Acta Protozoologica 2009 48/4Jagiellonian University Kraków, Poland© Jagiellonian UniversityActaProtozoologicaActa Protozool. (2009) 48: 327–332Acanthamoeba castellanii: Endocytic Structures Involved in the Ingestionof Diverse Target ElementsArturo González-Robles 1 , Mónica González-Lázaro 2 , Maritza Omańa-Molina 3 ,and Adolfo Martínez-Palomo 41Department of Infectomic and Molecular Pathogenesis; 2 Department of Cell Biology, Center for Research and Advanced Studies,Mexico City; Mexico 3 Faculty of Superior Studies, UNAM, Iztacala, Tlalnepantla State of Mexico, MexicoSummary. Co-incubation of Acanthamoeba castellanii trophozoites with diverse target elements such as MDCK epithelial cells, hamstercornea, 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 lyticaction. These endocytic structures are used to lyse and ingest different targets in a contact-dependent mechanism.Key words: Acanthamoeba castellanii, amoebostomes, endocytosis, PhagocytosisIntroduction* Address for correspondence: Dr. Arturo González-Robles, CIN-VESTAV-IPN, Av. Instituto Politécnico Nacional 2508 San Pedro Zacatenco07360 Mexico, D. F. Mexico. E-mail: goroa@cinvestav.mxAcanthamoeba are pathogenic free-living amoebaethat can produce granulomatous encephalitis (Martínez1991) and more commonly amoebic keratitis, a painfulvision-threatening infection present predominantly incontact lens users (Schaumberg et al.1998).Phagocytosis is a specific form of endocytosis involvingthe internalization of large particles (includingother cells) into the cell body. In the literature onfree-living amoebae some reports deal with this topicand describe diverse morphological structures that participatein this process such as amoebostomes (Diaz etal. 1991; John et al. 1984, 1985, 1989) food cups anddigipodia (Dove Pettit et al. 1996, Marciano-Cabral andJohn, 1983; Marciano-Cabral and Fulford 1986).The purpose of this report is to group the principalendocytic structures found in Acanthamoeba castellaniithat are involved in the in vitro ingestion of diverse targetelements.Material and methodsAmoebae and cell culturesA. castellanii trophozoites were originally isolated from a contactlens associated with a human case of keratitis at the Luis SánchezBulnes Hospital in Mexico City. Amoebae were grown and

Acta Protozoologica 2009 48/4Jagiellonian University Kraków, Poland© Jagiellonian University328A. González-Robles et al.maintained in axenic culture in 2% Bactocasitone culture medium(pancreatic digest of casein, Becton Dickinson, Sparks, MD) supplementedwith 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 logarithmicphase of growth by chilling the culture tubes in an ice-waterbath for 15 min. After centrifugation at 2000 rpm for 5 min, the pelletswere resuspended in culture medium.Trophozoites were interacted with various target elements asmonolayers or freshly trypsinized (individualized) MDCK epithelialcells, human erythrocytes, Acanthamoeba cysts, bacteria and hamstercornea, either in Bactocasitone or in a mixture of Bactocasitoneand Dulbecco’s modified Eagle medium in equal proportions. Theamoebae: target elemets ratio and incubation times varied accordingto each assay, as described as follows.Epithelial cells of the established MDCK line of canine kidneyorigin (Madin-Darby Canine Kidney) were grown to confluence on25 cm 2 cell culture flasks (Corning, Corning Incorporated, NY) inDulbecco’s modified Eagle’s medium (Microlab, Mexico) supplementedwith 10% fetal bovine serum (Gibco, Grand Islands, N.Y.)and antibiotics in a 5% CO 2atmosphere at 37°C. Afterwards, confluentmonolayers were incubated in a mixture of Bactocasitone andDulbecco’s modified Eagle’s medium in equal proportions and A.castellanii trophozoites were added in a 1:2 target cell:amoeba ratiofor 1–2 h. In a parallel assay, freshly trypsinized MDCK cells wereincubated for 1 h with the amoebae (1:1 ratio). Human erythrocyteswere freshly prepared following standard techniques. Briefly, extractedblood mixed with Alsever’s solution was centrifuged andwashed twice with PBS. Co-incubations were done in a 10:1 erytrocytes:amoebaratio for 30 and 60 min. at 37şC in 2% Bactocasitonemedium. Interactions with cysts were carried out incubating A.castellanii cysts and trophozoites in a 2:1 ratio in 2% Bactocasitonemedium at 28şC for 2 h.Interactions with bacteria (Enterobacter aerogenes) were donein a 9 cm diameter Petri dish covered with 1.5% non nutritive agarmedium containing 1×10 6 amoebae in 100 µl of Bactocasitone mediumfor 20 min. at 30şC and 0.1 ml of heat inactivated bacteria. Interactionswith hamster cornea were performed according to protocol002/02, approved by the Institutional Animal Care and Use Committee,in accordance with norm-062-Zoo-1999, based on the Guidefor the Care and Use of Laboratory Animals. After anesthesia withsodium pentobarbital (Sedatphorte; 4.72 mg/100 g of body weight),both corneas were removed. Each cornea contained a peripheral rimof scleral tissue, leaving the lens untouched. Corneas were placed in96-well styrene plates and incubated with 10 6 trophozoites in 0.2 mlof culture medium for 1 or 2 h.Scanning electron microscopyGlutaraldehyde-fixed samples were dehydrated with increasingconcentrations of ethanol, critical-point dried using a Samdri 780apparatus (Tousimis, Rockville Maryland, USA), coated with goldwith a JEOL JFC-1100 ion-sputtering device, and examined both ina XL-30 ESEM (FEI Company, Eindhoven, The Netherlands) and aZeiss DSM 982 Gemini (Carl Zeiss Electron Optics Division, Overkochen,Germany) scanning electron microscopes.Transmission electron microscopyAfter co-incubation, samples were fixed with 2.5% glutaraldehydein 0.1 M cacodylate buffer pH 7.2 at room temperature, postfixedwith 1% osmium tetroxide in cacodylate buffer, and dehydratedin increasing concentrations of ethanol. Samples were thentransferred to propylene oxide, and subsequently to a mixture ofpropylene oxide/epoxy resins (1/1) before being embedded in epoxyresins. Samples were observed in a Morgagni 268D transmissionelectron microscope (FEI Company, Eindhoven, The Netherlands).Results and discussionA. castellanii strain was identified by morphologicalcharacteristics according to Page (1988). Acanthamoebacastellanii trophozoites that interacted with diversecells created phagocytic structures of various shapesand sizes that ranged from huge amoebostomes as reportedby Khan (2001, 2003) to minute cell surface specializationsthat “pinch-off” small portions of target cellsurface. A peculiar finding was that these amoebae produceddifferent phagocytic structures whose morphology“adapted” according to the size and shape of the ingestedmaterial, e.g., when a large volume of biologicalmaterial was nearby a big amoebostome was formed.Previously, diverse authors have reported phagocyticstructures in free living amoebae with different namessuch as amebostomes (John et al. 1985, 1989; Diaz etal. 1991, Khan. 2001, 2003), sucker structures (Marciano-Cabraland John, 1983; John et al. 1984) or foodcups (Dove-Petit et al. 1986; Marciano-Cabral and Fulford1986) which essentially represent the same phagocyticarrangement.After co-incubation with MDCK cells, up to threedifferent phagocytic structures were observed in A.castellanii. With freshly trypsinized cells, trophozoitesformed large amebostomes, many of them clearlytrying to engulf the whole cell (Fig. 1 A) which wasfinally phagocyted (Fig. 2 A). In contrast, during theinteraction with low-platted MDCK cell cultures someamoebae formed slender cylindrical sucker structuresthat pulled out small portions of the target cell whichwere then introduced into the cell body (Figs 1 B and 2B). It was also observed that after two hours of interactionwith epithelial MDCK cell monolayers, the cellswere severely damaged and many thin remnants of targetcells were flanked by phagocytic membrane ruffleswith a jaw-like appearance (Fig 1 C) or by microchannels(Fig. 2 C).

Acta Protozoologica 2009 48/4Jagiellonian University Kraków, Poland© Jagiellonian UniversityEndocytic structures of A. castellanii 329Fig. 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 bymeans of a long slender sucker structure (arrowhead) ingests a portion of a contiguous target cell. Bar: 20 µm; C – after 1 h of interactionwith a MDCK epithelial cell monolayer, two amoebae hold a thin remnant portion of the target cell by means of a jaw-like cytoplasmicruffle (arrows). Bar: 10 µm; D – an Acanthamoeba castellanii trophozoite (Ac) interacting with hamster corneal stroma (Cs) exhibits ahuge amebostome. Bar: 10 µm; E – trophozoites in contact with hamster corneal epithelial cells. An amoeba (Ac) is engulfing a portion ofa corneal epithelial cell (Cec) by means of a slender sucker-like structure. Bar: 10 µm; F – two human erythrocytes are introduced into theamoeba by means of a large amebostome. Bar: 10 µm; G and H – light and scanning electron microscopy images of the interaction betweenA. 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.

Acta Protozoologica 2009 48/4Jagiellonian University Kraków, Poland© Jagiellonian University330A. 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. castellaniitrophozoite (Ac) is engulfing a potion of a MDCK epithelial cell by means of two amoebostomes that lack cellular organelles. Bar: 1µm; 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 cellpulling 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 portionof 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 processof bacteria, one of them lies inside a phagocytic vacuole. Bar: 0.5 µm.

Acta Protozoologica 2009 48/4Jagiellonian University Kraków, Poland© Jagiellonian UniversityEndocytic structures of A. castellanii 331Fig. 3. A–D – selected frames of video time lapse microcinematography which demonstrate how an amoeba pinches-off a portion of aMDCK cell; E – this pinching-off process is clearly observed by transmission electron microscopy. By means of a tiny endocytic structurea trophozoite engulfs a portion of the cytoplasm of a MDCK target cell through a slender narrow channel (double arrow). A portion of theendocyted material is observed inside a vacuole (arrow). Bar: 1 µm.

Acta Protozoologica 2009 48/4Jagiellonian University Kraków, Poland© Jagiellonian University332A. González-Robles et al.When amoebae were in contact with hamster cornea,both large amoebostomes and slender cylindricalsucker structures were present (Figs 1 D, E). Besides,large amoebostomes were formed when A. castellaniitrophozoites were co-incubated with human erythrocytes(Fig.1 F) which were lastly introduced into thecell soma (Fig. 2 E.). Acanthamoeba cysts (Figs 1 G,H) and bacteria (Figs. 1 I, 2 F) were also endocyted bymeans of large amoebostomes as well as by undersizedphagocytic structures. When Acanthamoeba castellaniiand freshly trypsinized MDCK epithelial cell co-incubationswere studied by time lapse video microscopy,the pinch-off manner of lytic action of the target cellsurface was clearly observed (Figs 3 A–D). In someamoebae, the pinching-off mechanism of small portionsof the target cell is a very dynamic event, with the subsequentlysis of the damaged cells since the amoeba iscapable to form microphagocytic channels and ingestportions of the cell surface (Fig. 3 E). This processtakes only few seconds in which the cortical actin cytoskeletonplays an important role as it is present in thephagocytic structures (González-Robles et al. 2008).Chambers and Wiser (1969) reported an analogous processof this pinching-off mechanism in the interactionof mouse tumor cells and macrophages. Similar observationshave been made in the parasitic protozoa Entamoebahistolytica (Martínez-Palomo et al. 1985) andTrichomonas vaginalis (González- Robles et al. 1995).In a recent report, our group demonstrated that actincytoskeleton in A.castellanii is largely formed by fibersand networks of actin located mainly in cytoplasmiclocomotion structures such as lamellipodia and in variousendocytic structures (González-Robles et al. 2008).The actin-rich cytoskeleton of the trophozoites allowsrapid morphological changes. These endocytic structuresalong with the movements of the amoebae play animportant function in the early events of the cytophaticeffect.Clearly, the mechanic participation of the endocyticprocess is used by A. castellanii to harm target cells andobtain different biological materials as an additionalfood source.Since phagocytic structures represent an importantmethod of cell damage as a contact-dependent mechanismit will be interesting to know why the trophozoitesproduce different morphological endocytic structuresaccording to the nature of the material to be engulfedand which are the signals that trigger these events.ReferencesChambers, V. C. and Weiser, R. S. (1969) The ultrastructure of targetcells and immune macrophages during their interaction in vitro.Cancer Res. 29: 301–317Diaz J., Osuna A., Rosales M. J., Cifuentes J., Mascaró C. (1991)Sucker-like structures in two strains of Acanthamoeba: scanningelectron microscopy study. Int J Parasitol. 31: 365–367Dove Pettit, D. A., Williamson, J., Cabral, G. A., Marciano-Cabral,F. (1996) In vitro destruction of nerve cells cultures by Acanthamoebaspp.: a transmission and scanning electron microscopystudy. J Parasitol. 82: 769–777González-Robles A., Lázaro-Haller A. G., Espinoza-Cantellano M.,Anaya-Veázquez F. Martínez-Palomo A. (1995) Trichomonasvaginalis: Ultrastructural bases of the cytophatic effect. J Eukaryot.Microbiol. 42: 641–651González-Robles A., Castańón G., Hernández-Ramírez V. I., Salazar-VillatoroL., González-Lázaro M., Omańa-Molina M., Talamás-RohanaP., Martínez-Palomo A. (2008) Acanthamoebacastellanii: Identification and distribution of actin cytoskeleton.Exp. Parasitol. 119: 411–417John D. T., Cole T. B. Jr., Marciano-Cabral. F. M. (1984) Sucker-likestructures on the pathogenic amoeba Naegleria fowleri. Appl.Environ. Microbiol. 47: 12–14John D. T., Cole T. B. Jr., Bruner R. A. (1985) Amoebostomes ofNaegleria fowleri. J Protozool. 32: 12–19John D. T., John R. A. (1989) Cytophatogenicity of Naegleria fowleriin mammalian cell cultures. Parasitol. Res. 76: 20–25Khan N. A. (2001) Pathogenicity, morphology and differentiation ofAcanthamoeba. Curr. Microbiol. 43: 391–395Khan N. A. (2003) Pathogenesis of Acanthamoeba infections. Microb.Pathogenesis. 34: 227–285Marciano-Cabral F., John D. T. (1983) Cytophatogenicity of Naegleriafowleri for rat neuroblastoma cell cultures: Scanningelectron microscopy study. Infect. Immun. 40: 1214–1217Marciano-Cabral F. M. Fulford D. E. (1986) Cytophatology ofpathogenic Naegleria species for cultured rat neuroblastomacells. Appl. Environ. Microb. 51: 1133–1137Martínez J. A. (1991). Infection of the central nervous system due toAcanthamoeba. Rev. Infect. Dis. 13: (Suppl. 5) S399–S402Martínez-Palomo A., González-Robles A., Chávez B., Orozco E.,Fernández-Castelo S. Cervantes A. (1985) Structural bases ofthe cytolytic mechanisms of Entamoeba histolytica. J. Protozool.32: 166–175Page F.C. (1988) A new key to freshwater and soil gymnamoebaewith instructions for culture. Culture Collections of Algae andProtozoa. Freshwater Biological Association. Ambleside, Cumbria,England, 92–97Schaumberg D. A., Snow K. K. Dana M. R. (1998) The epidemic ofAcanthamoeba keratitis: where do we stand? Cornea 17: 3–10

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