomatids has been adequately studied by electronmicroscopy using ultrathin sections. Although this technique gives important infor-mation concerning the organization and structure of cellular components, it does notpermit detailed examination of the structure and organization of cell membranes. Incontrast, in replicas of freeze-fractured specimens, the inner components of the cellmembrane are exposed permitting examination of the outer aspect of the inner

W. De Sonza, A. Martinez-Palomo and A. Gonzdlez-Robles

14Fig. 14. E (E) and P (P) faces of the cell membrane of bloodstream trypomastigotes,

showing pinocytotic vesicles (arrowheads), x 50 000.

Cell surface of T. cruzi 295

Figs. 15, 16. Region of attachment of the flagellum to the body of a bloodstream try-pomastigote. Linear clusters of particles (arrowheads) are seen both in the cell body(cb) and flagellar membrane. Fig. 15, x 64 000; Fig. 16, x 41 000.

296 W. De Souza, A. Martinez-Palomo and A. Gonzdlez-Rohles

membrane half (P face) and the inner aspect of the outer membrane half (E face)(Pinto da Silva & Branton, 1970; Branton et al. 1975). In most cells studied, fractureface P exhibits distinctly larger density of IMP than that observed on fracture face E.Usually IMP are randomly distributed over the cell membrane. IMP are assumed torepresent integral proteins located in the hydrophobic domain of the membrane lipidbilayer. A special pattern in the arrangement of the particles is usually interpreted asa functionally specialized region of the cell membrane.

Our results show that the plasma membrane of T. cruziis structurally more complexwhen compared with metazoan cell membranes such as those of fibroblasts, erythro-cytes, etc. In contrast to other membranes, the number of IMP in both inner faces ofthe T. cruzi plasma membrane is roughly similar. Furthermore, differentiationsof IMP distribution are characteristic of the epimastigote and trypomastigote stages ofT. cruzi cell cycle.

One of the specialized membrane regions of T. cruzi is the cytostome, found only inepimastigote forms. This structure plays an important role in the nutrition of epimasti-gote and intracellular spheromastigote forms. In the latter stage it participates in aprocess of intracellular phagotrophy (Meyer & De Souza, 1973). In freezc-fracture,the cytostome appears as a particle-poor region delimited by a pallisade-like row ofadjacent IMP localized close to the flagellar pocket (Martinez-Palomo et al. 1976). Inbloodstream trypomastigote forms, the cytostome is not present. Instead, a largenumber of pinocytic vesicles were observed. This fact can be understood in terms ofthe life cycle of T. cruzi. Intracellular spheromastigote forms always possess a cyto-stome. Trypomastigote forms, however, live in the blood of the vertebrate host andtake nutrients from the blood by diffusion or pinocytosis. The presence of the cyto-stome in the epimastigote forms can be explained by the fact that they represent anintermediate stage between sphero- and trypomastigotes. A cytostome has also beenfound in epimastigotes of T. mega, T. conorhini, and T. raiae (Steinert & Novikoff,i960; Brooker & Preston, 1967; Milder & Deane, 1969), in choanomomastigotes ofCritliidia fasciculata (Brooker, 1971) and in promastigotes of Herpetomonas samuel-pessoai (De Souza et al. 1976). In salivarian trypanosomatids, cytostomes were notfound in bloodstream forms of T. vivax, T. equinum and congolense, or in any of thedevelopmental stages of T. brucei (Brown, Armstrong & Valentine, 1963; Vickerman,1969a; Langreth & Balber, 1975).

Another specialized region of the T. cruzi plasma membrane is located on theflagellum. At the base of the flagellum of epimastigotes, we have found a collar-likearray of IMP similar to the ciliary necklace found in other flagella and cilia (Gilula &Satir, 1972; Linder & Staehelin, 1977). It has been suggested that the necklace may beinvolved in the control of local membrane permeability and therefore this region couldrepresent an energy-transducing zone of the flagellum. The specialized region ofT. en/.s7 trypanosomes is remarkable because the membrane rows of IMP appear to berelated to a localized differentiation of the surface coat at the base of the flagellum inthe form of hair-like surface projections.

The paucity of IMP on the flagellar membranes of both epi- and trypomastigoteforms has also been observed in Critliidia fasciculata (Easterbrook, 1971) and T. brucei

Cell surface of T. cruzi 297

(Smith et al. 1974; Hogan & Patton, 1976). These results suggest that, although theflagellar membrane is in continuity with the membrane of the cell body, it may havea different composition, particularly in terms of protein components.

It is well known that the flagellum of the epi- and trypomastigote forms runsattached to the cell body from its point of origin, ending in a free flagellum. There arecontacts between the flagellum and the cell membrane forming areas of specializedjunctions. This region has been extensively studied in ultrathin sections and byfreeze-fracture in T. brucei (Vickerman, 19696; Smith et al. 1974; Hogan & Patton,1976). In T. cruzi the small desmosome-like junctions as seen in sections are not asclear as those reported for T. brucei. In this last trypanosomatid the presence ofclusters of membrane particles spaced at intervals of 75-90 nm, formed by groups ofup to 6 particles was described as representing 'the only obvious specialization of thefractured surfaces of the cell body or flagellum that can be related to the junctionalcomplexes' (Smith et al. 1974). The junctions show specialized structures on the Pface of the flagellar membrane and not on the general cell membrane at the junctionalarea. However, in bloodstream trypomastigote forms of T. cruzi we observed regionsof the cell membrane which show a special pattern in the organization of membraneparticles on both E and P faces, as well as on both faces of the flagellar membrane. Thepresence of IMP on both faces of flagellar and body membrane tends to confirm thesuggestion that the rows of particles represent fractured protein components involvedin the adhesion of the flagellum to the cell body. This assertion is supported by thefact that the IMP rows are found in register where the fracture plane deviates from theflagellum to the body plasma membrane.

The cell membrane of bloodstream trypomastigote forms of T. cruzi present fewermembrane particles than epimastigote forms. Studies of the chemical nature ofmembrane particles in other membrane systems has demonstrated that the particlesrepresent, in general, membrane-intercalated proteins of the integral type. It has beensuggested that the frequency of the particles may be related to the physiologicalactivity of the cell membrane. Other studies have shown the existence of somedifferences in the cell surface of epi- and trypomastigote forms of T. cruzi. Examinationof net surface charge of epimastigotes from acellular cultures and bloodstream try-pomastigotcs show that trypomastigote forms exhibit a much higher electrophoreticmobility and a more intense binding of cationized ferritin than epimastigote forms.Studies of mixed populations in acellular cultures demonstrate that there is a definiteincrease in negative surface charge during development from epi- to trypomastigoteforms of T. cruzi (De Souza et al. 1977). Another difference is related to the suscepti-bility of T. cruzi to lysis induced by mammalian serum (Nogueira, Bianco & Cohn,1975): while epimastigote forms are lysed, the trypomastigote forms remain unaltered.

An interesting feature of the bloodstream forms of T. cruzi is the presence of asurface coat covering the entire plasma membrane. In T. brucei (Vickerman, 19696)the surface coat contains the variant antigens demonstrated by agglutinating reaction.Another characteristic of the surface coat of salivarian trypanosomes is its mobility inthe form of streamers that are shed into the blood, probably accounting for theexoantigen of immune serum. The surface coat may contain also absorbed host serum

298 W. De Sonza, A. Martinez-Palomo and A. Gonzdlez-Robles

proteins. The surface coat of salivarian components is of importance for the under-standing of host-parasite relationships, because antigenic variation of coat antigens,shedding of antigenic components, and adsorption of host serum proteins may allrepresent effective means by which the protozoon may avoid the immune response ofthe host. In turn, the nature of the coat found in T. cruzi bloodstream forms is notknown, except for the fact that carbohydrates are present (De Souza & Meyer, 1975).The coat is barely visible in culture forms of T. cruzi, where only specific cytochemicaltechniques selective for carbohydrates reveal a thin (5 nm) surface coat. The surfacecoat of bloodstream forms of T. cruzi was found to be more prominent than in cultureforms, although less so than in T. brucei (Vickerman, 19696). In view of the consider-able significance of the surface coat of salivarian trypanosomes, the elucidation of thenature and function of the surface coat of T. cruzi might be highly relevant to anunderstanding of the complex host-parasite interaction present in Chagas' disease.

This research was supported in part by grants from. Bayer Laboratories, Mexico City, givento W.S. and A.M.P. through the National Academy of Medicine, Mexico, from the NationalResearch Council of Brazil, to W.S., and from CONACYT, Mexico to A.M.P.

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{Received 30 January 1978)