Determination of Transcripts of the Three Different <i>Ehlgl</i> Genes by RT PCR

<p>Total RNA was prepared from freshly harvested G3 and RBV trophozoites. PCR was performed using antisense conserved primer for <i>Ehlgl1, Ehlgl2, and Ehlgl3</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>, primer XV) and a specific sense primer for each of the genes as indicated. Lanes: 1, primer specific for <i>Ehlgl1</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>, primer XI); 2, primer specific for <i>Ehlgl2</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>, primer XVI); 3, primer specific for <i>Ehlgl3</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>, primer XVII); 4, primers specific for <i>EhRPL21</i> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a> primers II + IX).</p

Silencing of Cysteine Proteinase 5 <i>(EhCP5)</i>

<div><p>(A) Diagram of the pAP-CP5 construct.</p><p>(B) Northern blot analysis. Lanes: 1, RNA from untransfected HM1:IMSS; 2, RNA from untransfected G3; 3, RNA from G3 transfected with pAP-CP5 (probes used are as indicated).</p></div

Induction of Capping of the Gal/GalNAc-Lectin to the Uroid Region of the Trophozoites

<p>Confocal microscopy of trophozoites; left photomicrographs, trophozoites incubated at 4 °C; right photomicrographs, trophozoites were incubated at 37 °C for 20 min to observe the induction of capping using two monoclonal antibodies against the heavy subunit of the Gal-lectin [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-b035" target="_blank">35</a>]. Each image shows the fluorescent Gal-lectin superimposed on a Nomarsky section. Samples are HM-1:IMSS, G3, and RBV (the plasmidless strain silenced in <i>Ehlgl1</i> and <i>Ehap-a</i>).</p

In Vivo Labeling of Cysteine Proteinases

<p>The different trophozoite cultures were grown for 18 h with the radiolabeled cysteine proteinase inhibitor Fmoc-[I¹²⁵]Tyr-Ala-diazomethylketone (10 μg/ml, 10 μCi/ml), harvested, washed, and lysed [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-b049" target="_blank">49</a>]. Samples of each cell lysate (25 μg) were separated on a 12% acrylamide reducing gel and exposed to X-ray film to reveal the different cysteine proteinase bands. Samples in the lanes on the left radiograph are: 1, untransfected HM-1:IMSS; 2, untransfected G3; 3, G3 transfected with pAP-CP5 and grown with 50 μg/ml of G418; 4, RB8 trophozoites after removal of the plasmid (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#s2" target="_blank">Results</a>); 5, HM-1:IMSS grown with a lower specific activity inhibitor (0.05 μCi/μg). Samples on the right radiograph demonstrate the CP-5 band location in HM-1:IMSS and its absence from the E. dispar culture [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-b050" target="_blank">50</a>].</p

Silencing of the <i>Ehlgl1</i> Gene

<div><p>(A) Diagrams of the <i>Ehlgl1</i>-containing plasmids pB33 and pAY. The 473 bp <i>Ehap-a</i> promoter, SINE and T-rich (Tr) regions are marked. Plasmid pAY includes 44 bp of the signal peptide (sp) of the <i>Ehap-a</i> gene. Both plasmids contain the ORF of <i>Ehlgl1</i> gene and the 3′ regulatory sequence from the <i>Ehactin</i> gene.</p><p>(B) Northern blot analysis of amoebic RNA extracts. Lanes: 1, untransfected HM1:IMSS; 2, HM-1:IMSS transfected with plasmid pB33; 3, HM-1:IMSS transfected with pAY; 4, untransfected clone G3; 5, clone G3 transfected with pB33; 6, clone G3 transfected with pAY. Blots were probed as indicated.</p><p>(C) Western blot analysis of protein lysates separated on SDS-PAGE (12%) showing the blot that reacted with the polyclonal antibodies against the Lgl1 protein. Lanes 1–6 are as (B). Lane 7 contains lysate from the plasmidless RBV trophozoites (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#s2" target="_blank">Results</a>).</p></div

Changes in the Transcription of Other Amoebapore-Like Genes in G3 Trophozoites as Determined by RT-PCR

<div><p>(A) Total RNA was prepared from freshly harvested HM1-IMSS and G3 trophozoites. The RNA was treated with RNase-free DNase, and reversed transcribed using oligo dT-adaptor primer (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>, primer I). PCR was then performed using a sense primer from the gene of interest (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>) and the adaptor primer (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020048#ppat-0020048-t001" target="_blank">Table 1</a>, primer II) as antisense for all the genes. Lanes: 1, <i>EhRPL21;</i> 2, <i>Ehap-a;</i> 3, <i>Ehap-b;</i> 4, <i>Ehap-c;</i> 5, <i>SAPLIP 1</i>; 6, <i>SAPLIP 5</i>; 7, <i>SAPLIP 14</i>.</p><p>(B) Sequence comparisons between the three genes that were silenced: I, <i>Ehap-a;</i> II, <i>Ehap-b;</i> III, <i>SAPLIP 1</i>.</p></div

Cell cytotoxicity during interaction between human colonic explants and <i>E. histolytica</i> or <i>E. dispar</i>.

<p>Mean LDH concentrations (IU/L) released into the supernatant of the organotypic culture after incubation with <i>E. histolytica</i> WT or <i>E. dispar</i> or in the absence of amoeba (control) from 1 to 7 hours. Data are from 8 individual experiments. <b>*</b> indicates a significant difference between WT and control (<i>p</i><0.03) and between WT and <i>E. dispar</i> (<i>p</i><0.05). <b>#</b> indicates a significant difference between WT and control (<i>p</i><0.001) and between WT and <i>E. dispar</i> (<i>p</i><0.02)</p

Migration through the lamina propria of <i>E. histolytica</i> sub-strains impaired in virulent functions.

<p>Representative images from three individual experiments are shown. Histological examination of colonic tissue sections after seven hours of incubation, with HGL2, G3, RBV and RB8. Transversal tissue slices were stained with haematoxylin-eosin. Trophozoites were immunostained with antibodies against the Gal/GalNAc lectin. Experiments with HGL2, G3 and RBV revealed that trophozoites were able to invade the mucosa, as described for the WT. In contrast, RB8 parasites were unable to penetrate deeper into the lamina propria and were blocked at the surface of the mucosa, although they were still able to disorganize and detach cells from the upper side of the mucosa.</p

Interaction between Entamoeba and the lumen surface of the human colonic explants.

<p><b>A</b>. Analyse by histology of the mucus layer at the surface of Human colonic fragments incubated for seven hours without <i>Entamoeba</i> (left panel) and with <i>E. histolytica</i> (right panel). The mucus layer covering the epithelium at the surface was observable after seven hours of organotypic culture but not in the presence of <i>E. histolytica</i>. <b>B</b>. Scanning electron micrographs of the luminal surface of the human colonic explants incubated with <i>E. histolytica</i> or <i>E. dispar</i>. Representative images from three individual experiments are shown. (a) <i>E. histolytica</i> trophozoites adhering to the mucus layer at time 0; (b) 2 hours after incubation, the mucus layer had been degraded by <i>E. histolytica</i> and the regular mucosal architecture of the colonic epithelium was visible. Holes corresponded to the crypts of Lieberkühn and abundant aggregates were seen in the interglandular regions. (c) The aggregates were composed of human cells and trophozoites, as seen in an enlargement of this region (d) After 4 hours, the epithelium was damaged and (e) <i>E. histolytica</i> trophozoites began to penetrate into the tissue (f) After 4 hours, <i>E. dispar</i> trophozoites were still adhering to the mucus but had not degraded it and (g) had not evoked the recruitment of cells to the interglandular region, as shown after manually scraping the mucus after SEM fixation procedure of the sample.</p

Pro-inflammatory cytokines secretion induced in the <i>ex-vivo</i> human colonic model by sub-strains of <i>E. histolytica</i>.

<p>Mean concentrations (pg/ml) of IL-1β, IL-8, IFN-γ and TNF secreted after 4 hours (grey bars) and 7 hours (black bars) of incubation of HGL2, NEO, RBV, G3, WT and RB8 trophozoites with 3 individual human colonic explants. NEO and HGL2 parasites induced significantly higher levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IFN-γ and TNF in the explants incubated for 4 hours (<i>p</i><0.05) and 7 hours (<i>p</i><0.02), compared with the control in the absence of amoeba. RBV, G3 and WT strains induced significantly higher levels of pro-inflammatory cytokines [IL-1β (<i>p</i><0.05,<0.01 and <0.01 respectively) IL-8 (<0.04,<0.01 and <0.01 respectively), IFN-γ(<0.009,<0.003 and <0,01 respectively) and TNF (<0.02,<0.02 and <0.02 respectively)] in the explants incubated for 4 hours and IL-8 (<0.009, <0.001 and <0.01 respectively), IFN-γ (<0.002,<0.0004 and <0.01 respectively) and TNF (<0.008,<0.008 and <0.02 respectively)] at 7 hours, compared with the control in the absence of amoeba. WT secreted IL1β (0.04/0.03), IL-8 (0.001/0.001), IFN-γ (0.001/0.002) and TNF (0.008/0.01) at 4 and 7 hours respectively, compared with RB8.</p