Comparison of NADP-dependent alcohol dehydrogenase activity in lysates of <i>E. dispar</i>, <i>E. histolytica</i> HM-1∶IMSS, and <i>E. histolytica</i> HM-1∶IMSS overexpressing EhADH3 (HAO).

<p>Units/mg represents the enzyme activity (conversion of 1 mmole NADPH/min/mg of lysate protein using ethanol as substrate) within the lysates.</p>1<p>P≤0.004 for the difference in activity between lysates from <i>E. dispar</i> and <i>E. histolytica</i> HM-1∶IMSS.</p>2<p>P<0.001 for the difference in activity between lysates from <i>E. histolytica</i> HM-1∶IMSS and HAO.</p

Live immuofluoresent surface staining of EhADH3 reveals its presence on the plasma membrane surface of <i>E. histolytica</i> HM-1∶IMSS.

<p>Amebae were harvested at 4°C, then blocked with blocking buffer for 10 min prior to staining with rabbit polyclonal anti-EhADH3 antibodies (panels A,B,C) or staining with antibodies pre-incubated with a molar excess of recombinant EhADH3 (panels D,E,F). Panels A and D show staining with the AlexaFlour secondary antibody, panels B and E the brightfield image, and panels C and F are a merge of the two. Magnification 63×.</p

Representative 2D-DIGE gel of <i>E. histolytica</i> and <i>E. dispar</i> lysates demonstrating the extent of differences between species.

<p>One representative gel image of DIGE comparisons between <i>E. histolytica</i> and <i>E. dispar</i> highlights the measured differences in fluorescently labeled protein abundance between these two species. Yellow identifies protein spots that were proportionately higher in <i>E. dispar</i>; blue represents increased abundance in <i>E. histolytica</i>; white represents equal signal. Proteins were identified as follows (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000415#pntd-0000415-t001" target="_blank">Table 1</a>): 1: ADH2- Higher in <i>E. histolytica</i> 6.1×; 2: ADH3- Higher in <i>E. histolytica</i> 5.8×; 3: Grainin 2- Higher in <i>E. dispar</i> 9.6×; 4: LIM domain protein- Higher in <i>E. histolytica</i> 12.6×.</p

Peptide data from observed proteomic differences between <i>E. histolytica</i> and <i>E. dispar</i>.

<p>Peptide data from observed proteomic differences between <i>E. histolytica</i> and <i>E. dispar</i>.</p

2D-DIGE comparison of two <i>E. histolytica</i> HM-1∶IMSS strains.

<p>2852 spots were identified in this gel representing whole cell lysates from two strains of <i>E. histolytica</i> HM-1∶IMSS separately prepared. One was maintained in Saint Louis, Missouri, USA, and the other in London, England. Using a three-fold cutoff, only 6 labeled protein spots were found to fluoresce at different levels, suggesting limited biological variation exists between preparations and isolates. White spots are indicative of identical protein amounts; blue represents increased abundance in the Saint Louis isolate, while yellow represented increased abundance in the London isolate.</p

The gel area and spot representing ADH3 from one representative DIGE gel.

<p>The right panel is a fluorescent intensity scan of <i>E. histolytica</i>; the identical region from <i>E. dispar</i> is on the left. The outlined spot was identified as ADH3 by mass spectrometry. The demarcated region was used to calculate the signal fold difference between the species' ADH3 protein abundance, which was 5.82-fold higher in <i>E. histolytica</i> than <i>E. dispar</i> in this gel.</p

Recombinant EhADH3 prefers straight chain alcohols as a substrate.

<p>An enzymatic substrate preference assay was conducted to determine the optimal substrate for recombinant EhADH3. Butanol was demonstrated to be the preferred substrate, followed by shorter straight-chain alcohols. Branched alcohols were not detectably processed by recombinant EhADH3.</p

Peroxisomal machinery genes in selected organisms.

<p>Black circles indicate the gene is present. Grey circles indicate multiple isoforms.</p

Spliceosomal introns in <i>Blastocystis</i>.

<p>(A) Sequences flanking the predicted exon–intron junctions in subtype (ST) 1 were aligned separately for each intron category and visualized with WebLogo3 (<a href="http://weblogo.threeplusone.com/" target="_blank">http://weblogo.threeplusone.com/</a>). The category and number (<i>N</i>) of each spliceosomal intron type are shown on the right. (B) Distribution of intron size in 3 sequenced <i>Blastocystis</i> ST genomes. Data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003769#pbio.2003769.s032" target="_blank">S11 Data</a>.</p

Randomized Axelerated Maximum Likelihood (RAxML) tree of kinases identified from <i>Blastocystis</i> subtypes (STs) 1, 4, and 7.

<p>Blue lines correspond to proteins from ST1, black from ST4, and red from ST7. Kinase families are indicated on the periphery.</p