Acta Protozoologica 2009 48/4
Jagiellonian University Kraków, Poland
© Jagiellonian University
Acta Protozool. (2009) 48: 327–332
Acta
Protozoologica
Acanthamoeba castellanii: Endocytic Structures Involved in the Ingestion
of Diverse Target Elements
Arturo González-Robles1, Mónica González-Lázaro2, Maritza Omańa-Molina3,
and Adolfo Martínez-Palomo4
1
Department of Infectomic and Molecular Pathogenesis; 2Department of Cell Biology, Center for Research and Advanced Studies,
Mexico City; Mexico 3Faculty of Superior Studies, UNAM, Iztacala, Tlalnepantla State of Mexico, Mexico
Summary. Co-incubation of Acanthamoeba castellanii trophozoites with diverse target elements such as MDCK epithelial cells, hamster
cornea, human erythrocytes, Acantamoeba cysts and bacteria clusters produced morphologically different endocytic structures. In general,
up to three different types of endocytic structures were observed after the interaction between A. castellanii and the different target elements:
large amoebostomes, slender cylindrical sucker-like channels and minute cell surface specializations, some of them with a pinch-off lytic
action. These endocytic structures are used to lyse and ingest different targets in a contact-dependent mechanism.
Key words: Acanthamoeba castellanii, amoebostomes, endocytosis, Phagocytosis
Introduction
Acanthamoeba are pathogenic free-living amoebae
that can produce granulomatous encephalitis (Martínez
1991) and more commonly amoebic keratitis, a painful
vision-threatening infection present predominantly in
contact lens users (Schaumberg et al.1998).
Phagocytosis is a specific form of endocytosis involving the internalization of large particles (including other cells) into the cell body. In the literature on
free-living amoebae some reports deal with this topic
and describe diverse morphological structures that par-
ticipate in this process such as amoebostomes (Diaz et
al. 1991; John et al. 1984, 1985, 1989) food cups and
digipodia (Dove Pettit et al. 1996, Marciano-Cabral and
John, 1983; Marciano-Cabral and Fulford 1986).
The purpose of this report is to group the principal
endocytic structures found in Acanthamoeba castellanii
that are involved in the in vitro ingestion of diverse target elements.
Material and methods
Amoebae and cell cultures
* Address for correspondence: Dr. Arturo González-Robles, CINVESTAV-IPN, Av. Instituto Politécnico Nacional 2508 San Pedro Zacatenco 07360 Mexico, D. F. Mexico. E-mail: [email protected]
A. castellanii trophozoites were originally isolated from a contact lens associated with a human case of keratitis at the Luis Sánchez Bulnes Hospital in Mexico City. Amoebae were grown and
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© Jagiellonian University
328 A. González-Robles et al.
maintained in axenic culture in 2% Bactocasitone culture medium
(pancreatic digest of casein, Becton Dickinson, Sparks, MD) supplemented with 10% fetal bovine serum (Gibco, Grand Islands N.
Y.). Cultures were incubated at 26°C in borosilicate tubes (Pyrex,
Mexico). Trophozoites were harvested after 3 days during the logarithmic phase of growth by chilling the culture tubes in an ice-water
bath for 15 min. After centrifugation at 2000 rpm for 5 min, the pellets were resuspended in culture medium.
Trophozoites were interacted with various target elements as
monolayers or freshly trypsinized (individualized) MDCK epithelial
cells, human erythrocytes, Acanthamoeba cysts, bacteria and hamster cornea, either in Bactocasitone or in a mixture of Bactocasitone
and Dulbecco’s modified Eagle medium in equal proportions. The
amoebae: target elemets ratio and incubation times varied according
to each assay, as described as follows.
Epithelial cells of the established MDCK line of canine kidney
origin (Madin-Darby Canine Kidney) were grown to confluence on
25 cm2 cell culture flasks (Corning, Corning Incorporated, NY) in
Dulbecco’s modified Eagle’s medium (Microlab, Mexico) supplemented with 10% fetal bovine serum (Gibco, Grand Islands, N.Y.)
and antibiotics in a 5% CO2 atmosphere at 37°C. Afterwards, confluent monolayers were incubated in a mixture of Bactocasitone and
Dulbecco’s modified Eagle’s medium in equal proportions and A.
castellanii trophozoites were added in a 1:2 target cell:amoeba ratio
for 1–2 h. In a parallel assay, freshly trypsinized MDCK cells were
incubated for 1 h with the amoebae (1:1 ratio). Human erythrocytes
were freshly prepared following standard techniques. Briefly, extracted blood mixed with Alsever’s solution was centrifuged and
washed twice with PBS. Co-incubations were done in a 10:1 erytrocytes:amoeba ratio for 30 and 60 min. at 37şC in 2% Bactocasitone medium. Interactions with cysts were carried out incubating A.
castellanii cysts and trophozoites in a 2:1 ratio in 2% Bactocasitone
medium at 28şC for 2 h.
Interactions with bacteria (Enterobacter aerogenes) were done
in a 9 cm diameter Petri dish covered with 1.5% non nutritive agar
medium containing 1×106 amoebae in 100 µl of Bactocasitone medium for 20 min. at 30şC and 0.1 ml of heat inactivated bacteria. Interactions with hamster cornea were performed according to protocol
002/02, approved by the Institutional Animal Care and Use Committee, in accordance with norm-062-Zoo-1999, based on the Guide
for the Care and Use of Laboratory Animals. After anesthesia with
sodium pentobarbital (Sedatphorte; 4.72 mg/100 g of body weight),
both corneas were removed. Each cornea contained a peripheral rim
of scleral tissue, leaving the lens untouched. Corneas were placed in
96-well styrene plates and incubated with 106 trophozoites in 0.2 ml
of culture medium for 1 or 2 h.
Scanning electron microscopy
Glutaraldehyde-fixed samples were dehydrated with increasing
concentrations of ethanol, critical-point dried using a Samdri 780
apparatus (Tousimis, Rockville Maryland, USA), coated with gold
with a JEOL JFC-1100 ion-sputtering device, and examined both in
a XL-30 ESEM (FEI Company, Eindhoven, The Netherlands) and a
Zeiss DSM 982 Gemini (Carl Zeiss Electron Optics Division, Overkochen, Germany) scanning electron microscopes.
Transmission electron microscopy
After co-incubation, samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 at room temperature, postfixed with 1% osmium tetroxide in cacodylate buffer, and dehydrated in increasing concentrations of ethanol. Samples were then
transferred to propylene oxide, and subsequently to a mixture of
propylene oxide/epoxy resins (1/1) before being embedded in epoxy
resins. Samples were observed in a Morgagni 268D transmission
electron microscope (FEI Company, Eindhoven, The Netherlands).
Results and discussion
A. castellanii strain was identified by morphological
characteristics according to Page (1988). Acanthamoeba castellanii trophozoites that interacted with diverse
cells created phagocytic structures of various shapes
and sizes that ranged from huge amoebostomes as reported by Khan (2001, 2003) to minute cell surface specializations that “pinch-off” small portions of target cell
surface. A peculiar finding was that these amoebae produced different phagocytic structures whose morphology “adapted” according to the size and shape of the ingested material, e.g., when a large volume of biological
material was nearby a big amoebostome was formed.
Previously, diverse authors have reported phagocytic
structures in free living amoebae with different names
such as amebostomes (John et al. 1985, 1989; Diaz et
al. 1991, Khan. 2001, 2003), sucker structures (Marciano-Cabral and John, 1983; John et al. 1984) or food
cups (Dove-Petit et al. 1986; Marciano-Cabral and Fulford 1986) which essentially represent the same phagocytic arrangement.
After co-incubation with MDCK cells, up to three
different phagocytic structures were observed in A.
castellanii. With freshly trypsinized cells, trophozoites formed large amebostomes, many of them clearly
trying to engulf the whole cell (Fig. 1 A) which was
finally phagocyted (Fig. 2 A). In contrast, during the
interaction with low-platted MDCK cell cultures some
amoebae formed slender cylindrical sucker structures
that pulled out small portions of the target cell which
were then introduced into the cell body (Figs 1 B and 2
B). It was also observed that after two hours of interaction with epithelial MDCK cell monolayers, the cells
were severely damaged and many thin remnants of target cells were flanked by phagocytic membrane ruffles
with a jaw-like appearance (Fig 1 C) or by microchannels (Fig. 2 C).
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Endocytic structures of A. castellanii 329
Fig. 1. A–C. Interaction between A. castellanii and MDCK epithelial cells. A – During the interaction with freshly trypsinized MDCK cells,
a trophozoite (Ac) is seen phagocyting a whole cell. Note some flattened cell projections that do not correspond to the typical acanthopodia
(arrows). Bar: 10 µm; B – co-incubation with low platted MDCK epithelial cells. A trophozoite (Ac) lies over two epithelial cells and by
means of a long slender sucker structure (arrowhead) ingests a portion of a contiguous target cell. Bar: 20 µm; C – after 1 h of interaction
with a MDCK epithelial cell monolayer, two amoebae hold a thin remnant portion of the target cell by means of a jaw-like cytoplasmic
ruffle (arrows). Bar: 10 µm; D – an Acanthamoeba castellanii trophozoite (Ac) interacting with hamster corneal stroma (Cs) exhibits a
huge amebostome. Bar: 10 µm; E – trophozoites in contact with hamster corneal epithelial cells. An amoeba (Ac) is engulfing a portion of
a corneal epithelial cell (Cec) by means of a slender sucker-like structure. Bar: 10 µm; F – two human erythrocytes are introduced into the
amoeba by means of a large amebostome. Bar: 10 µm; G and H – light and scanning electron microscopy images of the interaction between
A. castellanii trophozoites and a cyst of the same species. Images demonstrate how an amoeba (Ac) is phagocyting an A. castellanii cyst.
Bar: 10 µm; I – by means of a small sucker structure (Ss), bacteria (B) are engulfed. Bar: 2 µm.
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© Jagiellonian University
330 A. González-Robles et al.
Fig. 2. Transmission electron microscopy of the interaction between Acanthamoeba castellanii trophozoites and different target elements. A
– after 2 h of interaction, a freshly trypsinized MDCK cell is observed in a digestive vacuole of a trophozoite. Bar: 50 µm. B – an A. castellanii trophozoite (Ac) is engulfing a potion of a MDCK epithelial cell by means of two amoebostomes that lack cellular organelles. Bar: 1
µm; C – cross section of a portion of a confluent MDCK epithelial cell monolayer in which an amoeba (Ac) is on the surface of the target cell
pulling out a thin portion of the cell surface after 2 h of co-incubation. Bar: 50 µm; D – an A. castellanii trophozoite is phagocyting a portion
of a hamster corneal epithelial cell (Cec). Some cellular fibrogranular projections of the amoeba are also interacting with another target cell.
Bar: 5 µm; E – an ingested human erythrocyte is observed inside an amoeba. Bar: 1.0 µm; F – high magnification of the endocytosis process
of bacteria, one of them lies inside a phagocytic vacuole. Bar: 0.5 µm.
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© Jagiellonian University
Endocytic structures of A. castellanii 331
Fig. 3. A–D – selected frames of video time lapse microcinematography which demonstrate how an amoeba pinches-off a portion of a
MDCK cell; E – this pinching-off process is clearly observed by transmission electron microscopy. By means of a tiny endocytic structure
a trophozoite engulfs a portion of the cytoplasm of a MDCK target cell through a slender narrow channel (double arrow). A portion of the
endocyted material is observed inside a vacuole (arrow). Bar: 1 µm.
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332 A. González-Robles et al.
When amoebae were in contact with hamster cornea, both large amoebostomes and slender cylindrical
sucker structures were present (Figs 1 D, E). Besides,
large amoebostomes were formed when A. castellanii
trophozoites were co-incubated with human erythrocytes (Fig.1 F) which were lastly introduced into the
cell soma (Fig. 2 E.). Acanthamoeba cysts (Figs 1 G,
H) and bacteria (Figs. 1 I, 2 F) were also endocyted by
means of large amoebostomes as well as by undersized
phagocytic structures. When Acanthamoeba castellanii
and freshly trypsinized MDCK epithelial cell co-incubations were studied by time lapse video microscopy,
the pinch-off manner of lytic action of the target cell
surface was clearly observed (Figs 3 A–D). In some
amoebae, the pinching-off mechanism of small portions
of the target cell is a very dynamic event, with the subsequent lysis of the damaged cells since the amoeba is
capable to form microphagocytic channels and ingest
portions of the cell surface (Fig. 3 E). This process
takes only few seconds in which the cortical actin cytoskeleton plays an important role as it is present in the
phagocytic structures (González-Robles et al. 2008).
Chambers and Wiser (1969) reported an analogous process of this pinching-off mechanism in the interaction
of mouse tumor cells and macrophages. Similar observations have been made in the parasitic protozoa Entamoeba histolytica (Martínez-Palomo et al. 1985) and
Trichomonas vaginalis (González- Robles et al. 1995).
In a recent report, our group demonstrated that actin
cytoskeleton in A.castellanii is largely formed by fibers
and networks of actin located mainly in cytoplasmic
locomotion structures such as lamellipodia and in various endocytic structures (González-Robles et al. 2008).
The actin-rich cytoskeleton of the trophozoites allows
rapid morphological changes. These endocytic structures along with the movements of the amoebae play an
important function in the early events of the cytophatic
effect.
Clearly, the mechanic participation of the endocytic
process is used by A. castellanii to harm target cells and
obtain different biological materials as an additional
food source.
Since phagocytic structures represent an important
method of cell damage as a contact-dependent mechanism it will be interesting to know why the trophozoites
produce different morphological endocytic structures
according to the nature of the material to be engulfed
and which are the signals that trigger these events.
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