Trogocytosis by Entamoeba histolytica Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum

Acquisition of human cell membrane proteins is inhibited with cytochalasin D treatment. CMFDA-labeled amoebae were pretreated with either cytochalasin D (Cyto. D) or DMSO (Control) and were then combined with Hoechst labeled human cells. Immediately after coincubation, cells were placed on ice to halt ingestion and stained with fluorescently conjugated streptavidin. Samples were quantitatively analyzed using imaging flow cytometry, with 10,000 images collected for each sample. (A) Gate used to identify single amoebae from total cells. Focused cells were gated on single amoebae using the aspect ratio and intensity of CMFDA fluorescence. (B) Representative plots of images with and without human cell nuclei (high- and low-Hoechst populations) are shown. The high-Hoechst population contained images of amoebae with human cells, and the low-Hoechst population contained images of amoebae without human cells. (C) The overlap of biotin and CMFDA fluorescence was measured, and biotin-positive images were gated. Representative plots of DMSO- and cytochalasin D-treated samples are shown. (D) Quantification of plots from panel B. DMSO-treated samples are shown in blue, and cytochalasin D-treated samples are shown in orange. (E) Quantification of plots from panel C. (F) Representative images of the populations shown in panel C. Amoebae are shown in green, cell nuclei are shown in blue, and biotin is shown in magenta. Arrows indicate patches of transferred biotin. Whole human cells with stained nuclei are marked with asterisks. Six replicates across 3 independent experiments were performed.

Interaction with human cells leads to protection from lysis by human serum. (A) CMFDA-labeled amoebae were incubated alone or in the presence of DiD-labeled human cells for 1 h. Cells were then exposed to either active human serum, heat-inactivated human serum, or M199s medium. Following exposure to serum, samples were stained with Live/Dead violet, and viability was quantified using imaging flow cytometry, with 10,000 images collected for each sample. (B) Representative plots showing internalization of human cells from amoebae incubated with human cells or in the absence of human cells. (C) Representative plots comparing amoebic death from the conditions shown in panel B. (D) Quantification of amoebic death for all experimental conditions. Cells exposed to M199s medium are shown in gray, to heat-inactivated (HI) human serum in red, and to active human serum in blue. Percentages of dead amoebae were normalized to numbers of dead amoebae in the amoeba-alone samples that were treated with active human serum. (E) Representative images of live and dead amoebae from amoebae coincubated with human cells and exposed to active human serum. Amoebae are shown in green, human cell membranes in red, and dead cells in violet. (F) Representative histograms showing the mean fluorescence intensity (MFI) of DiD in live and dead amoebae from samples exposed to human serum. (G) Quantification of the DID MFI shown in panel F. Ten replicates across 5 independent experiments were performed. ns, not significant.

Protection from human serum lysis is dependent on contact with human cells. (A) Depiction of each transwell condition used in panels B to C. CMFDA-labeled amoebae and DiD-labeled human cells were incubated alone, together, or separately under four different transwell conditions. Condition 1, amoebae alone in the lower chamber; condition 2, amoebae and human cells together in the lower chamber; condition 3, human cells in the upper chamber and amoebae in the lower chamber; and condition 4, amoebae and human cells together in the upper chamber and amoebae in the lower chamber. Cells were coincubated in transwells for 1 h, and then cells from the lower chambers were harvested, exposed to human serum, and analyzed. Viability was assessed using Live/Dead violet dye and imaging flow cytometry (B) Quantification of human-cell-positive amoebae under conditions 1 to 4 from panel A. (C) Quantification of amoebic death under conditions 1 to 4. Percentages of dead amoebae were normalized to numbers of dead amoebae under condition 1 (amoebae alone). Ten replicates across 5 independent experiments were performed.

Protection from human serum is actin dependent. CMFDA-labeled amoebae were incubated alone or in the presence of DiD-labeled human cells for 1 h and then exposed to active human serum. Samples were then stained with Live/Dead violet viability dye and analyzed by imaging flow cytometry. (A) Amoebae were either pretreated with cytochalasin D (dark gray) or DMSO (light gray) for 1 h. The internalization of human cells was quantified. (B) The quantification of amoebic death is shown. Percentages of dead amoebae were normalized to the numbers of dead amoebae in the amoeba-alone DMSO-treated samples. Six replicates across 3 independent experiments were performed.

Protection is associated with trogocytosis but not phagocytosis of human cells. (A) Human cells were pretreated with staurosporine (Stauro; dark gray) or DMSO (light gray). The human cell viability before coincubation is shown. (B) Quantification of human cell internalization by amoebae. (C) Quantification of amoebic death. Percentages of dead amoebae were normalized to the numbers of dead amoebae in the amoeba-alone samples. Eight replicates across 4 independent experiments were performed.

EhROM1 knockdown mutants defective in phagocytosis but not trogocytosis are protected from serum lysis. Amoebae were stably transfected with an EhROM1 knockdown plasmid (EhRom1) or a vector control plasmid (Control). (A) Silencing of EhROM1 was verified by using reverse transcriptase (RT) PCR. RT was included (+) or omitted (–) as a control. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used to control for loading. (B) EhROM1 and vector control transfectants were incubated on ice with live human cells for 1 h and then fixed and analyzed using confocal microscopy. The percentage of amoebae with 3 or more attached human cells for each condition is displayed; a vector control is shown with open bars, and the EhROM1 knockdown mutant is shown with blue bars. Four replicates across 2 independent experiments were performed. Twenty images were collected per slide, and 195 to 252 individual amoebae were counted per condition. (C) Representative images from panel B. Amoebae are shown in green, and human cells are shown in red. The arrow indicates an amoeba with a rosette of attached human cells. (D) CMFDA-labeled EhROM1 knockdown mutants (blue circles) or vector control transfectants (open circles) were incubated alone or in the presence of live DiD-labeled human cells for 0, 5, 20, 40, or 80 min. Internalization of human cell material was quantified using imaging flow cytometry. Twenty replicates across 10 independent experiments were performed. (E) CMFDA-labeled EhROM1 knockdown mutants (blue circles) or vector control transfectants (open circles) were incubated alone or in the presence of heat-killed CTDR-labeled human cells for 0, 5, 20, 40, or 80 min. Internalization of human cell material was quantified using imaging flow cytometry. Four replicates across 2 independent experiments were performed. (F) EhROM1 (blue bar) or vector control (open bar) amoebae were coincubated with live human cells for 1 h and then exposed to human serum. Viability was assessed using Live/Dead violet dye and imaging flow cytometry. Percent protection was calculated by subtracting the total lysis of amoebae coincubated with human cells from the total lysis of amoebae incubated alone. Nine to 10 replicates across 5 independent experiments were performed. Protection data are means of results from 2 replicates per experiment from all 5 experiments.