Article Figures & Data
Figures
- FIG 1
Wortmannin is toxic to E. histolytica. Trophozoites (2 × 104 cells) were exposed to a range of concentrations of wortmannin or its diluent, dimethyl sulfoxide (DMSO), or an equivalent volume of media (control) and grown for 3 days. Cell viability was assessed and expressed as a percentage of untreated control cells. The data represent the means ± SD from 3 trials (***, P < 0.001). Treatment with 2 mM or higher concentrations of wortmannin resulted in the death of nearly 100% of the cells.
- FIG 2
Domains found in EhCoactosin and sequence comparison of coactosin from E. histolytica to those of other species. (A) EhCoactosin is an F-actin binding protein with a postulated molecular mass of 16.2 kDa (148 amino acids). It is predicted to have an actin depolymerization factor homology (ADF-H) domain (orange hexagon), an N-myristoylation site (gray vertical line labeled M), 4 casein kinase II phosphorylation sites (red flags labeled C), and a protein kinase C phosphorylation site (red flag labeled P). (B) The predicted amino acid sequence of EhCoactosin was aligned with coactosin from other organisms using ClustalW multiple sequence alignment, v1.4 (MacVector v9.0). Conserved amino acids are shaded, and the consensus sequence is indicated below the aligned sequence (#, conservation but no consensus). The E. histolytica ADF-H domain is indicated by the red line. Asterisks represent the lysine residues Lys75 and Lys131 of human coactosin that are essential for binding to F-actin and 5-lipoxygenase, respectively.
- FIG 3
RT-qPCR confirms overexpression of EhCoactosin and SREHP. RNA from untransfected (wild type; WT) log-phase E. histolytica trophozoites or from trophozoites transfected with an expression vector encoding SREHP or EhCoactosin was used for RT-qPCR analysis of expression. The ssRNA gene was used as a loading control. SREHP and EhCoactosin were expressed at approximately 3.6 (±0.5)-fold and 7.1 (±1.8)-fold, respectively, above wild-type levels. The data represent the means ± SD from 2 biological replicates and 3 technical replicates per biological replicate (**, P < 0.01; ***, P < 0.001).
- FIG 4
SREHP and EhCoactosin overexpressors are less susceptible to wortmannin and LY294002 toxicity. Transgenic trophozoites were exposed to 1 mM or 2 mM wortmannin (A), 50 μM LY294002 (B), or 25 μM LY294002 (C) for 3 days, after which viability was assessed. The data are reported as a percentage of diluent-treated control amoebae and represent the means ± SD from 3 trials (*, P < 0.05). (A) SREHP- and EhCoactosin-overexpressing cells exhibited increased survival in the presence of wortmannin compared to untransfected wild-type (WT) cells or another transgenic cell line overexpressing an irrelevant gene, EhLimA, from the same expression vector. (B) SREHP-overexpressing cells exhibited enhanced survival in the presence of 50 μM LY294002. (C) EhCoactosin-overexpressing cells exhibited enhanced survival in the presence of 25 μM LY204002.
- FIG 5
Cycloheximide is toxic to E. histolytica cells overexpressing SREHP and EhCoactosin. Transgenic trophozoites were exposed to 100 nM cycloheximide for 48 h, and viability was assessed. The data are presented as a percentage of untreated control cells and represent the means ± SD from 3 trials. Cycloheximide was toxic to both wild-type (WT) and transgenic amoebae. Therefore, cells overexpressing SREHP and EhCoactosin may not have a multidrug resistance phenotype.
- FIG 6
Cells overexpressing EhCoactosin display increased cell motility. A motility assay was performed on EhCoactosin overexpressors or untransfected wild-type (WT) cells. Images of cells moving under agar were captured using confocal microscopy. The maximum distance migrated by lead trophozoites was measured. The data represent the means ± SD from 3 trials (***, P < 0.001).
- FIG 7
GFP-PHBtk overexpressors display altered PI(3,4,5)P3 levels. Phosphoinositides were extracted from whole-cell lysates, and PI(3,4,5)P3 levels were measured using dot blots with antibodies specific to PI(3,4,5)P3. (A) A typical dot blot is shown. (B) Blot densities were analyzed by ImageJ software (version 1.42q). The density for the untransfected wild type (WT) was arbitrarily set to 1, and all other data are reported as a ratio to the WT. The data represent the means ± SD from 3 trials. PI(3,4,5)P3 levels were higher in GFP-PHBtk-expressing cells (*, P < 0.05) than in WT cells or in a control cell line expressing an irrelevant protein, luciferase (Eh209).
- FIG 8
GFP-PHBtk overexpressors possess increased levels of EhCoactosin and SREHP transcripts. RNA from untransfected (wild type; WT) log-phase E. histolytica trophozoites, from a cell line expressing a GFP-tagged PH domain (GFP-PHBtk), and from a cell line expressing an irrelevant protein, luciferase (Eh209), was used for qPCR analysis of expression. The ssRNA gene was used as a loading control. (A) SREHP was expressed in the GFP-PHBtk cell line at a level that was approximately 1.65 (±0.07)-fold (**, P < 0.01) higher than that in WT cells. (B) EhCoactosin was expressed in the GFP-PHBtk cell line at a level that was approximately 3.4 (±0.57)-fold (***, P < 0.001) higher than that in WT levels cells. Neither SREHP nor EhCoactosin was expressed at a higher level in the control cell line (Eh209), suggesting that the increase seen in the GFP-PHBtk cell line was authentic. The data represent the means ± SD from ≥3 biological replicates.
Tables
- TABLE 1
Isolation of cDNAs from E. histolytica overexpressers with and without selection with 2 mM wortmannin
GI no. Gene name No. of isolatesa Control Selected XM_645834.2 Actin binding protein, EhCoactosin 2 13 AY141199.1 Ehapt2 retrotransposon 1 12 XM_648457.2 40S ribosomal protein S14, putative 4 9 XM_646071.2 Ras family GTPase, EhRap2 1 8 XM_647007.2 60S ribosomal protein L10, putative 1 7 XM_645248.2 60S ribosomal protein L7, putative 5 7 XM_647452.2 60S ribosomal protein L17, putative 3 5 M80910.1 SREHP 1 4 XM_650080.1 Alcohol dehydrogenase, putative 7 3 XM_650825.1 40S ribosomal protein S25, putative 4 3 XM_644144.2 Hypothetical protein, H644 6 2 AY141200.1 Ehapt2 retrotransposon 2 2 XM_646441.2 40S ribosomal protein S12, putative 0 2 XM_652064.2 40S ribosomal protein S7, putative 0 1 XM_647340.1 60S ribosomal protein L7a, putative 0 1 X90911.1 Profilin 0 1 XM_651795.2 60S ribosomal protein L7, putative 0 1 XM_650280.2 Hypothetical protein, BAR and SH3 domain-containing protein 0 1 XM_644485.1 40S ribosomal protein S25, putative 1 1 XM_651024.2 60S ribosomal protein L4, putative 1 1 XM_645170.2 Serine protease inhibitor, putative 2 1 XM_644082.2 60S ribosomal protein L14, putative 2 1 XM_645695.2 60S ribosomal protein L13, putative 4 1 XM_644069.2 Enolase, putative 4 1 XM_643252.2 60S ribosomal protein L15, putative 5 1 XM_647370.2 60S ribosomal protein L9 6 1 Z48752.1 Alcohol dehydrogenase 3 6 0 XM_645775.2 Hypothetical protein 9 0 XM_646205.2 60S ribosomal protein L23, putative 1 0 XM_651193.2 40S ribosomal protein S9, putative 1 0 XM_650207.2 C2 domain containing protein 3 0 XM_651611.2 Vacuolar ATP synthase subunit E, putative 1 0 XM_643162.2 Serine-rich protein 1 0 XM_651764.2 60S ribosomal protein L11, putative 1 0 XM_648038.2 Hypothetical protein, mRNA 1 0 XM_001913740.1 40S ribosomal protein S7, putative 1 0 XM_645636.1 Rho GTPase activating protein, putative 1 0 XM_652029.2 Hypothetical protein 1 0 Total 89 90 ↵a E. histolytica overexpressors were left untreated (control) or were subjected to selection with 2 mM wortmannin (selected).
