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 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

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

CLS induction and stool samples positive for <i>E</i>. <i>histolytica</i>.

<p>A) Samples used in this study. From left to right: <i>E</i>. <i>histolytica</i> 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 <i>E</i>. <i>histolytica</i> from <i>E</i>. <i>dispar</i>. Samples 1 and 4 were positive to <i>E</i>. <i>dispar</i>, sample 3 to <i>E</i>. <i>histolytica</i> and sample 2 negative. <i>E</i>. <i>histolytica</i> and <i>E</i>. <i>dispar</i> trophozoite DNA were used as positive controls (Eh+ and Ed+).</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

Quantitative multiplexed proteomics of <i>Taenia solium</i> cysts obtained from the skeletal muscle and central nervous system of pigs

<div><p>In human and porcine cysticercosis caused by the tapeworm <i>Taenia solium</i>, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, <i>i</i>.<i>e</i>., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.</p></div