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Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

ABSTRACT: Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

SUBMITTER: Min X

PROVIDER: S-EPMC4732598 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

Min Xiangyang X Feng Meng M Guan Yue Y Man Suqin S Fu Yongfeng Y Cheng Xunjia X Tachibana Hiroshi H

PLoS neglected tropical diseases 20160129 1

<h4>Background</h4>Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affect ...[more]

PMID: 26824828

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Project description:Entamoeba histolytica is an intestinal protozoan parasite with remarkable ability to kill and phagocytose host cells, causing amoebic colitis and extraintestinal abscesses. The intermediate subunit (Igl) of galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-specific lectins is considered an important surface antigen involved in the pathogenesis of E. histolytica. Here, we applied mass spectrometry-based quantitative proteomics technology to analyze the protein expression profile changes occurring in host Caco2 cells incubated with E. histolytica trophozoites or stimulated by purified native Igl protein. The expression levels of 1,490 and 489 proteins were significantly altered in the E. histolytica-treated and Igl-treated groups, respectively, among 6,875 proteins totally identified. Intriguingly, central carbon metabolism of host cells was suppressed in both E. histolytica-treated and Igl-treated groups, with evidence of decreased expression levels of several key enzymes, including pyruvate kinase muscle type 2, presenting a Warburg-like effect in host cells. Besides, Igl had potential physical interactions with central carbon metabolism enzymes and the proteolytic degradation family members proteasome subunit alpha and beta, which may be responsible for the degradation of key enzymes in carbon metabolism. These results provided a novel perspective on the pathogenic mechanism of E. histolytica and compelling evidence supporting the important role of Igl in the virulence of E. histolytica. IMPORTANCE Metabolic reprogramming is considered a hallmark of some infectious diseases. However, in amoebiasis, a neglected tropical disease caused by protozoan parasite E. histolytica, metabolic changes in host cells have yet to be proven. In this study, advanced data-independent acquisition mass spectrometry-based quantitative proteomics was applied to investigate the overall host cellular metabolic changes as high-throughput proteomics could measure molecular changes in a cell or tissue with high efficiency. Enrichment analysis of differentially expressed proteins showed biological processes and cellular pathways related to amoeba infection and Igl cytotoxicity. Specifically, central carbon metabolism of host cells was dramatically suppressed in both E. histolytica-treated and Igl-treated groups, indicating the occurrence of a Warburg-like effect induced by trophozoites or Igl from E. histolytica. Distinct differences in ubiquitin-mediated proteolysis, rapamycin (mTOR) signaling pathway, autophagy, endocytosis, and tight junctions provided novel perspectives on the pathogenic mechanism of E. histolytica.

Project description:Adherence of Entamoeba histolytica trophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin, hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 enhanced immunoreactivity (P < 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P < 0.001) and cysteine-rich (P = 0.005) Hgl2 to CHO cells was galactose dependent and competitively inhibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml). The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc(19)BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P < 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (without hgl2 lectin mRNA) bound GalNAc(19)BSA. Binding to GalNAc(19)BSA was shown to be dependent on the concentration of GalNAc(19)BSA coated in each well or (35)S-lectin added (K(D) = 0.85 +/- 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P < 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.

Dataset Information

Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

Publications

Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

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