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. 2015 Apr-Jun;10(2):213-29.

Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein

Entsar Saheb  1 , Wendy Trzyna  2 , Katherine Maringer  3 , John Bush  3
Affiliations

Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein

Entsar Saheb et al. Iran J Parasitol. 2015 Apr-Jun.

Abstract

Background: Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host's immune response. Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein.

Methods: The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr), were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr.

Results: Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings.

Conclusion: Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii.

Keywords: Acanthamoeba; Development; Dictyostelium; Metacaspase; Phagocytosis.

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Figures

Fig. 1:
Fig. 1:
Cells Over-expressing Acmcp in Dictyostelium has higher rates of phagocytosis during a period of 180 minutes as compared to control cell line. (A) Graphical representation with standard error showing differences in RITC latex bead phagocytosis rates in Acmcp (○) and Acmcp-dpr (∆) cell lines over the WT (♦) and pDneo2a-GFP cells (■). Data are presented as relative fluorescence to AX4, which is considered 100%. After 180 minutes, cells over-expressing Acmcpdpr had a high rate of phagocytosis compared to the other cell lines. (B) Overlaid images of bright field with visualization of red bead in Acmcp and Acmcp-dpr mutant cell lines show an increase in bead uptake compared to the WT-AX4 and pDneo2a-GFP, confirming the increased rates of phagocytosis observed in the graphical representation
Fig. 2:
Fig. 2:
The rate of endocytosis increased in cells over-expressing Acmcp or Acmcp-dpr versus control cell lines. Data are presented as relative fluorescence to WT, which is considered 100%. (A and B) Significant differences are shown in endocytosis rates. Cells expressing Acmcp and Acmcp–dpr over 120 minutes loaded with RITC- dextran showed significant differences in the rate of endocytic uptake. (C) The vesicles of the early endocytic system from endosome to lysosome were shown in red by visualization of RITC-dextran after 60 minutes of treatment. There does appear to be a difference for RITC-dextran in cell lines that express Acmcp and Acmcp–dpr mutant (indicated by arrows) compared to the controls. (D) Overlaid images of bright field with visualization of red RITC-dextran in Acmcp and Acmcp-dpr mutant cell lines show an increase in uptake of RITC-dextran compared to the WT and pDneo2a-GFP
Fig. 3:
Fig. 3:
No significant difference detected in the rates of exocytosis or fluid marker recycling in the mutant cell lines as compared to control cells. (A and B) After 180 minutes of accumulation, cells were washed and re-suspended in a growth medium. At the indicated times, the remaining intracellular RITC- dextran was measured. Acmcp and Acmcp-dpr cells did not show a significant change in the exocytosis process. (C) Graphical representation with standard error of RITC- Dextran recycling over 120 minutes showed no significant variation in recycling amongst all cell lines
Fig. 4:
Fig. 4:
Over-expression of the Acmcp-dpr protein in Dictyostelium causes severe defect in developmental process of these transformed cells. (A) Representative photomicrographs of streaming after 8 hours incubation at 22 °C on KK2 plates. WT and pDneo2a-GFP cells show the normal streaming event of Dictyostelium as the cells move to a central location before slug formation. The Acmcp cells showed fewer streaming events compared to the control cells. Acmcp-dpr over-expressing cells failed to stream under the same conditions. (B) Representative series photomicrographs of aggregation territories in absence of nutrition. After incubation for 16 hours, WT cells and pDneo2a-GFP show the normal aggregation event of Dictyostelium as the cells move to a central location before slug formation. Acmcp cells showed wider aggregation compared to the controls. A large quantity of non-aggregating cells was observed in Acmcp-dpr mutants. (C) Developmental representative series after 24 hours at 22°C. WT and pDneo2a-GFP cell lines developed into fruiting bodies in the absence of nutrition. The fruiting bodies of Acmcp expressing cells appear the same as those of the wild type and pDneo2a-GFP. Acmcp-dpr cells failed to develop or form fruiting bodies.
Fig. 5:
Fig. 5:
Measurement of the chemotaxis activity in all cell lines using 5μM cAMP as chemo-attractant. After 4 hours, the Dictyostelium cells moved beyond one field of view on the agarose in a 60 × 15 mm Petri dish. The amoeboid control cells (WT and pDneo2a-GFP) clearly show trails toward the source of cAMP. Cells with Acmcp over-expression showed faster movement, while cells with Acmcp-dpr mutant scarcely moved towards the source of cAMP.
Fig. 6:
Fig. 6:
Cell death quantification in Dictyostelium using flow cytometry analysis. WT, pDneo2a-GFP, Acmcp, and Acmcp-dpr cells were subjected to starvation and cAMP, then incubated with or without DIF for 6 hours. Cells were next stained with 1μg ml−1 Propidium Iodide for 10 minutes. Fluorescent PI-positive cells were quantified using flow cytometry. (A) Dot plot data with side scatter and forward scatter shows the dead cells distinct from living cells. (B) Quantification for DIF treatment cells undergoing apoptosis. Cells over-expressing Acmcp-dpr showed a lower rate of apoptosis compared to the other cell lines.
Fig. 7:
Fig. 7:
Acmcp-dpr over-expressing cells have increased survival ratios and metabolic activity as compared to controls. (A) Survival ratios of the mutant cells after 72 hours of treatment with DIF. Cells over-expressing Acmcp showed lower survival rates while cells over-expressing Acmcp-dpr had a higher survival rate compared to the other cell lines. (B) To measure the metabolic activity, luminescence was recorded 10 minutes after the CellTiter-Glo® reagent was added to cells in a 96-well plate. The luminescent signal from Acmcp-dpr cells was two times greater than the signal from the other cell lines (WT, pDneo2a-GFP, and Acmcp).
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