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

Saheb E, Trzyna W, Maringer K, Bush J - Iran J Parasitol (2015 Apr-Jun)

Bottom Line: Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations.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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, College of Sciences, University of Baghdad, Baghdad, Iraq.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: The severe defect in Acmcp-dpr cell line development and its inability to respond to the cAMP signal could be due the fact that deletion of the proline rich region from metacaspase protein may affect signal transduction pathway. Starving wild-type Dictyostelium cells and treating them with the differentiation factor (DIF) will cause autophagic cell death (51). To determine if the Acmcp-dpr over-expression altered the cell death process, all cell lines were starved, subjected to DIF treatment and PI staining. Cell death was then quantified. Results of a representative experiment showed that the Acmcp-dpr cell line had less dead cells in response to DIF compared to other cell lines (Fig. 6A).


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

Saheb E, Trzyna W, Maringer K, Bush J - Iran J Parasitol (2015 Apr-Jun)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: The severe defect in Acmcp-dpr cell line development and its inability to respond to the cAMP signal could be due the fact that deletion of the proline rich region from metacaspase protein may affect signal transduction pathway. Starving wild-type Dictyostelium cells and treating them with the differentiation factor (DIF) will cause autophagic cell death (51). To determine if the Acmcp-dpr over-expression altered the cell death process, all cell lines were starved, subjected to DIF treatment and PI staining. Cell death was then quantified. Results of a representative experiment showed that the Acmcp-dpr cell line had less dead cells in response to DIF compared to other cell lines (Fig. 6A).

Bottom Line: Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations.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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, College of Sciences, University of Baghdad, Baghdad, Iraq.

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.

No MeSH data available.


Related in: MedlinePlus