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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

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
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Figure 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

Mentions: In D. discoideum, the membrane transport system connects the endo-lysosomal and CV system (48). We previously showed that Acmcp and Acmcp-dpr localize in the CV bladder and have a role in regulating the function of the CV complex in D. discoideum (46). To determine if the over-expression of Acmcp would alter the phagocytosis rates within the cells, Acmcp and Acmcp-dpr over-expressing cell lines and the control cells were subjected to phagocytosis assay using RITC-latex beads for over 180 minutes. The rate of solid particle uptake into the cells was measured at sample time points (Fig. 1A). Over the course of 60 minutes, cells expressing Acmcp had an increased rate of bead uptake (the fluorescence intensity of intracellular RITC-latex bead was 750 nm) which was about 3.5 times higher than the WT, pDneo2a-GFP, and Acmcp-dpr cells (their uptake levels were near 200 nm). Interestingly, after 60 minutes, the control cells (WT and pDneo2a-GFP) showed a decrease in the rate of phagocytosis of RITC-beads, whereas Acmcp cells showed a significant increase in phagocytosis rate over the course of 90 minutes (around 1861 nm), after which the rate began to decrease.


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)

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
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4522297&req=5

Figure 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
Mentions: In D. discoideum, the membrane transport system connects the endo-lysosomal and CV system (48). We previously showed that Acmcp and Acmcp-dpr localize in the CV bladder and have a role in regulating the function of the CV complex in D. discoideum (46). To determine if the over-expression of Acmcp would alter the phagocytosis rates within the cells, Acmcp and Acmcp-dpr over-expressing cell lines and the control cells were subjected to phagocytosis assay using RITC-latex beads for over 180 minutes. The rate of solid particle uptake into the cells was measured at sample time points (Fig. 1A). Over the course of 60 minutes, cells expressing Acmcp had an increased rate of bead uptake (the fluorescence intensity of intracellular RITC-latex bead was 750 nm) which was about 3.5 times higher than the WT, pDneo2a-GFP, and Acmcp-dpr cells (their uptake levels were near 200 nm). Interestingly, after 60 minutes, the control cells (WT and pDneo2a-GFP) showed a decrease in the rate of phagocytosis of RITC-beads, whereas Acmcp cells showed a significant increase in phagocytosis rate over the course of 90 minutes (around 1861 nm), after which the rate began to decrease.

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