Table 1.
Oligonucleotides used for RT-PCR of Entamoeba histolytica cyst wall proteins.
Fig 1.
CLS induction and stool samples positive for E. histolytica.
A) Samples used in this study. From left to right: E. histolytica trophozoites, CLS (induced from trophozoites after a 4 h treatment with H2O2 4 mM), CLS stained with calcofluor white and a cysts partially purified from a fecal sample and stained with Lugol. B) Representative agarose gel of the PCR products differentiating E. histolytica from E. dispar. Samples 1 and 4 were positive to E. dispar, sample 3 to E. histolytica and sample 2 negative. E. histolytica and E. dispar trophozoite DNA were used as positive controls (Eh+ and Ed+).
Fig 2.
Comparison of proteomic data obtained from E. histolytica trophozoites, cysts and CLS.
A) Proteome comparison using the BioVenn software. B) Percentage of annotated and hypothetical proteins identified in each sample. C) Global protein association between samples, labeled as total, annotated, or hypothetical proteins as determined by correlation test. R-values closer to 1 indicate a closer association. T: Trophozoite; CLS: cyst-like structure; C: cyst.
Table 2.
Proteins shared among trophozoites, cysts, and CLS as identified by LC-MS/MS.
Table 3.
Proteins identified as unique with highest peptide-hit score in trophozoites, CLS, and cysts.
Fig 3.
Functional classification of proteins identified in trophozoites, cysts, and CLS total extracts.
The number of proteins (left y-axis) in each of 19 functional groups is shown.
Fig 4.
Comparison of functional categories among trophozoites, CLS and cysts.
A functional comparison among the three cell types is represented in a Venn diagram. Intersections show shared functions among the analyzed samples.
Fig 5.
Validation of proteins identified by RT-PCR.
Five proteins were selected for RT-PCR validation. Left above: malic enzyme. Left middle: F1-6BA, fructose 1,6-bisphosphate aldolase. Left below: GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Right above: Gal/GalNAc lectin LC3 fragment. Right middle: peroxiredoxin. Right below: ARF, ADP-ribosylation factor, used as loading control. T: trophozoite, CLS: cyst-like structure.
Fig 6.
RT-PCR of transcripts encoding for cyst wall proteins in trophozoites and CLS.
RNA was isolated from trophozoites and CLS samples and used to perform a RT-PCR for a number of cyst wall specific proteins: Jessie 1 (J1), Jessie 2 (J2), Jessie 3 (J3), Jacob, and chitin synthase (CS). RT-PCR of the ADP-ribosylation factor (ARF) was used as an internal control. Odd numbers: trophozoites samples; even numbers: CLS samples.
Fig 7.
Jacob protein expression in CLS and cyst.
A)15 μg of sample protein were resolved on 12% acrylamide gel (Coomassie blue-stained) and transferred to a PVDF membrane. Anti-Jacob 1:1000 and a goat HRP-conjugated anti-rabbit 1:50,000 antibodies were used. Unique bands of around 30-kDa and 62-kDa were observed in CLS and cyst samples, respectively, but not in a trophozoites sample. T: Trophozoite, CLS: Cyst-like structure, C: Cyst. B) Immunofluorescence on fixed and permeabilized cyst and CLS using anti-Jacob 1:200 and a goat FITC-conjugated anti-rabbit 1:200 antibodies.
Table 4.
Proteins identified in cysts in both studies.
Table 5.
List of functional categories, both biological and molecular, identified with Argot2 for hypothetical cyst proteins.