Quantitative Proteomics Reveals Metabolic Reprogramming in Host Cells Induced by Trophozoites and Intermediate Subunit of Gal/GalNAc Lectins from Entamoeba histolytica

Abstract

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.

Keywords: Entamoeba histolytica; Gal/GalNAc lectins; metabolic reprogramming; pyruvate kinase; quantitative proteomics.

FIG 1

Quality control of the mass spectrometry platform and overview of protein expression landscape. (A) Correlations between peak areas of any two samples from four groups. The bottom-left half of the panel represents the pairwise Pearson correlation coefficients of the samples, and the top-right half of the panel depicts the pairwise scatterplots from the same comparison. (B and C) Average for differentially expressed genes (B) and principal-component analysis (C) in the four groups analyzed in quantitative proteomics. Igl control group, Igl-treated group, E. histolytica control group, and E. histolytica-treated group are marked in blue, red, brown, and yellow, respectively.

FIG 2

Quantitative proteomics analysis of Caco2 cells after infection with E. histolytica or stimulation by Igl. Heat map (A) and volcano plots (B) of differentially expressed genes in Caco2 cells stimulated with E. histolytica. Heat map (C) and volcano plots (D) of differentially expressed genes in Caco2 cells stimulated with Igl. Upregulated genes are shown in red, and downregulated genes are shown in green.

FIG 3

Enrichment analysis of altered expressed proteins in Caco2 cells infected with E. histolytica. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed proteins in Caco2 cells infected with E. histolytica, including the enriched clusters of biological process (A), cell components (B), molecular function (C), and KEGG pathways (D). TCA, tricarboxylic acid.

FIG 4

Enrichment analysis of altered expressed proteins in Caco2 cells stimulated with Igl. GO and KEGG enrichment analysis of differentially expressed proteins in Caco2 cells stimulated with Igl, including enriched clusters of biological process (A), cell components (B), molecular function (C), and KEGG pathways (D).

FIG 5

Metabolic reprogramming of host cells induced by E. histolytica trophozoites and purified Igl protein. (A) Bar charts display the expression changes of proteins associated with central carbon metabolism induced by E. histolytica trophozoites (Student’s t test), including pyruvate kinase (PKM), ATP-dependent 6-phosphofructokinase (PFKM), 6-phosphogluconolactonase (PGLS), succinyl-CoA synthetase (SUCLG), phosphoglycerate kinase 1 (PGK1), and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT). (B) Representative immunohistochemical images showing that PKM2 was decreased in intestinal tissue from the mouse model of amoebic colitis. Scale bar, 100 μm. Western blotting showed that PKM2 was decreased in both Caco2 cells (C) and CHO cells (D) after 0- (control), 30-, or 60-min E. histolytica stimulation. (E) Bar charts display the expression changes of proteins associated with central carbon metabolism induced by purified Igl protein (Student’s t test), including PKM, PFKM, PGLS, SUCLG1, PGK1, and DLAT. (F and G) Western blotting showed that PKM2 was decreased in both Caco2 cells (F) and CHO cells (G) after 12- or 24-h Igl stimulation. Control: Caco2 or CHO cells without Igl stimulation.

FIG 6

Representative confocal images of host cells incubated with E. histolytica trophozoites for 30 min. Green, PKM2; red, Igl; blue, DAPI. Scale bar, 10 μm.

FIG 7

Functional analysis of proteins obtained by Igl pulldown assay. (A) KEGG enrichment analysis of proteins obtained by Igl pulldown assay. (B) The top 10 hub genes identified in the protein-protein interaction network.