I . Protozoal., 26(2), 1979, pp.

186-195
0 1979 by the Society of Protozoologists
Microscopic Observations on the Filopodia of Entamoeba histoZytica*
WILLIAM B. LUSHBAUGH and FRED E. PITTMAN
Veterans Administration Hospital and Department of Medicine,
Medical University of South Carolina, Charleston, South Carolina 29403
SYNOPSIS. Intact critical point
dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by
scanning electron microscopy (SEM) . Half and quarter pm thick sections of epoxy-embedded trophozoites were examined by
HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 pm in length. The cytoplasm of
filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called
surface-active lysosomes with trigger, dendritic plasmalemmal extensions, and extra-amebic vesicles by previous investi-
gators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal
occurrence in E. histolytica and may function in: ( a) endocytosis or pinocytosis; ( b) exocytosis; (c) attachment to substratum;
(d) penetration of tissue; ( e ) release of cytotoxic substances; or ( f ) contact cytolysis of host cells.
Index Key Words: Entamoeba histolytica; filopodia; high voltage electron microscopy; scanning electron microscopy.
Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy.
HE surface of Entamoeba histolytica has been implicated in
T the contact cytolysis of leukocytes ( 9) and mammalian tissue
culture cells (5, 11). Eaton et al. ( 5) proposed that vermiforni
extensions or triggers seen in thin sections on the surface
plasmalemma of trophozoites could be responsible for the ob-
served cytolysis when associated with hydrolytic enzyme-con-
taining vacuoles they termed surface-active lysosomes. The
existence of structures resembling triggers on the surface of E.
histolytica was confirmed in subsequent ultrastructural studies
(13, 14, 19, 20) . Vesicular structures associated with amebae in
human colon biopsy material were identified as extra-amebic
vesicles (18). Deas & Miller ( 3) recently demonstrated in serial
thin sections of infected human tissues that the triggers ( 5)
and the extra-amebic vesicles (18) of E. histolytica were short
portions of longer structures they called dendritic plasmalemmal
extensions. The plasmalemmal extensions were characterized as
relatively rare, partly tubular, multibranched extensions of the
plasmalemma several pm in length. Early scanning electron
microscope (SEM) studies (5, 6, 20) of the E. histolytica surface
failed to corroborate the presence of surface extensions on this
ameba. More recently, McCaul & Bird ( 16), using improved
preparative technics, were able to visualize with SEM a limited
number of short (2-3 pm) microfilopodia on E. histolytica
cultivated with crithidia.
In the present investigation we studied the 3-dimensional sur-
face structure of E. histolytica from axenic cultures.
MATERIALS AND METHODS
Cultures.-Entamoeba histozytica strains HM-1:IMSS (for-
merly ABRM) and HK-9 (7, 17), were cultivated axenically in
15-ml test tubes of TP-S-1 medium ( 4) . All trophozoites ex-
amined were obtained from cultures in the logarithmic growth
phase.
Light Microscopy.-Cultures were removed from a 37 C in-
cubator and maintained at 25 C in a vertical position until the
trophozoites collected at the bottom of the tube. Trophozoites
were transferred to slides in a small volume of medium, covered
* This investigation was supported by the Medical Research Ser-
vice of the Veterans Administration; Research Grant #AI-12649
from the National Institute of Allergy and Infectious Diseases,
U.S. Public Health Service; the John A. Hartford Foundation, Inc.,
New York, New York; and a grant from the Division of Research
Resources, National Institutes of Health, U.S. Public Health
Service, for the use of the high voltage electron microscope at the
University of Colorado, Boulder.
with a coverglass, and observed in a phase-contrast microscope.
Photomicrographs were recorded on Kodak Tri-X Pan@ film.
Preparation of Intact Cells for Examination by HVEM and
SEM.-Trophozoites were removed from test-tube cultures and
aseptically transferred to petri dishes containing Formvar-coated
gold finder grids immersed in TP-S-1 medium. Petri-dish cul-
tures were incubated 2 h at 37 C. At this time, trophozoites ob-
served by phase-contrast microscopy were actively motile and
present on open areas of the grids. Petri-dish cultures were fixed
by the drop-wise addition of sufficient volumes of glutaraldehyde
to yield a final concentration of 2% (v/v) glutaraldehyde in
TP-S-1. Fixation was begun at 37 C. After 10 min, cultures were
removed from the incubator and fixation was continued at 25 C
for 50 min. Trophozoites affixed to the Formvar-coated grids
by glutaraldehyde fixation were rinsed in 0.1 M cacodylate-HC1
buffer (pH 7. 2), then exposed to 1% (w/v) OsO, in the same
buffer for 10 min. After a rinse in distilled water, the trophozoites
were stained with 2% (w/v) aqueous uranyl acetate for 10
min and dehydrated through an ascending series of ethanols.
Grids were critical point dried from liquid COz (Polaron Instru-
ments, Watford, England). The dried whole trophozoites were
first observed with a high voltage JEOL JSM-1000 transmission
electron microscope operated at an accelerating voltage of 1000
kV. The same grids were then mounted on SEM stubs with silver
paint and, in some instances, sputter-coated with gold-palladium
to reduce charging artefacts. This material was examined in a
Coates & Welter high resolution field emission scanning electron
microscope operated at an accelerating voltage of 16 kV.
Preparation of Sections for Examination by Transmission
Electron Microscopy.-Trophozoites were fixed 1 h at 25 C by
addition of sufficient volumes of glutaraldehyde to culture tubes to
result in 2% glutaraldehyde in TP-S-1. Cells were further pro-
cessed and embedded as a pellet in Spurr resin as previously de-
scribed (10). Thin and thick (0.25 and 0.5 pm) sections were
cut with glass knives on a Sorvall MT-2-B ultramicrotome. Thin
sections were routinely double stained. Thick sections were
stained for 3 h at 60 C in an aqueous solution of 4% (w/v) uranyl
magnesium acetate. After cooling to room temperature, the grids
were washed with distilled water and stained for 30 min in lead
citrate at 25 C. Thin sections were examined in a Hitachi
HU-12A transmission electron microscope operated at an acceler-
ating voltage of 75 kV, or in a Zeiss 9-S-2 operated at 60 kV.
Thick sections were observed at different degrees of specimen
tilt in a JEOL JSM-I000 high voltage transmission electron
microscope operated at 1000 kV.
186
FILOPODIA OF E. histolytica
All figures are of Entamoeba histolytica.
Figs. 1-5. [Phase-contrast photomicrographs.] 1. Trailing end of a motile ameba drawn out into a slender filament (arrows) by its
attachment to the substrate. x 800. 2. Actively motile trophozoite with numerous short filopodia (arrows) at the posterior end.
x 1,700. 3, 4. Nonmotile rounded trophozoites (center of figure) with tufts of fibpodia (arrows) arising from restricted areas of their
surfaces. Fig. 3, x 1,900; Fig. 4, x 1,100. 5. Numerous filopodia (arrows) randomly distributed on the surfaces of nonmotile organisms.
x 700.
OBSERVATIONS other cells (Fig. 1 ) . More commonly, numerous short (several
pm) filopodia were seen in a restricted area at the posterior
surface of actively motile forms (Fig. 2) . Rounded trophozoites
often had a tuft of multiple filopodia arising from a small surface
area (Figs. 3, 4). More commonly, filopodia could be seen ran-
domly distributed on any part of the cell surface (Fig. 5). The
length of filopodia and the number/cell were variable. Many of
Li ght Microscopy.-Filopodial extensions were frequently Seen
on living trophozoites of E. histolytica examined by phase-contrast
microscopy (Figs. 1-5). In some progressively motile forms, the
trailing ends of the trophozoites were pulled out to extreme length
(>l o0 pm) by attachment of filopodia to the substrate or to
188 FILOPODIX OF E. histolytica
Figs. 6, 7. [High voltage transmission electron micrographs of whole trophozoites fixed in situ and critical point dried.] 6. Part of an
ameba (AM) with several filopodia ( F) . The lengths of 2 of the filopodia, from the cell surfaces to the tips, are indicated in pm.
X 4,500. 7. Parts of 2 trophozoites, the surfaces of which are seen as black images with irregular elevations, i.e. blebs and vesicles ( V) .
Fusiform swellings (S) and branching ( B) are evident in some of the filopodia ( F) . x 11,000.
FILOPODIA OF E. histolytica
189
Figs. 8, 9. [Scanning electron micrographs of trophozoites.] 8. Cell with several lobopodia (L) and a long filopodium ( F) . Irregularly
spaced vesicles (arrows) are seen on the uneven surface. x 3,000. 9. Part of an ameba fixed while progressing on the substrate. Note
the numerous filopodia (arrows) in the area of the uroid. Many of these filopodia were attached to the substrate, pulling out the poste-
rior end of the organism into a tail. x 4,000.
the shorter filopodia (e.g. Fig. 3 ) were inflated at the tip. Oc-
casionally, vesicles formed at the tips and could be seen as they
moved down the filopodia, finally disappearing at the cell surface.
HVEM and SEM of Whol e Cells.-HVEM has proved useful
in the study of the interior of whole mammalian tissue culture
cells ( 22 ) . We learned, however, that the cytoplasmic structure
of intact critical point dried E. histolyiica trophozoites could not
be visualized by this technic, as the amebae were too thick to be
penetrated by the 1000 kV electrons. I t was possible, however, to
see that the periphery of whole trophozoites had irregular bulges,
vesiculations, and surface filopodia (Figs. 6, 7) . These fixed filo-
podia ranged in length from several to over 30 pm. Some were
branched and some had fusiform swellings at various points
(Figs. 6, 7, 14).
When seen in the scanning electron microscope, the surface of
E. hirtozytica trophozoites was undulating, finely wrinkled and
covered at irregular intervals by protruding vesicles or blebs of
various sizes (Figs. 8, 9, 11-13, 15). Lobopodia (Figs. 8, 11 ) and
filopodia were the most prominent structures on the trophozoite
surface. Some filopodia were quite long (Figs. 8, 9). In tropho-
zoites which appeared to have been fixed while progressively
motile (Fig. 9), the largest numbers of filopodia were associated
with the uroid area. The uroid illustrated in Fig. 9 was a thin,
fan-shaped shelf of cytoplasm from which about 60 filopodia
arose. The majority of these filopodia were adherent to the
Formvar substrate and appeared to be drawn out behind the
advancing ameba. The cell shown in Fig. 9 was examined by
HVEM before the finder grid was mounted on the SEM stub. Its
filopodia, as shown by HVEM (Fig. l o) , were - 0.1 pm in
diameter and bounded by a unit membrane with a glycocalyx
(0.025 pm deep) indistinguishable from that of the cell surface.
Many of these filopodia terminated in knobs. The cytoplasm of
filopodia was devoid of organelles and had the mesh-like
trabecular appearance described as typical of critical point dried
cytoplasm in intact mammalian cells ( 2 2 ) .
Some trophozoites, fixed while apparently at rest, had no
distinct tail or uroid region (Figs. 11, 12), but possessed a tuft
of many filopodia. In these cases, the filopodia in the tufts arose
from a restricted area of the surface, - 5 pm2, and were not at-
tached to the substrate. Other filopodia, not associated with either
the uroid or tufts, were branched and had fusiform inflations at
their tips or along their length (Figs. 13, 15). Some of these
fiiopodia protruded singly from the upper surface of trophozoites
(Figs. 13, 14); others were grouped along the lower surface
near the point of contact with the substrate (Fig. 15). The base
of the latter type was bilaterally flattened and bifurcated at a
point - % of the way from the trophozoite surface to the tip.
Many of these bifurcated and branched filopodia were inflated at
their tips.
HVEM Examination of Thick Sections.-Thick sections of
epoxy-embedded trophozoites were photographed at different
specimen-tilt angles and viewed stereoscopically. Trophozoites
with a distinct uroid region (Figs. 16, 17) had a protruding portion
of the cytoplasm invested with large numbers of filopodia, most of
which appeared as elongated vesicular structures. I n several in-
stances, continuity between the vesicles could be demonstrated
stereoscopically, as could also continuity between short lengths
of filopodia and the plasmalemma. One of the filopodia shown
E. histolytica
in Fig. 16 ends in a knob, has a side branch, and is continuous
with the cell surface in the plane of section of an adjacent sub-
plasmalemmal vacuole. This structural arrangement is similar
to that described previously by Eaton et al. (5) as the surface-
active lysosome with trigger. Short filopodia were seen around
the periphery of trophozoites (Fig. 17) . These filopodia re-
sembled those seen on surfaces of intact cells fixed by other
methods (Figs. 12-15).
DISCUSSION
Filopodia are a filamentous type of pseudopodium seen in
many testaceans and in the genus Ameba (12). With the ex-
ception of one recent SEM study (16), filopodia have not been
adequately demonstrated in E. histolytica. This may be because
the majority of these filopodia are too small to resolve with the
light microscope, and because technics used in earlier SEM
studies of E. histolytica were unlikely to preserve such delicate
structures. In these SEM studies (5, 6) , trophozoites were chilled,
washed, centrifuged, and either freeze-dried or air-dried from
acetone. I n situ fixation and critical point drying appear to
preserve surface structures adequately. In addition to suffering
some of the technical inadequacies of the earlier SEM studies,
transmission electron microscopy has not permitted accurate
visualization of filopodia, as these structures are too long and
irregular to be seen in their entirety in single thin sections.
In the present study we compared data obtained by several
different methods used to study surface structure of axenically
grown E. histolytica trophozoites. Phase-contrast microscopy,
HVEM of thick sections and whole cells, and SEM of whole cells
led us to conclude that the surface of E. histozytica is convoluted
and wrinkled rather than smooth and rounded, and that filopodia
are a normal surface feature of these amebae. The filopodia are
numerous and may be found at any site on the cell surface. The
majority of filopodia large enough to be visualized by light
microscopy were associated with the uroid. Although filopodia
may vary considerably in length, their ultrastructure, as seen with
HVEM, is identical, regardless of location. I t was ascertained by
stereopair analysis of thick sections that structures previously
interpreted in single thin sections as extra-amebic vesicles were
often continuous with one another and with the cell surface. This
corroborates observations made by Deas & Miller from serial
thin sections ( 3) . We propose that such extra-amebic vesicles
as well as triggers and dendritic plasmalemmal extensions
represent parts of E. histolytica filopodia that can be visualized
in their entirety only by ultrastructural examination of whole
cells. I n situ fixation and critical point drying are probably
essential to preserve these delicate surface structures and prevent
artefactual distortion.
Gradual fixation in situ of whole motile trophozoites, without
3
Fig. 10. High voltage transmission electron micrograph of intact filopodia in the uroid region of the cell shown in Fig. 9. The filopodia
have swollen tips and each is bounded by a unit membrane ( U) with a glycocalyx ( G) . The trabecular ( T) appearance of the cyto-
plasm is similar in all filopodia. x 125,000.
Fig. 11. Scanning electron micrograph of a trophozoite with lobopodia ( L) , surface vesicles (arrows), and a tuft of filopodia ( F) on
one side of the cell. x 3,500.
Figs. 12-15. [Except for Fig. 14, a high voltage transmission electron micrograph, the figures are scanning electron micrographs.] 12.
Tuft of filopodia (T) arising from a limited area of the cell surface. X 3,000. 13. Filopodia not arranged in tufts and not associated with
the uroid. Some of these filopodia (arrows on the left of Figure) were solitary; others ( F) formed groups in the areas in which the cell
surface met the substrate. x 18,000. 14. Group of branched filopodia, with several fusiform swellings (arrows) separated by constric-
tions, is seen arising from a small area of the cell surface ( AM) . Note a part of a surface vesicle (V) to the left of the filopodia.
x 50,000. 15. Filopodia, several bifurcated (arrows) near their tips, on the cell surface ( AM) in the area where it meets the substrate.
x 25,000.
FILOPODIA OF E. histolytica 191
192
FILOPODIA OF E. histolytica
FILOPODIA OF E. histolytica 193
194 FILOPODIA OF
washing and centrifugation, also permitted visualization of cells
with lobopodia and a distinct tail or uroid region. As early as
1946, Hopkins & Warner (8) observed that the uroid of E.
histolytica was sticky and adhered to the substratum SO firmly
that it can be drawn out into a very long slender filament. We
suggest rather that the attachment of numerous short filopodia to
the substrate may be responsible for pulling out the trailing end
of motile amebae to form a tail. We noted that most tropho-
zoites which lacked obvious directional orientation at the time of
fixation had a tuft of short filopodia. We hypothesize that this
tuft is a constant feature associated with a restricted area of
the surface, the uroid, and that this area forms a tail only when
the filopodia are attached to the substrate and the cell is engaged
in active directional locomotion ( 1 ) .
McCaul & Bird (16) observed no filopodia on the upper sur-
faces of amebae. Unlike these authors, we observed filopodia
over the entire surface of E. histolytica trophozoites. McCaul &
Bird speculated that filopodia are involved in motility of E.
histoZytica and in its attachment to a substrate. As noted above,
such attachment may account for the formaion of a uroid in E.
hisolytica.
Entamoeba histolytica filopodia may participate in endo-
cytosis and/or exocytosis. It is known that these structures in
testaceans are active in endocytosis ( 12) . In the present study,
fixed, critical point dried filopodia observed on intact cells
often had swollen tips and fusiform swellings at various points
along their length. The swellings could represent vesicles in
transit. In the uroid region of living trophozoites viewed by phase-
contrast microscopy, we occasionally observed the formation of
vesicles at the tips of filopodia and movement of vesicles into the
cell. Similar observations were allegedly made by Brug ( 2)
according to a report of van Thiel (21). Hopkins & Warner
(8) and Zaman ( 23) , however, in discussing the uroid of E. histo-
lytica, proposed that this region was primarily excretory in func-
tion. They were unable to see the numerous intact short filopodia,
but observed many vesicles within and just outside of the cyto-
plasm in the uroid region. Vesicles outside the plasmalemma
could represent cross sections of filopodia. Those within the
cytoplasm could, of course, have been formed by endocytic or
exocytic processes. Small particles which can be seen in an
electron microscope might be used in culture media as markers to
help determine whether these filopodia function in an exocytic or
endocytic manner.
Of special interest is the question whether or not filopodia play
a role in pathogenicity of E. histolytica. Eaton & Meerovitch ( 5)
hypothesized that the plasmalemma of the filopodia they termed
triggers depolarized upon contact with another cell to premit
release of acid phosphatase-positive material present within
vacuoles (surface-active lysosomes ) at the base of the filopodia.
An alternative interpretation of these data is possible-vacuoles
containing acid phosphatase-positive material are seen near the
surface of amebae in association with filopodia because they
represent digestive vacuoles formed by the addition of digestive
enzymes to vesicles which had been endocytosed by surface filo-
podia.
Although many previous authors (3, 5, 16) agree that filopodia
E. histolytica
(triggers, plasmalemmal extensions, etc.) could play a role in
contact-cytolysis of target cells by intact amebae, there is no con-
clusive evidence to support such a hypothesis. Isolation of a cyto-
toxic protein capable of producing the same cytopathogenic effects
as living trophozoites on monolayers has recently been reported
from this laboratory ( 15). Immunocytochemical localization of
the cytotoxin could help to determine if the filopodia and/or
surface vesiculations participate in the release of toxic substances.
ACKNOWLEDGEMENTS
We thank Drs. John Wolosewick, Jerome Paulin, Douglas Mohr,
and Walter Humphreys, and George Wray for technical advice;
Ann Hofbauer, Beth Howell, Linda Sears, and Andrew Kairalla
for technical assistance; Dr. Joseph C. Ross for his encouragement
and support; and Drs. Gordon Hennigar and Harvey Bank for
providing the use of the scanning electron microscope.
LITERATURE CITED
1. Bird RG. 1956. A constant momholo&&cal feature in the
trophozoite stage of Entamoeba histolytica. Tyans. R. SOC. Trop.
Med. Hy g. 5 0 , 302.
2. Brug SL. 1928. Observations on a culture of E. histolytica.
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3. Deas JE, Mill% JH. 1977. Plasmalemmal modifications
of Entamoeba histolytica in vivo. J . Parasitol. 6 3 , 25-31.
4. Diamond LS. 1968. Techniques of axenic cultivation of
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5. Eaton RDP, Meerovitch E, Costerton JW. 1970. The
functional mor~holow of Dathogenicitv in Entamoeba histolytica.
Ann. Trop. Mi d . Paiasitol. -64, f99-30.1.
6. Feria-Velasco A, Gonz6lez del Pliego M, Tapia-Arizmendi G.
1976. Comparative study of Entamoeba histolytica trophozoites by
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8. Hopkins DL, Warner KL. 1946. Functional cytology of
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10. Kairalla AB, Hofbauer AF, Pittman JC, Lushbaugh WB,
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1 1. Knight R, Bird RG, McCaul TF. 1975. Fine structural
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face. Ann. Trop. Me d. Parasitol. 69, 197-202.
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Springfield, Illinois.
13. Lushbaugh WB, Miller JH. 1974. Fine structural topo-
chemistry of Entamoeba histolytica Schaudinn, 1903. J . Parasitol.
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14. ___ , Pence DB, Deas JE, Miller JH. 1972. In viuo
acid phosphatase activity of Entamoeba histolytica, in Arceneaux
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Publishing Division, Baton Rouge, Louisiana, pp. 154-5.
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210-3.
c
16. Section (0.25 pm thick) through the uroid region, including
parts of many filopodia ( F) , a large proportion of which appear as extra-amebic vesicles. One filopodium, continuous with the cell
surface near a subplasmalemmal vacuole ( V) , has a swollen tip and a side branch (arrows). x 37,500. 17. Section (0. 5 pm thick)
through 2 trophozoites. I n the ameba on the right, note the uroid with numerous filopodia ( F) and in the one on the left, filopodia
(arrows) distributed along its periphery. X 27,000.
Figs. 16, 17. [High voltage transmission electron micrographs.]
FILOPODIA OF E. histolytica
1 95
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changes after trophozoite contact-bservations by scanning electron
microscopy. I nt . J. Parasitol. 7, 383-8.
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enterology 65, 588-603.
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surface active lysosome in the trophozoites of Entamoeba histolytica
from the human colon. Ann. Trop. Med. Parasitol. 66, 339-42.
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strain NIH-260. Znt. I. Parasitol. 3, 457-60.
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I. Protozool., 26(2),. 1979, pp. 195-199
0 1979 by the Society of Protozoologists
Ultrastructural Damage to the Malaria Parasite in the Sickled Cell
MILTON J. FRIEDMAN
The Rockefeller University, New York, New York 10021
SYNOPSIS. The process by which malaria parasites are killed in sickled erythrocytes was studied by electron microscopy. Zn
vitro cultures of Plasmodium falciparum in sickle cell hemoglobin (HbS) homozygous (SS) and heterozygous (SA) red cells
were deoxygenated for up t o 6 h and fixed under anaerobic conditions. Parasites in SS cells appeared to be disrupted by
intrusions of needle-like deoxyHbS aggregates ; disintegration of cytoplasm and membranes followed. I n SA red cells, the para-
sites were generally not disrupted. Instead, extensive vacuolization occurred, a sign of metabolic inhibition. The resistance of
HbS gene carriers to malaria results partly from these causes of intracellular parasite death.
Index Key Words: Plasmodium falciparum ; sickle cell hemoglobin; resistance to infection.
HE causal agent of malaria, Plasmodium, is for much of its
T life cycle an intra-erythrocytic parasite, well protected from
most of the bodys defenses. If the intracellular environment of
the parasite could be altered, protection against malaria might
ensue. That this does occur in nature is indicated by the high
frequency of abnormal hemoglobins and red cell enzyme poly-
morphisms in areas endemic for malaria ( 6) . Sickle cell hemo-
globin ( HbS) , the most common of the hemoglobin variants,
carries a &chain mutation which decreases the solubility of the
deoxygenated form. When HbS-containing red cells are exposed
to a low 0, tension, HbS forms filamentous aggregates which
distort the cell into a sickle shape and markedly increase intra-
cellular viscosity (4). This alteration might be expected to in-
hibit the growth of an intracellular organism.
I t has been shown that deoxygenation of red cells containing
HbS leads to the death of intracellular Plasmodium falciparum
( 3 ) . Parasite death was closely correlated with sickling of the
erythrocyte when 0, tension was varied and when sickling was
inhibited with cyanate. Thus, the resistance of HbS gene carriers
to malaria may be a direct result of deoxyHbS aggregation. I n
homozygous (SS) variant cells, with almost all HbS, the complete
lysis of both parasites and host cells occurred during the first 24 h
of deoxygenation. I n heterozygous (SA) cells, however, which
contain less than 50% HbS, dead parasites were not lysed, but
remained as condensed bodies in intact erythrocytes. In addition,
the oldest and youngest of the parasites in SA cells survived the
first day at low 0,, while all parasites were killed in SS cells.
The indication of different mechanisms of killing in these 2 cell
types and the dramatic disappearance of infected cells in the SS
red cell culture suggested this study of the effect of sickling on
parasite ultrastructure.
MATERIALS AND METHODS
Culture Conditions.-Plasmodium falciparum (FCR-3/FMG)
was cultivated i n settled layers of human red cells as described
previously (10). The culture medium was RPMI-1640 (GIBCO)
with 25 mM Hepes (Sigma), 10% human serum (type AB), and
40 pg/ml gentamicin (Schering) . Variant and normal eryth-
rocytes were obtained, analyzed, and stored ( 3 ) . For the present
study, an initial red cell suspension of 5% (v/v) was inoculated
with infected, homologous (SS, SA, or AA) cells to obtain a
parasitemia of 1%. Petri-dish cultures were incubated at 37 C
under 18% O,, 3% CO,, and the medium was changed daily for
2 days and then twice daily for 1 to 2 days until a parasitemia of
20% was reached.
Deoxygenation.-Infected red cells (AA, SA, and SS) were re-
suspended in fresh medium and transferred to flasks through
which a humidified mixture of 1% O,, 5% CO,, and 94% N,
was flowing and which were mounted on a rocking platform at
37 C. After 0.25, 2, and 6 h, 0.5 ml of cell suspension was
removed with a glass syringe and injected into 5 ml fixative under
anaerobic conditions.
Fixation.-The fixative for electron microscopy was a deaerated
mixture of 2% (v/v) glutaraldehyde, 4% (w/v) sucrose, and