Phagocytosis-like Mechanism Research Articles

A hybrid breast cancer/mesenchymal stem cell population enhances chemoresistance and metastasis.

Patients with triple negative breast cancer remain at risk for metastatic disease despite treatment. The acquisition of chemoresistance is a major cause of tumor relapse and death, but the mechanisms are far from understood. We have demonstrated that breast cancer cells (BCCs) can engulf mesenchymal stem/stromal cells (MSCs), leading to enhanced dissemination. Here, we show that clinical samples of primary invasive carcinoma and chemoresistant breast cancer metastasis contain a unique hybrid cancer cell population co-expressing pan-cytokeratin and the MSC marker fibroblast activation protein-alpha. We show that hybrid cells form in primary tumors and that they promote breast cancer metastasis and chemoresistance. Using single cell microfluidics and in vivo models, we found that within the hybrid cell population are polyploid senescent cells that contribute to metastatic dissemination. Our data reveal that WNT5A plays a crucial role in supporting the chemoresistance properties of hybrid cells. Furthermore, we identify that WNT5A mediates hybrid cell formation through a phagocytosis-like mechanism that requires BCC-derived Interleukin 6 and MSC-derived C-C Motif Chemokine Ligand 2. These findings reveal hybrid cell formation as a novel mechanism of chemoresistance and suggest that interrupting this mechanism may be a potential strategy to overcome breast cancer drug resistance.

Interorgan transfer of intact micron-sized lipid droplets to macrophages during the Drosophila immune response

Abstract Macrophages are white blood cells of the immune system in both mammals and Drosophila. In addition to their roles in engulfing and killing microbial invaders by phagocytosis, these cells play important roles in embryonic development, tissue repair, metabolism, and maintenance of homeostasis. Under certain inflammatory conditions, mammalian macrophages accumulate lipids in the form of lipid droplets, including in infection, atherosclerosis, and obesity; however, neither the mechanisms nor purpose of this accumulation are well-understood. We have found that fly macrophages also accumulate lipid droplets, with astonishing speed following bacterial infection: filling up to 30% of cell volume within 3 hrs of infection. We have discovered a completely novel mechanism for this inflammatory lipid accumulation: instead of synthesizing lipids from fatty acids or glucose taken up from the blood, fly macrophages obtain intact, micron-sized lipid droplets from the adipose tissue, through a phagocytosis-like mechanism we term “phagoliposis”. By genetically blocking the metabolism of these lipid droplets, we have discovered some important inflammatory mechanisms fueled by these lipids. Supported by grants from NIH (SC2AI133653)

Abstract P5-06-06: Hybrid cells generated by Mesenchymal Stem/Stromal Cell Engulfment enhance breast cancer metastasis upon Doxorubicin treatment in mouse model

Abstract Introduction: Our previous findings evidenced that BCCs engulf MSCs in clinical samples of breast cancer metastasis. Among the BCC-engulfing MSCs, we observed that some of them retain some markers of MSCs, resulting in the generation of hybrid cancer cell population. However, the mechanisms of hybrid cancer cell formation and the phenotypic features of hybrid cancer cells are still unclear. Here, we tested the hypothesis that hybrid cancer cells may acquire in vivo a dormant phenotype, with an increased survival advantage and chemoresistant features. Method: MSC labeled with DsRED (DsRED-MSC) were cultured with MDA-MB-231 labeled with GFP (GFP-231) to generate a hybrid cell-enriched co-culture. Live imaging microscopy, flow cytometry, cytokine array and western blot were used to characterize the hybrid cancer cells. GFP-231 were incubated with phRodo labeled MSC and subjected to phagocytosis assay. In the co-culture of DsRED-MSC with GFP-231 treated with Doxorubicin (Doxo) or untreated, we analyzed the percentage of DsRED+/GFP+ hybrid population. Hybrid-enriched co-culture or GFP-231 in single culture labeled with firefly luciferase were intracardially injected in NOD/SCID mice and monitored for metastases by bioluminescence imaging (BLI), upon Doxo treatment. After collecting the tissues at necropsy, metastases were identified by GFP fluorescence microscopy. Results: In co-cultures, DsRED+/GFP+ hybrid cells had a higher percentage of Ki-67 low in G1 and polyploidy compared to GFP-231+ cells. Hybrid cancer cells have a distinct cytokine profile than control BCCs with increased levels of senescence-associated secretory phenotype (SASP) factors. Hybrid cancer cell formation occurs through a phagocytosis-like mechanism, which involves WNT5A and MSR1. Doxo treatment increased the percentage of DsRED+/GFP+ hybrid cells, whereas reduced the percentage of GFP-231+ cells. In animal study, we observed a lower qualitative BLI intensity in mice injected with hybrid cell-enriched co-cultures compared to control. Doxo treatment increased the metastatic burden in mice inoculated with hybrid cell-enriched co-cultures compared to untreated Conclusions: MSC engulfment by BCCs results in a hybrid multinucleated cell population. Hybrid cells acquire a dormant phenotype characterized by a higher percentage of Ki-67low cells in G1 and a senescent phenotype compared to controls. Hybrid-cell-enriched co-cultures established less metastasis compared to control in vivo, but became resistant and acquire the ability to form metastasis upon doxo treatment. Citation Format: Giuseppina Augimeri, Maria E. Gonzalez, Daniela Bonofiglio, Sebastiano Andò, Celina G. Kleer. Hybrid cells generated by Mesenchymal Stem/Stromal Cell Engulfment enhance breast cancer metastasis upon Doxorubicin treatment in mouse model [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-06.

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.

Phagocytosis-like cell engulfment by a planctomycete bacterium

Phagocytosis is a key eukaryotic feature, conserved from unicellular protists to animals, that enabled eukaryotes to feed on other organisms. It could also be a driving force behind endosymbiosis, a process by which α-proteobacteria and cyanobacteria evolved into mitochondria and plastids, respectively. Here we describe a planctomycete bacterium, ‘Candidatus Uab amorphum’, which is able to engulf other bacteria and small eukaryotic cells through a phagocytosis-like mechanism. Observations via light and electron microscopy suggest that this bacterium digests prey cells in specific compartments. With the possible exception of a gene encoding an actin-like protein, analysis of the ‘Ca. Uab amorphum’ genomic sequence does not reveal any genes homologous to eukaryotic phagocytosis genes, suggesting that cell engulfment in this microorganism is probably not homologous to eukaryotic phagocytosis. The discovery of this “phagotrophic” bacterium expands our understanding of the cellular complexity of prokaryotes, and may be relevant to the origin of eukaryotic cells.

Gulp1 controls Eph/ephrin trogocytosis and is important for cell rearrangements during development.

Trogocytosis, in which cells nibble away parts of neighboring cells, is an intercellular cannibalism process conserved from protozoa to mammals. Its underlying molecular mechanisms are not well understood and are likely distinct from phagocytosis, a process that clears entire cells. Bi-directional contact repulsion induced by Eph/ephrin signaling involves transfer of membrane patches and full-length Eph/ephrin protein complexes between opposing cells, resembling trogocytosis. Here, we show that the phagocytic adaptor protein Gulp1 regulates EphB/ephrinB trogocytosis to achieve efficient cell rearrangements of cultured cells and during embryonic development. Gulp1 mediates trogocytosis bi-directionally by dynamic engagement with EphB/ephrinB protein clusters in cooperation with the Rac-specific guanine nucleotide exchange factor Tiam2. Ultimately, Gulp1's presence at the Eph/ephrin cluster is a prerequisite for recruiting the endocytic GTPase dynamin. These results suggest that EphB/ephrinB trogocytosis, unlike other trogocytosis events, uses a phagocytosis-like mechanism to achieve efficient membrane scission and engulfment.

Labeling of endothelial cells with magnetic microbeads by angiophagy.

Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism 'angiophagy'. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5min, as compared to gravitational settling (p < 0.0001). We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.

Rac1/WAVE2 and Cdc42/N-WASP Participation in Actin-Dependent Host Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi.

This study evaluated the participation of host cell Rho-family GTPases and their effector proteins in the actin-dependent invasion by Trypanosoma cruzi extracellular amastigotes (EAs). We observed that all proteins were recruited and colocalized with actin at EA invasion sites in live or fixed cells. EA internalization was inhibited in cells depleted in Rac1, N-WASP, and WAVE2. Time-lapse experiments with Rac1, N-WASP and WAVE2 depleted cells revealed that EA internalization kinetics is delayed even though no differences were observed in the proportion of EA-induced actin recruitment in these groups. Overexpression of constitutively active constructs of Rac1 and RhoA altered the morphology of actin recruitments to EA invasion sites. Additionally, EA internalization was increased in cells overexpressing CA-Rac1 but inhibited in cells overexpressing CA-RhoA. WT-Cdc42 expression increased EA internalization, but curiously, CA-Cdc42 inhibited it. Altogether, these results corroborate the hypothesis of EA internalization in non-phagocytic cells by a phagocytosis-like mechanism and present Rac1 as the key Rho-family GTPase in this process.

Engulfment of the midbody remnant after cytokinesis in mammalian cells.

The midbody remnant (MBR) that is generated after cytokinetic abscission has recently attracted a lot of attention, because it might have crucial consequences for cell differentiation and tumorigenesis in mammalian cells. In these cells, it has been reported that the MBR is either released into the extracellular medium or retracted into one of the two daughter cells where it can be degraded by autophagy. Here, we describe a major alternative pathway in a variety of human and mouse immortalized cells, cancer cells and primary stem cells. Using correlative light and scanning electron microscopy and quantitative assays, we found that sequential abscissions on both sides of the midbody generate free MBRs, which are tightly associated with the cell surface through a Ca(2+)/Mg(2+)-dependent receptor. Surprisingly, MBRs move over the cell surface for several hours, before being eventually engulfed by an actin-dependent phagocytosis-like mechanism. Mathematical modeling combined with experimentation further demonstrates that lysosomal activities fully account for the clearance of MBRs after engulfment. This study changes our understanding of how MBRs are inherited and degraded in mammalian cells and suggests a mechanism by which MBRs might signal over long distances between cells.

Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells

Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.

Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes

Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.

Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium

In vitro endocytosis of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE) was studied, the latter being an interesting target for ocular gene therapy. We found that cationic CBA ABOL DNA polyplexes attach to cell surface proteoglycans of these RPE cells and get subsequently internalized via a phagocytosis-like mechanism, as well as Flotillin dependent endocytosis. Introduction Proper delivery of therapeutic genes to designated cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. To this end, typically sub-micron sized particles are made by combining the therapeutic genes with a carrier material, such as cationic polymers, that aid in delivering the genes to the target site. In this work, for the first time, we have evaluated the ability of the highly promising bioreducible polymer carrier cystamin bisacrylamid aminobutanol (CBA ABOL) [1] (Fig. 1) to deliver plasmid DNA in cultured cells of the retinal pigment epithelium (ARPE-19) and characterized in vitro the cellular interactions with these target cells. These studies are of crucial importance since the further design and functionalization of polymeric gene carriers depend strongly on our understanding of the mechanisms involved in cellular adhesion, intracellular uptake and intracellular processing of the polyplexes. Experimental methods ARPE-19 cells (retinal pigment epithelial cell line; ATCC number CRL-2302) were cultured in DMEM:F12 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 2% penicillin-streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. CBA ABOL gene complexes with an average hydrodynamic diameter of 130 nm and an average zeta potential of + 45 mV in 20 mM HEPES buffer were obtained by adding the polymer in a mass ratio of 48/1 in 20 mM HEPES to the plasmid and vortexing the mixture for 10 min. For every transfection, fresh polyplexes were prepared and applied to the cells within 30 min after complexation. For all uptake studies, YOYO-1™ (λex = 491 nm, λem = 509 nm, Molecular Probes, Merelbeke, Belgium) labeled pGL4.13 plasmid (Promega, Leiden, The Netherlands) was used. For all transfection studies, gWiz™GFP plasmid (Aldevron, Freiburg, Germany) was used. siRNAs were all purchased from Dharmacon and transfected in cells with the help of LipofectaminRNAiMAX (Invitrogen, Merelbeke, Belgium). Protein knockdown was assessed on Western Blot. Uptake of polyplexes or endocytic markers or GFP expression was measured on a FACS Calibur Flow Cytometer (Beckton Dickinson, Erembodegem, Belgium). For genetic labeling of endosomes, cells were transfected with GFP-fusion proteins. For fluorescence colocalization studies with GFP labeled cellular structures, cells were transfected with GFP-fusion proteins using Lipofectamin2000 (Invitrogen, Merelbeke, Belgium) and 24 h later exposed to red fluorescent labeled polyplexes. For this, CBA ABOL was complexed with pGL4.13 plasmid, covalently labeled with Cy5 (Label IT Nucleic Acid Labeling Kit, Mirus Bio Corporation, WI, USA). Live cell fluorescence colocalization was then performed on a custom built laser epi-fluorescence microscope set-up. A Nikon Plan Apo VC 100× 1.4 NA oil immersion objective lens (Nikon Belux, Brussels, Belgium) was used for imaging. GFP and Cy5 were excited with 491 nm and 636 nm laser light and emission was detected on an EMCCD camera (Roper Scientific, Nieuwegein, The Netherlands). For live cell imaging the cells were placed in a stage top incubation chamber (Tokai Hit, Shizuoka, Japan), set at 37 °C, 5% CO2 and 100% humidity. Result and discussion First, we found evidence that these net positively charged CBA ABOL polyplexes adhere to the negatively charged heparan sulfate proteoglycans (HSPGs) at the cell surface and that polyplex internalization is blocked by antibodies against Toll-like receptor 9

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.

Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus

Invertebrates harbouring endosymbiotic chemoautotroph bacteria are widely distributed in a variety of reducing marine habitats, including deep-sea hydrothermal vents. In these species mechanisms of symbiont transmission are likely to be key elements of dispersal strategies that remained partially unresolved because the early life stages are not available for developmental studies. To study cessation and re-establishment of symbiosis in the host gill a laboratory experiment was conducted over 45 days in a controlled set-up (LabHorta) that endeavour re-creation of the hydrothermal vent chemical environment. Our animal model was the vent bivalve Bathymodiolus azoricus from the Menez Gwen vent site of the Mid Atlantic Ridge (MAR). Animals were exposed to conditions lacking inorganic S supply for 30 days, which is vital for their symbionts, and then re-acclimatized in sulphide-supplied seawater for an additional 15 days. Gradual disappearance of bacteria from the symbiont-bearing gill cells was observed in animals kept in seawater free of dissolved sulphide for up to 30 days, and was evidenced by histological, ultrastructural observations and Polymerase Chain Reaction tests. Following re-acclimatisation in S-supplied seawater, proliferation of sulphur-bacteria in the gill bacteriocytes confirms the functionality of our sulfide-feeding system in supporting chemoautotrophic symbionts. It may also indicate a horizontal endosymbiont acquisition, i.e. from the environment to the host by means of phagocytosis-like mechanism involving special “pit-like” structures on the apical cell membrane. The present work reports the first laboratory set-up successfully used to maintain the hydrothermal vent bivalve B. azoricus for prolonged periods of time by supplying inorganic sulphur as an energy source for its bacterial endosymbionts. Survival of symbiont bacteria is a critical factor influencing the host physiology and thus the methods reported here represent great potential for future studies of host-symbiont dynamics and for post-capture experimental investigations.

Adhesion of leptospires to mouse fibroblasts (L929) and its enhancement by specific antibody.

The adhesion of leptospires (Leptospira interrogans serovar. copenhageni L45) to mouse L-cells was studied by microscopic observations. Within 3 h of infection of monolayers many leptospires adhered to 95-100% of the cells, and intracellular leptospires were demonstrated by electron microscopy. No specific site of attachment on the cells or the leptospires was observed. Avirulent or dead leptospires adhered poorly but attachment of the saprophytic leptospire L. biflexa serovar. patoc occurred on cell and glass surfaces. After adhesion, microvilli on the cell surfaces disappeared within 6 h of infection and cell damage was observed after 12 h. The adhesion was greatly enhanced by the presence of specific antiserum at a subagglutinating concentration. No direct penetration by leptospires of the host cells was observed with transmission and scanning electron microscopy. It appears that (1) adhesion of leptospires to L-cells precedes cell damage, and (2) leptospires may enter cells either through damaged membranes, or by a phagocytosis-like mechanism.

OBSERVATIONS BY IMMUNOFLUORESCENCE MICROSCOPY AND ELECTRON MICROSCOPY ON THE CYTOPATHOGENICITY OF NAEGLERIA FOWLERI IN MOUSE EMBRYO-CELL CULTURES

The destruction of secondary mouse-embryo (ME) cells by Naegleria fowleri was studied by indirect immunofluorescence with ME-cell antiserum as a specific label to trace the fate of mammalian-cell cytoplasm. The appearance of naegleria-induced cytopathic effect in the cultures coincided with the accumulation of discrete particles containing granules of ME-cell antigen within the cytoplasm of amoebae, suggesting that the organisms ingested host-cell material. In cultures containing cytochalasin B, a non-lethal inhibitor of phagocytosis by N. fowleri trophozoites failed to acquire any granular fluorescence and were not cytopathogenic. The engulfment of mammalian-cell cytoplasm by the organisms was confirmed when thin sections of naegleria-infected ME-cell cultures were examined by electron microscopy. Amoebae were seen in the process of detaching portions of cytoplasm from whole ME cells by means of distinctive ingesting pseudopodia, and fragments of mammalian-cell cytoplasm were identified within the food vacuoles of trophozoites. There was no evidence for cytotoxic disruption of ME cells before or during engulfment of these fragments. It is concluded that N. fowleri trophozoites attack and destroy cultured ME cells by a phagocytosis-like mechanism alone, without the aid of any amoeba-associated cytotoxic or cytolytic agents. The possible significance of these findings with respect to the in-vivo pathocity of N. fowleri is discussed.