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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

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.
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Figure 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.

Mentions: The unexpected effect of the over-expressing Acmcp on phagosomes allowed investigation into the possibility that this mutation may ultimately affect the developmental program of Dictyostelium. This program was triggered by starvation conditions. To address whether the Acmcp may have a role in mediating the developmental pathways, we set up a developmental assay scheme using the wild type and the mutant cell lines. First, to determine the mutation effects on streaming, cells were washed with developmental buffer and spread evenly in a KK2 plate. The cells were then monitored microscopically and photographed after 8 hours. Fig. 4A illustrates that there was normal streaming among the control cells (AX4 and pDneo2a-GFP) and Acmcp cell lines. In the Acmcp-dpr mutant cells, there were little to no streaming cells. This observation suggests that deletion of the proline rich region inhibits the signal transduction and cells fail to stream toward a specific center. Next, to investigate the mutation effects on development behavior, cell aggregation was monitored.


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)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: The unexpected effect of the over-expressing Acmcp on phagosomes allowed investigation into the possibility that this mutation may ultimately affect the developmental program of Dictyostelium. This program was triggered by starvation conditions. To address whether the Acmcp may have a role in mediating the developmental pathways, we set up a developmental assay scheme using the wild type and the mutant cell lines. First, to determine the mutation effects on streaming, cells were washed with developmental buffer and spread evenly in a KK2 plate. The cells were then monitored microscopically and photographed after 8 hours. Fig. 4A illustrates that there was normal streaming among the control cells (AX4 and pDneo2a-GFP) and Acmcp cell lines. In the Acmcp-dpr mutant cells, there were little to no streaming cells. This observation suggests that deletion of the proline rich region inhibits the signal transduction and cells fail to stream toward a specific center. Next, to investigate the mutation effects on development behavior, cell aggregation was monitored.

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