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Research Article | Host-Microbe Biology

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

Hannah W. Miller, Rene L. Suleiman, Katherine S. Ralston
Patricia J. Johnson, Editor
Hannah W. Miller
Department of Microbiology and Molecular Genetics, University of California, Davis, California, USA
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Rene L. Suleiman
Department of Microbiology and Molecular Genetics, University of California, Davis, California, USA
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Katherine S. Ralston
Department of Microbiology and Molecular Genetics, University of California, Davis, California, USA
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Patricia J. Johnson
University of California Los Angeles
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DOI: 10.1128/mBio.00068-19
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  • FIG 1
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    FIG 1

    Following interaction with human cells, human cell membrane proteins are displayed by amoebae. (A) Human cell membrane proteins were labeled with biotin prior to coincubation with CMFDA-labeled amoebae. Cells were coincubated for 5 min and immediately fixed. Following fixation, samples were labeled with fluorescently conjugated streptavidin and DAPI. (B) Representative images of amoebae incubated alone or coincubated with biotinylated human cells. Amoebae are shown in green, and streptavidin is shown in red. Nuclei are shown in blue. Arrow indicates a patch of biotin-streptavidin localized to the amoeba surface. (C) Three-dimensional rendering of Z stack images taken from panel B. The arrow indicates transferred biotin. (D) Human cells were labeled with CellTracker deep red (CTDR) prior to coincubation with CMFDA-labeled amoebae. Cells were coincubated for 5 min and immediately fixed. Following fixation, samples were labeled with DAPI and MHC I molecules were detected using immunofluorescence. (E) Representative images of amoebae incubated alone or coincubated with CTDR-labeled human cells. Amoebae are shown in green, human cell cytoplasm is shown in red, MHC I molecules are shown in white, and nuclei are shown in blue. The arrow indicates a patch of MHC I molecules localized to the amoeba surface. (F) Three-dimensional rendering of Z stack images taken from panel E. The arrow indicates transferred MHC I. For panels B to F, images were collected from 4 independent experiments. For biotin experiments, 76 images of amoebae with human cells and 21 images of amoebae alone were collected. For MHC I experiments, 83 images of amoebae with human cells and 40 images of amoebae alone were collected.

  • FIG 2
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    FIG 2

    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.

  • FIG 3
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    FIG 3

    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.

  • FIG 4
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    FIG 4

    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.

  • FIG 5
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    FIG 5

    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.

  • FIG 6
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    FIG 6

    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.

  • FIG 7
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    FIG 7

    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.

  • FIG 8
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    FIG 8

    Proposed model of protection from serum lysis. Amoebae encounter live human cells while invading the intestine or disseminating in the bloodstream and perform trogocytosis. Trogocytosis leads to acquisition and display of human cell membrane proteins on the amoeba surface. One potential mechanism for the acquisition and display of human cell membrane proteins is through fusion of the amoebic and human cell plasma membranes during trogocytosis (during nibble). Human cell proteins might be directly transferred to the amoeba surface through membrane fusion at the site of trogocytosis without being first internalized. Another potential mechanism is through internalization of bites during trogocytosis (after nibble). The ingested membrane proteins might then be trafficked to the amoeba surface. Display of human cell membrane proteins then protects the amoebae from lysis in the blood by inhibiting the complement cascade.

Supplemental Material

  • Figures
  • FIG S1

    Gating strategy used to quantify transferred biotin. Gating strategy used to quantify biotin-positive amoebae. Focused cells were gated from all collected events. Next, single cells were gated, and then single amoebae were gated. Single amoebae were divided into high-Hoechst and low-Hoechst populations to identify images with and without human cell nuclei. Finally, biotin-positive amoebae were gated from images with and without human cell nuclei. Download FIG S1, TIF file, 1.3 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Analysis used to quantify the background level of phagocytosis in trogocytosis assays. The imaging flow cytometry data from the experiments shown in Fig. 2 were used to quantify the level of phagocytosis in trogocytosis assays. CMFDA-labeled control DMSO-treated amoebae were 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) Single amoebae were gated from the total number of cells. Next, biotin-positive amoebae were gated. (B) Human cell nuclei (asterisks) that were surrounded by a biotin/streptavidin ring (arrow) were considered extracellular, while human cell nuclei that lacked a biotin ring were considered internalized. Thus, amoebae that were associated with extracellular human cells were considered phagocytosis negative, while amoebae associated with internalized human cells were considered phagocytosis positive. Amoebae that were biotin positive (arrowhead) without associated human cell nuclei were considered phagocytosis negative. Some amoebae were out of focus or were associated with too many human cells to be reliably scored; thus, these images were left unscored. Representative images of phagocytosis-positive, phagocytosis-negative, and unscored amoebae are shown. (C) Among three independent experiments, the average level of phagocytosis was 3% (range of 2 to 5%). (D) Table showing the raw data for the analysis in panel C. One hundred images each from three separate experiments (300 total scored images, plus unscored images as indicated) were counted. Images were counted independently by two different researchers, and the counts were averaged. Download FIG S2, TIF file, 2.0 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    Optimization of complement assay. The ability of unsupplemented human serum from different vendors to lyse amoebae was tested at various concentrations for 30 min, 1 h, and 2 h at 35°C. Samples were labeled with the viability dye Live/Dead violet and percentages of dead amoebae were determined using imaging flow cytometry. The percentage of dead amoebae was not normalized. (A) Sigma male AB serum. Note that serum was stored at −20°C instead of −80°C. (B) Sigma complement serum human lyophilized powder. (C) Innovative Research pooled normal human complement serum. (D) Valley Biomedical human complement (serum). (E, F) The lysis of increasing concentrations of serum from Innovative Research and Valley Biomedical was tested with the addition of 150 µM CaCl2 and 150 µM MgCl2 for 1 h at 35°C. Download FIG S3, TIF file, 2.0 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S4

    Gating strategy used in the serum lysis assay. Focused cells were gated from all collected events. Next, focused events were divided into gates that contained either debris and human cells or single amoebae. Single amoebae positive for human cells were gated, and then internalization of human cells was measured. The percentage of dead amoebae was gated from single amoebae. Download FIG S4, TIF file, 2.1 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S5

    Nonnormalized data from the serum lysis assay shown in Fig. 3. (A and B) Amoebic lysis was varied and fell into two groups, low lysis (A) and high lysis (B). (C) Lysis from all nonnormalized data. (D) Lysis from all data normalized to the condition with amoebae incubated in the absence of human cells and with exposure to active human serum. Download FIG S5, TIF file, 1.2 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S6

    Centrifugation does not rescue the defect in cytochalasin D-treated amoebae. The experiments shown in Fig. 5 were repeated with the addition of a centrifugation step to force contact between amoebae and human cells at the start of the coincubation. CMFDA-labeled amoebae and DiD-labeled human cells were centrifuged together at 400 × g for 8 min and then coincubated for 1 h, or amoebae were centrifuged and incubated in the absence of human cells as a control. Samples were then exposed to active human serum for 1 h, stained with Live/Dead violet viability dye, and quantitatively analyzed using imaging flow cytometry. Ten thousand images were collected for each sample. (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. The percentages of dead amoebae were normalized to the number of dead amoebae in DMSO-treated, amoeba-alone samples. Eight replicates across 4 independent experiments were performed. Download FIG S6, TIF file, 0.9 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S7

    Internalization of human cells and amoebic death, as well as additional data, from the serum lysis assay shown in Fig. 7. (A) Internalization of human cells by vector control transfectants (white bar) or EhROM1 knockdown mutants (purple bar); (B) percentages of normalized amoeba death under the conditions where amoebae were incubated alone; (C) nonnormalized amoebic death from all conditions. Download FIG S7, TIF file, 0.6 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S8

    (A) Gating strategy used in the trogocytosis and phagocytosis assays shown in Fig. 7. Shown are sample data from a trogocytosis assay, where CMFDA-labeled amoebae were incubated with live DiD-labeled human cells. For phagocytosis assays, CMFDA-labeled amoebae were incubated with CTDR-labeled heat-killed human cells. Focused cells were gated from all collected events. Next, single cells were gated, and then single amoebae were gated. Amoebae positive for human cells were gated, and internalization of human cells was measured. (B) Sample data from a trogocytosis assay, with representative plots from the vector control condition showing internalization of human cells over time as well as representative images collected at each time point. Download FIG S8, TIF file, 1.3 MB.

    Copyright © 2019 Miller et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Trogocytosis by Entamoeba histolytica Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum
Hannah W. Miller, Rene L. Suleiman, Katherine S. Ralston
mBio Apr 2019, 10 (2) e00068-19; DOI: 10.1128/mBio.00068-19

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Trogocytosis by Entamoeba histolytica Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum
Hannah W. Miller, Rene L. Suleiman, Katherine S. Ralston
mBio Apr 2019, 10 (2) e00068-19; DOI: 10.1128/mBio.00068-19
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KEYWORDS

Entamoeba histolytica
trogocytosis
complement
immune evasion
amoebiasis
phagocytosis
endocytosis

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