Phagocytosis is reduced in <i>E. histolytica</i> cells over-expressing H644 or EhLimA.

<p>Wild-type (WT) or transgenic cells were incubated with erythrocytes (hRBC:amoeba ratio; 100∶1) for 10 minutes, lysed, and spectrophotometrically analyzed for internalized heme at 405 nm. The data represent the mean ± S.D. of 4 experiments (**<i>P</i><0.01; *<i>P</i><0.05). Amoebae over-expressing H644 or EhLimA exhibit reduced phagocytosis of hRBCs.</p

qPCR confirms over-expression of H644 and EhLimA.

<p>RNA from untransfected (wild-type; WT) log phase <i>E. histolytica</i> trophozoites or from trophozoites transfected with an expression vector encoding H644 or EhLimA was used for qPCR analysis of expression. The ssRNA gene was used as a loading control. H644 and EhLimA were expressed at approximately 3.1 (±0.0)- and 5.6 (±0.4)-fold, respectively, over wild-type levels.</p

Tubercidin-loaded erythrocytes are less toxic to <i>E. histolytic</i> cells over-expressing H644 or EhLimA.

<p>Transgenic trophozoites were exposed to tubercidin-loaded erythrocytes (3 treatments) and viability was assessed 48 hours after treatments. The data are reported as a percent of the starting number of viable amoebae before treatment. The data represent the mean ± S.D of 3 trials. <i>E. histolytica</i> transgenic cells exposed to tubercidin-charged hRBCs displayed increased survival as compared to untransfected wild-type (WT) cells.</p

Tubercidin-loaded erythrocytes are less toxic to an <i>E. histolytica</i> cell line with a phagocytosis defect.

<p>Trophozoites over-expressing a GFP-tagged version of a pleckstrin homology (PH) domain from mammalian Bruton’s tyrosine kinase (GFP-PH^Btk), which exhibit reduced phagocytosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043025#pone.0043025-Byekova1" target="_blank">[22]</a> or a control cell line over-expressing GFP alone (GFP) were exposed to tubercidin-loaded erythrocytes three times and viability was assessed 48 hours after treatment. The data are reported as a percent of the starting number of viable amoebae before treatment. The data represent the mean ± S.D of 3 trials. The phagocytic mutant was insensitive to treatment with tubercidin-charged hRBCs as evidenced by >100% survival (growth) in the presence of selection. This indicates that the selection scheme may be used to enrich for phagocytosis mutants from a population of cells.</p

Cycloheximide is toxic to <i>E. histolytica</i> cells over-expressing H644 or EhLimA.

<p>Transgenic trophozoites were exposed to 100 nM cycloheximide for 48 hours after which viability was assessed. The data are reported as a percent of untreated control amoebae. The data represent the mean ± S.D of 3 trials. Cycloheximide was toxic to both untransfected wild-type (WT) amoebae and <i>E. histolytica</i> transgenic cells. Therefore, cells over-expressing H644 or EhLimA do not exhibit a multidrug resistance phenotype.</p

Fluid-phase endocytosis is not reduced in <i>E. histolytica</i> cells over-expressing H644 or EhLimA.

<p>Wild-type (WT) or transgenic cells were incubated with the fluid-phase marker, FITC-dextran for 30 minutes, lysed, and analyzed for internalized FITC-dextran using spectrofluorimetry. The data represent the mean ± S.D. of ≥3 experiments. Amoebae over-expressing H644 or EhLimA exhibit normal uptake of fluid-phase marker suggesting that these cell lines do not have wide-spread defects in endocytosis.</p

Tubercidin-loaded erythrocytes are toxic to <i>E. histolytica</i>.

<p>Trophozoites were exposed to tubercidin-loaded erythrocytes two or three times (# of Treatments) and viability was assessed 24 hours or 48 hours after treatment. The data are reported as a percent of the starting number of viable amoebae before treatment. The data represent the mean ± S.D of ≥3 trials (***<i>P</i><0.001). Three treatments of <i>E. histolytica</i> cells with tubercidin-charged hRBCs resulted in the death of nearly 100% of the cells 48 hours after application of selection.</p

Isolation of cDNAs from <i>E. histolytica</i> over-expressors without (Control) and with (Selected) selection with tubercidin-charged erythrocytes.

<p>Isolation of cDNAs from <i>E. histolytica</i> over-expressors without (Control) and with (Selected) selection with tubercidin-charged erythrocytes.</p

Schematic of domains found in the hypothetical protein H644 and EhLimA.

<p>A. H644 is a hypothetical protein with a postulated molecular weight of 33.8 kDa (304 amino acids). It is predicted to have a lysine-rich region (blue hexagon), a glycosylation site (gray vertical line), a N-myristoylation site (purple vertical line), 3 casein kinase II phosphorylation sites (red flags labeled C), 5 protein kinase C phosphorylation sites (red flags labeled P), 2 tyrosine kinase phosphorylation sites (red flags labeled Y), and 2 cAMP/cGMP-dependant protein kinase phosphorylation sites (red flags labeled G). B. EhLimA has a molecular weight of 15.9 kDa (145 amino acids). It has an N-terminal LIM domain (orange pentagon), a C-terminal glutamic acid-rich region (green circle), 3 protein kinase C phosphorylation sites (red flags labeled P), and a N-myristoylation site (purple vertical line).</p

<i>eh</i>Dmc1 binds DNA.

<p><b>A.</b> Increasing concentrations of <i>eh</i>Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA (³²P-labeled H3 ssDNA). <b>B.</b> The mean binding percentages were graphed for three independent experiments from <b>A</b>. Error bars represent SEM. <b>C.</b> Increasing concentrations of <i>eh</i>Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA (³²P-labeled H3 annealed to H3c). <b>D.</b> The mean binding percentages were graphed for three independent experiments from <b>C</b>. Error bars represent SEM. Lane 1 for <b>A</b> and <b>C</b> is devoid of protein, and lane 6 for <b>A</b> and <b>C</b> was SDS/PK (S/P) treated containing the highest concentration of <i>eh</i>Dmc1.</p