Proteomic Study of Entamoeba histolytica Trophozoites, Cysts, and Cyst-Like Structures.

The cyst stage of Entamoeba histolytica is a promising therapeutic target against human amoebiasis. Our research team previously reported the production in vitro of Cyst-Like Structures (CLS) sharing structural features with cysts, including rounded shape, size reduction, multinucleation, and the formation of a chitin wall coupled to the overexpression of glucosamine 6-phosphate isomerase, the rate-limiting enzyme of the chitin synthesis pathway. A proteomic study of E. histolytica trophozoites, cysts, and in vitro-produced CLS is reported herein to determine the nature of CLS, widen our knowledge on the cyst stage, and identify possible proteins and pathways involved in the encystment process. Total protein extracts were obtained from E. histolytica trophozoites, CLS, and partially purified cysts recovered from the feces of amoebic human patients; extracts were trypsin-digested and analyzed by LC-MS/MS. In total, 1029 proteins were identified in trophozoites, 550 in CLS, and 411 in cysts, with 539, 299, and 84 proteins unique to each sample, respectively, and only 74 proteins shared by all three stages. About 70% of CLS proteins were shared with trophozoites, even though differences were observed in the relative protein abundance. While trophozoites showed a greater abundance of proteins associated to a metabolically active cell, CLS showed higher expression of proteins related to proteolysis, redox homeostasis, and stress response. In addition, the expression of genes encoding for the cyst wall proteins Jessie and Jacob was detected by RT-PCR and the Jacob protein identified by Western blotting and immunofluorescence in CLS. However, the proteomic profile of cysts as determined by LC-MS/MS was very dissimilar to that of trophozoites and CLS, with almost 40% of hypothetical proteins. Our global results suggest that CLS are more alike to trophozoites than to cysts, and they could be generated as a rapid survival response of trophozoites to a stressful condition, which allows the parasite to survive temporarily inside a chitin-like resistant cover containing Jacob protein. Our findings lead us to suggest that encystment and CLS formation could be distinct stress responses. In addition, we show that cysts express a high number of genes with unknown function, including four new, highly antigenic, possibly membrane-located proteins that could be targets of therapeutic and diagnostic usefulness

Proteins identified in cysts in both studies.

<p>Proteins identified in cysts in both studies.</p

Comparison of proteomic data obtained from <i>E</i>. <i>histolytica</i> trophozoites, cysts and CLS.

<p>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.</p

Validation of proteins identified by RT-PCR.

<p>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.</p

Proteins shared among trophozoites, cysts, and CLS as identified by LC-MS/MS.

<p>Proteins shared among trophozoites, cysts, and CLS as identified by LC-MS/MS.</p

Jacob protein expression in CLS and cyst.

<p>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.</p

List of functional categories, both biological and molecular, identified with Argot2 for hypothetical cyst proteins.

<p>List of functional categories, both biological and molecular, identified with Argot2 for hypothetical cyst proteins.</p

Proteins identified as unique with highest peptide-hit score in trophozoites, CLS, and cysts.

<p>Proteins identified as unique with highest peptide-hit score in trophozoites, CLS, and cysts.</p

RT-PCR of transcripts encoding for cyst wall proteins in trophozoites and CLS.

<p>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.</p