Entamoeba histolytica: Quantitative Proteomics Analysis Reveals Putative Virulence-Associated Differentially Abundant Membrane Proteins

Abstract

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.

Figure 1.

(A) Venn diagram indicating the two sets of proteins that were analyzed independently by LC-MALDI-TOF/TOF and LC-ESI-MS/MS. (B) Membrane protein prediction using TOPCONS on the total identified proteins analyzed by LC-MALDI-TOF/TOF and LC-ESI-MS/MS. LC-ESI-MS/MS = liquid chromatography coupling electrospray ionization tandem mass spectrometry.

Figure 2.

(A) Membrane protein prediction using TOPCONS on total protein identified from cytosolic fraction. (B) Venn diagram indicating the comparison of total identified proteins from membrane fraction and cytosolic fraction.

Figure 3.

(A) Membrane protein prediction using TOPCONS on the differentially abundant proteins that exhibit ≥ 2-fold changes in virulent versus avirulent variants. (B) Differentially abundant membrane proteins identified by LC-MALDI-TOF/TOF and LC-ESI-MS/MS. M1 and M2 represent two replicates detected from LC-MALDI-TOF/TOF and E1 and E2 represent two replicates detected from LC-ESI-MS/MS. (C) Differentially abundant membrane proteins identified by LC-MALDI-TOF/TOF in two technical replicates represented by M1 and M2. (D) Differentially abundant membrane proteins identified by LC-ESI-MS/MS in two technical replicates represented by E1 and E2.

Figure 4.

Radar plot of increased abundance (A) and decreased abundance (B) membrane proteins in virulent versus avirulent variants.

Figure 5.

Pie chart representation of the Protein ANalysis THrough Evolutionary Relationships functional classification of increased abundance membrane proteins in virulent versus avirulent variants according to their molecular function (A), biological process (B), and protein class (C) involvement.

Figure 6.

Pie chart representation of the Protein ANalysis THrough Evolutionary Relationships functional classification of decreased abundance membrane proteins in virulent versus avirulent variants according to their molecular function (A), biological process (B), and protein class (C) involvement.

Figure 7.

Interaction network of differentially abundant membrane proteins generated with STRING. Nodes highlighted with red rings represent increased abundance membrane proteins involved in (A) molecular function and (B) biological process and decreased abundance membrane proteins involved in (C) molecular function and (D) biological process according to gene ontology analysis via Protein ANalysis THrough Evolutionary Relationships. This figure appears in color at www.ajtmh.org.