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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

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
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Figure 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

Mentions: Functional analysis was used to determine the exocyotsis rates (Fig. 3A and B). Cells were pre-loaded with RITC-dextran for 180 minutes. Samples were then taken from between 0 and 180 minutes of chase, and the amount of released (exocytose) RITC-dextran in the growth medium was determined. In the wild type and mutant cells, nearly all of the preloaded RITC-dextran had released by 180 minutes of tracking.


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)

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
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4522297&req=5

Figure 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
Mentions: Functional analysis was used to determine the exocyotsis rates (Fig. 3A and B). Cells were pre-loaded with RITC-dextran for 180 minutes. Samples were then taken from between 0 and 180 minutes of chase, and the amount of released (exocytose) RITC-dextran in the growth medium was determined. In the wild type and mutant cells, nearly all of the preloaded RITC-dextran had released by 180 minutes of tracking.

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