Figure 1.
Detection of Entamoeba histolytica cysts in Percoll purified cyst samples by immunofluorescence to the Jacob protein.
E. histolytica cysts were purified using the Percoll density gradient centrifugation [26]–[28], and about 50 µL of the purified cysts were fixed in microscopic slides by adding 4% formaldehyde solution for 1 hour at 37°C. After washing with 1× PBS containing 0.1% Triton X-100, cells were treated with either 1∶200 diluted post-immune or pre-immune antisera against E. histolytica cyst-wall specific Jacob protein raised in rabbits. They were then labeled with 1∶200 diluted FITC-conjugated goat anti-rabbit secondary antibody and examined by immunofluorescence microscopy. (a) Anti-Jacob antisera stained the cyst wall (shown by white arrows), (b) while pre-immune antisera failed to stain cyst wall. White bar is approximately 10 microns.
Figure 2.
Total number of E. histolytica, human and other proteins detected in 5 cyst samples.
In 5 cyst samples, the total number of proteins detected by MS/MS analysis ranged from 982 (in sample 4268) to 1725 (in sample AM951). The maximum number of E. histolytica proteins detected in a sample was 167 (sample AM797), while the least number of E. histolytica protein was 75 (sample 4268). The percentages of total proteins belong to E. histolytica are shown inside the bar graphs for each sample, which ranged from 6.9% (for sample CMS33-7133 to 10.2% (for sample AM797).
Figure 3.
Venn diagram for 3-way comparison between the proteins of this study, proteins from the trophozoite proteome and the EST data on trophozoite.
Overlaps between the E. histolytica cyst proteomic data (N = 417), trophozoite proteomic data (N = 1825), and the trophozoite specific EST data (N = 1223) were checked. A total of 191 proteins overlapped between the cyst proteomic and trophozoite proteomic datasets. A total of 109 proteins overlapped between the cyst proteomic and the trophozoite EST datasets. Seventy eight proteins overlapped between all three datasets. A total of 195 proteins of cyst proteomic study did not show any overlaps with other two datasets derived from trophozoites of E. histolytica, suggesting these are E. histolytica cyst-specific proteins.
Figure 4.
Categories of all 417 proteins detected in 5 cyst samples.
E. histolytica proteins that were present in 1 or more cyst preparations fell into functional categories based on their annotations in the database. Out of 417 proteins, 117 did not have known function and are designated as hypothetical proteins. Four E. histolytica cyst-wall specific proteins (such as Jacob, two chitinases and chitin synthetase) were present in 1 or more cyst preparations. A total of 133 various enzymes (mostly protein kinases and GTPases) were also detected. Among other proteins, 18 transcription or translation related proteins (important for understanding of encystation biology), 12 surface associated proteins, 15 ribosomal proteins, 10 protein-protein interacting proteins, 12 vesicle coating or trafficking protein, 10 calcium signal related proteins, and 6 DNA repair proteins are notable.
Figure 5.
Categories of 195 unique cyst specific proteins.
E. histolytica proteins that were present in 1 or more cyst preparations and those that were not found in trophozoite proteome or trophozoite EST datasets were categorized in various functional categories based on their annotations in the database. Out of a total 195 proteins, 77 did not have any known function, and they are designated as hypothetical proteins. Four E. histolytica cyst-wall specific proteins (such as Jacob, 2 chitinases and chitin synthetase) were present in 1 or more cyst preparations. A total of 60 various enzymes (including 19 protein kinases, 10 GTPases, 2 chitinase and a chitin synthetase) were also detected. Among other proteins, 8 vesicle coating or trafficking protein, 4 DNA repair proteins, 5 transcription or translation related proteins, 4 surface associated proteins, are notable.
Table 1.
E. histolytica cyst proteins that show 80% or less identity with E. dispar proteins.
Table 2.
E. histolytica cyst proteins detected in 3 or more cyst samples.
Table 3.
E. histolytica cyst proteins with more than 10 spectrum counts detected in MS/MS experiments.