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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

The rate of endocytosis increased in cells over-expressing Acmcp or Acmcp-dpr versus control cell lines. Data are presented as relative fluorescence to WT, which is considered 100%. (A and B) Significant differences are shown in endocytosis rates. Cells expressing Acmcp and Acmcp–dpr over 120 minutes loaded with RITC- dextran showed significant differences in the rate of endocytic uptake. (C) The vesicles of the early endocytic system from endosome to lysosome were shown in red by visualization of RITC-dextran after 60 minutes of treatment. There does appear to be a difference for RITC-dextran in cell lines that express Acmcp and Acmcp–dpr mutant (indicated by arrows) compared to the controls. (D) Overlaid images of bright field with visualization of red RITC-dextran in Acmcp and Acmcp-dpr mutant cell lines show an increase in uptake of RITC-dextran compared to the WT and pDneo2a-GFP
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Figure 2: The rate of endocytosis increased in cells over-expressing Acmcp or Acmcp-dpr versus control cell lines. Data are presented as relative fluorescence to WT, which is considered 100%. (A and B) Significant differences are shown in endocytosis rates. Cells expressing Acmcp and Acmcp–dpr over 120 minutes loaded with RITC- dextran showed significant differences in the rate of endocytic uptake. (C) The vesicles of the early endocytic system from endosome to lysosome were shown in red by visualization of RITC-dextran after 60 minutes of treatment. There does appear to be a difference for RITC-dextran in cell lines that express Acmcp and Acmcp–dpr mutant (indicated by arrows) compared to the controls. (D) Overlaid images of bright field with visualization of red RITC-dextran in Acmcp and Acmcp-dpr mutant cell lines show an increase in uptake of RITC-dextran compared to the WT and pDneo2a-GFP

Mentions: It was shown that RITC-dextran is good fluid phase marker as it is internalized and does not degrade in Dictyostelium (55). The rates of fluid phase uptake (Fig. 2) were higher compared to that of the control cells. Wild type and mutant cells were fed RITC-dextran in HL5 growth media for over 120 minutes. Measurements of fluorescence intensity of RITC-dextran inside sampled cells were obtained and the data plotted as shown in Figures 2A and B. The level of total fluorescence intensity of the wild type cells was assigned a value of 100% for these assays. The results show that there was a significant difference in the endocytic rates between wild type and mutant cells. Cells over-expressing Acmcp or the Acmcp-dpr mutant had a significantly higher rate of RITC-dextran uptake at times past 15 minutes of feeding (36% and 33%, respectively, vs. the wild type value of 15%), and this trend was magnified until the 2 hour sample point (197% and 170% vs. 70 and 100% for AX4 cells). Compared to the levels of the GFP vector only cell line up-take, the mutants still had a higher level of endocytosis, as well.


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)

The rate of endocytosis increased in cells over-expressing Acmcp or Acmcp-dpr versus control cell lines. Data are presented as relative fluorescence to WT, which is considered 100%. (A and B) Significant differences are shown in endocytosis rates. Cells expressing Acmcp and Acmcp–dpr over 120 minutes loaded with RITC- dextran showed significant differences in the rate of endocytic uptake. (C) The vesicles of the early endocytic system from endosome to lysosome were shown in red by visualization of RITC-dextran after 60 minutes of treatment. There does appear to be a difference for RITC-dextran in cell lines that express Acmcp and Acmcp–dpr mutant (indicated by arrows) compared to the controls. (D) Overlaid images of bright field with visualization of red RITC-dextran in Acmcp and Acmcp-dpr mutant cell lines show an increase in uptake of RITC-dextran compared to the WT and pDneo2a-GFP
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4522297&req=5

Figure 2: The rate of endocytosis increased in cells over-expressing Acmcp or Acmcp-dpr versus control cell lines. Data are presented as relative fluorescence to WT, which is considered 100%. (A and B) Significant differences are shown in endocytosis rates. Cells expressing Acmcp and Acmcp–dpr over 120 minutes loaded with RITC- dextran showed significant differences in the rate of endocytic uptake. (C) The vesicles of the early endocytic system from endosome to lysosome were shown in red by visualization of RITC-dextran after 60 minutes of treatment. There does appear to be a difference for RITC-dextran in cell lines that express Acmcp and Acmcp–dpr mutant (indicated by arrows) compared to the controls. (D) Overlaid images of bright field with visualization of red RITC-dextran in Acmcp and Acmcp-dpr mutant cell lines show an increase in uptake of RITC-dextran compared to the WT and pDneo2a-GFP
Mentions: It was shown that RITC-dextran is good fluid phase marker as it is internalized and does not degrade in Dictyostelium (55). The rates of fluid phase uptake (Fig. 2) were higher compared to that of the control cells. Wild type and mutant cells were fed RITC-dextran in HL5 growth media for over 120 minutes. Measurements of fluorescence intensity of RITC-dextran inside sampled cells were obtained and the data plotted as shown in Figures 2A and B. The level of total fluorescence intensity of the wild type cells was assigned a value of 100% for these assays. The results show that there was a significant difference in the endocytic rates between wild type and mutant cells. Cells over-expressing Acmcp or the Acmcp-dpr mutant had a significantly higher rate of RITC-dextran uptake at times past 15 minutes of feeding (36% and 33%, respectively, vs. the wild type value of 15%), and this trend was magnified until the 2 hour sample point (197% and 170% vs. 70 and 100% for AX4 cells). Compared to the levels of the GFP vector only cell line up-take, the mutants still had a higher level of endocytosis, as well.

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