UVM Theses and Dissertations
Format:
Print
Author:
Vaithilingam, Archana
Dept./Program:
Cell and Molecular Biology Program
Year:
2011
Degree:
Ph. D.
Abstract:
Entamoeba histolytica is a protozoan parasite that causes amebic dysentery. Following adherence to the target cell, the trophozoites induce apoptosis and eventually phagocytose the apoptotic cell. Because amebic trophozoites can phagocytose apoptotic cells better than healthy or necrotic cells, we hypothesized that there are receptors present on the amebic surface that help in recognition of apoptotic cells. Previous studies in our lab have established arole for C1q, a complement protein, in opsonization of the target apoptotic cell. Clq is present in intestinal secretions and binds to calreticulin on the surface of macrophages and this interaction stimulates phagocytosis of the target cell in macrophages. A proteomic study ofphagosomes isolated from Entamoeba histolytica, identified a large amount of calreticulin in the preparations. Because of the role ofcalreticulin in macrophages as a receptor for C1q, and recovery in phagosomal preparations we hypothesized that calreticulin plays a role as areceptor on the trophozoite surface.
In this study, we have shown native E.histolytica calreticulin present on the surface of the trophozoite at the phagocytic cup both by microscopy and a biochemical approach. Over expression of epitope tagged calreticulin in E.histolytica results in selective translocation of the recombinant protein to the surface only on stimulation with lymphocytes. Also, induced expression of epitope tagged calreticulin, increases the phagocytic ability of trophozoites. Collectively, these results indicate that calreticulin is a potential receptor on the surface of E.histolytica and participates in phagocytosis. We have also shown binding of calreticulin to Clq using several approaches. This binding is competitively inhibited by addition of collectin proteins surfactant protein-A and mannose binding lectin, showing that they share a common mechanism. Mapping studies showed that the C domain of calreticulin, that has calcium binding function, was involved in C1q binding. Surprisingly, induced expression of the C domain also increased adherence and phagocytic ability of the parasites.
In an effort to look at the difference in gene expression between trophozoites that were competent or not to phagocytose CIq beads, we developed a sorting protocol to sort populations based on their phagocytosis ability and did a microarray analysis to identify any differences. 107 genes were upregulated in the phagocytosis positive population. Further characterization of some of these proteins will shed more light on the phagocytosis process in E.histolytica.
In this study, we have shown native E.histolytica calreticulin present on the surface of the trophozoite at the phagocytic cup both by microscopy and a biochemical approach. Over expression of epitope tagged calreticulin in E.histolytica results in selective translocation of the recombinant protein to the surface only on stimulation with lymphocytes. Also, induced expression of epitope tagged calreticulin, increases the phagocytic ability of trophozoites. Collectively, these results indicate that calreticulin is a potential receptor on the surface of E.histolytica and participates in phagocytosis. We have also shown binding of calreticulin to Clq using several approaches. This binding is competitively inhibited by addition of collectin proteins surfactant protein-A and mannose binding lectin, showing that they share a common mechanism. Mapping studies showed that the C domain of calreticulin, that has calcium binding function, was involved in C1q binding. Surprisingly, induced expression of the C domain also increased adherence and phagocytic ability of the parasites.
In an effort to look at the difference in gene expression between trophozoites that were competent or not to phagocytose CIq beads, we developed a sorting protocol to sort populations based on their phagocytosis ability and did a microarray analysis to identify any differences. 107 genes were upregulated in the phagocytosis positive population. Further characterization of some of these proteins will shed more light on the phagocytosis process in E.histolytica.