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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

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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Figure 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).

Mentions: ATP is a nucleotide correlates to the intra-cellular energy. When the ATP levels decrease, the cell performs basic apoptotic functions and dies. Our data showed that Acmcp-dpr cell line had less dead cells compare to the other cell lines. To test the metabolic activity of these cells, ATP levels were measured using CellTiter-Glo® Luminescent Cell Viability Assay. Fig. 7A shows that the ATP levels in cells over-expressing Acmcp-dpr were two times greater than the ATP levels in the other cell lines (wild type, pDneo2a-GFP and Acmcp).


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)

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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Related In: Results  -  Collection

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Figure 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).
Mentions: ATP is a nucleotide correlates to the intra-cellular energy. When the ATP levels decrease, the cell performs basic apoptotic functions and dies. Our data showed that Acmcp-dpr cell line had less dead cells compare to the other cell lines. To test the metabolic activity of these cells, ATP levels were measured using CellTiter-Glo® Luminescent Cell Viability Assay. Fig. 7A shows that the ATP levels in cells over-expressing Acmcp-dpr were two times greater than the ATP levels in the other cell lines (wild type, pDneo2a-GFP and Acmcp).

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