Plasminogen (PLMG) activation by schistosomes in the presence of tissue plasminogen activator (tPA).

<p>(<b>A</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 60 min) detected in the presence (+) or absence (-) of live schistosomula (1000 parasites per well, n≥5). (<b>B</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 60 min) detected in the absence (white bar) or presence of male (black bar) or female (gray bar) adult schistosomes. In each case, ≥5 adult worms were evaluated individually. (<b>C</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 30 min) detected in the presence of different numbers of male parasites as indicated, n≥5. (<b>D</b>) Plasmin activity (mean OD₄₀₅ value +/- SD; 60 min) detected in the absence (white bar) or presence of individual male <i>S</i>. <i>japonicum</i> (Sj), <i>S</i>. <i>haematobium</i> (Sh) or <i>S</i>. <i>mansoni</i> (Sm), n ≥10. Significant differences from control conditions (reagents themselves without parasites) are denoted by *, p<0.05, and ***, p <0.001.</p

Heterologous expression, purification and kinetics of r<i>Sm</i>Eno.

<p>(<b>A</b>) Heterologous expression of r<i>Sm</i>Eno in <i>E</i>. <i>coli</i> BL21 Star (DE3). Coomassie-stained gel showing SDS-PAGE resolution of lysates of <i>E</i>. <i>coli</i> bacteria harboring pTrcHisB::<i>Sm</i>Eno before (Gel, left lane) or 4 hours after (Gel, right lane) protein expression induction. The arrow indicates r<i>Sm</i>Eno in the induced lane. In western blot analysis of these lysates probed with monoclonal anti-His tag antibody, a prominent ~50 kDa protein (<i>Sm</i>Eno) is detected (Western Blot, anti-His, arrow). Recombinant <i>Sm</i>Eno protein was purified from bacterial lysate by immobilized metal affinity chromatography. A single ~50 kDa pure protein (<i>Sm</i>Eno) is resolved following Coomassie brilliant blue staining of an SDS-PAGE gel and this protein binds anti-ENO antibody, as determined by western blot analysis (right lane, arrow). Positions of migration of molecular mass markers are indicated on the left (kDa). Michaelis-Menten kinetic curve generated using PGA as substrate (<b>B</b>, catalyzing the forward reaction) or using PEP as substrate (<b>C</b>, catalyzing the reverse reaction). The apparent Km and Vmax values shown represent the mean +/- SD of three independent experiments. (<b>D</b>) Recombinant <i>Sm</i>Eno activity in a buffer system covering the pH range 5.5–9.5. Enzymatic activity is maximal at pH 7.5. (<b>E</b>) Impact of divalent ion (Mg²⁺ or Ca²⁺, as indicated) concentration on mean r<i>Sm</i>Eno activity (± SD). The highest activity value (at 100 mM Mg²⁺) was set at 100% and relative activities were calculated and are presented. Significant differences relative to equivalent measurements containing Ca²⁺ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid. (<b>F</b>) r<i>Sm</i>Eno activity in the presence of increasing concentrations of NaF (white bars) compared to its activity in the absence of inhibitors (set at 100%, black bar). Significant differences relative to the untreated control are denoted by *** for p <0.001. (<b>G</b>) Influence of increasing concentrations of mefloquine (MFQ) on r<i>Sm</i>Eno activity (left bars) or on schistosomula lysate enolase activity (right bars). Activity measured in the absence of MFQ was set at 100% and relative activities were calculated. Significant differences relative to the untreated control are denoted by * for p<0.05 and ** for p<0.01. In E-F, bars represent mean relative activity ± SD, n = 3.</p

Recombinant <i>Sm</i>Eno enhances plasminogen (PLMG) activation.

<p><b>(A)</b> Plasmin activity (mean OD₄₀₅ value +/- SD, n = 3) detected in the presence (“+”, right gray bars) or absence (“-”, left gray bars) of r<i>Sm</i>Eno. BSA served as negative control, (white bar). tPA is tissue plasminogen activator <b>(B)</b> Plasmin activity (mean OD₄₀₅ value +/- SD, n = 3) detected in the presence of increasing concentrations of r<i>Sm</i>Eno (black bars) or control protein (BSA) (gray bars) or no protein (white bar). Significant differences from control conditions (reagents themselves without protein) are denoted by ***, p <0.001.</p

Plasminogen (PLMG) interaction with rSmEno and with schistosome lysates (A) by ELISA or (B) by western blotting.

<p>(<b>A)</b> ELISA plates were coated with r<i>Sm</i>Eno (0.5 μg/well) or the indicated parasite extracts (1.0 μg/well each) and wells were incubated with increasing concentrations of PLMG (0–1 μg) in triplicate. As a negative control, some wells were coated with BSA (0.5 μg/well). Anti-PLMG antibody was used to detect PLMG following standard ELISA conditions. The lines represent the mean absorbance values at OD 450 nm (± SD). (<b>B</b>) Detection by western blot of schistosome PLMG-binding proteins (left panel). Lanes contain extracts from males (M), females (F) and schistosomula (S), as well as pure r<i>Sm</i>Eno (E), BSA (“-”, negative control) and commercially-obtained PLMG (“+”, a control for anti-PLMG antibody binding). Multiple bands in the schistosome extracts bind PLMG. The arrow indicates the position of migration of <i>Sm</i>Eno, here revealed to be a PLMG-binder. No binding to the negative control protein (BSA) is seen. The membrane was stripped and re-probed with anti-ENO1 antibody (right panel). A single, prominent ~47-kDa SmEno band is detected (arrow) in the extracts of males (M), females (F) and schistosomula (S) and in the case of purified rSmEno (E). Images are representative of three replicate experiments.</p

Schistosome enolase activity (mean PEP generated (μg/ml) +/- SD).

<p>(<b>A</b>) Enolase activity exhibited by live adult male or adult female worms (individuals) or schistosomula (~1000 parasites/sample) (n ≥ 10 replicates/sample). (<b>B</b>) Enolase activity exhibited by live <i>S</i>. <i>japonicum</i> (Sj), <i>S</i>. <i>haematobium</i> (Sh) and <i>S</i>. <i>mansoni</i> (Sm) adult males after 4 hours, n ≥ 12 replicates/sample. (<b>C</b>) Enolase activity exhibited by live schistosomula (~1,000/sample, black squares) versus total schistosomula lysate (open squares) over 2 hours, n ≥ 5/condition. (<b>D</b>) Enolase activity exhibited by live schistosomula (~1,000/sample, black circles) versus 2 h (open diamond) or 48 h (closed diamond), conditioned medium, n ≥ 5/condition. (<b>E</b>) Effect of varying divalent ion (Mg²⁺ or Ca²⁺) concentration (1, 10 or 50 mM as indicated) on mean relative enolase activity. The highest activity value (in 50 mM Mg²⁺) was set at 100% and activities relative to this are presented (n ≥ 5/condition). Significant differences relative to equivalent measurements containing Ca²⁺ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid.</p

<i>SmEno</i> gene suppression using RNA interference.

<p>(<b>A</b>) Mean level of <i>SmEno</i> gene expression (+/-SD, n = 3) in cultured adult schistosome males (left), females (center) or schistosomula (right) at 72 hours after treatment with control, irrelevant siRNA (“Cont” black bars, set at 100%) or siRNA targeting <i>Sm</i>Eno (“Eno”, white bars), as determined by qRT-PCR. (<b>B</b>) Detection by western blot of <i>Sm</i>Eno protein (top row), in extracts prepared from parasites 72 h after treatment with <i>Sm</i>Eno (Eno) or control (Cont) siRNAs. Diminished levels of <i>Sm</i>Eno protein is seen in the first lane of each group of samples. Western blot analysis detecting a control schistosome protein (lower row) shows roughly equivalent protein amounts per lane. (<b>C</b>) Mean (+/-SD, n = 3) surface <i>Sm</i>Eno enzyme activity in live adult male (left) or female (center) parasites or schistosomula (right) after treatment with control siRNA (“Cont”, black bars) or siRNA targeting <i>Sm</i>Eno (“Eno”, white bars). Significant differences between suppressed compared to control parasites are denoted by ***, p <0.001. (<b>D</b>) Plasmin activity (mean OD₄₀₅ value +/- SD, n = 3) detected in live adult male (left) or female (center) parasites or schistosomula (right) after treatment with control siRNA (“Cont”, black bars) or siRNA targeting <i>Sm</i>Eno (“Eno”, white bars). All conditions contain plasminogen (PLMG), and tissue plasminogen activator (tPA).</p

Immunolocalization of <i>Sm</i>Eno in <i>S</i>. <i>mansoni</i> adult worms and schistosomula.

<p>Indirect immunofluorescent labeling of native <i>Sm</i>Eno protein in sections of (<b>A</b>) an adult male and (<b>B</b>) a whole fixed schistosomulum using polyclonal anti-ENO1 antibody (and secondary anti-rabbit IgG antibody conjugated to Alexa 488 (green)). Enlargements of the areas shown in white boxes in the top row are presented in the middle row. Arrows indicate clear tegumental staining. As a control, secondary antibody alone was used on sections of <b>(C)</b> adult males and <b>(D</b>), whole fixed schistosomula. Scale bars = 100 μm or 50 μm in insets (middle row).</p

Expression profile of <i>SmEno</i> at different stages in the <i>S</i>. <i>mansoni</i> life cycle.

<p>Quantitative RT-PCR data showing relative expression level (mean +/- SD) of <i>SmEno</i> at different stages in the <i>S</i>. <i>mansoni</i> life cycle: eggs, cercariae, schistosomula (7-day cultured larvae), adult female worms, and adult males (set at 100%). Results are representative of two independent experiments. Significant differences between male adult worms and other life stages is denoted by ***, p <0.001.</p

Layout of the alimentary tract.

<p><b>A</b>. Male <i>S. haematobium</i> showing the distribution of black hemozoin pigment that delineates the lumen of the gut caecum (c). The mouth at the base of the oral sucker (os) opens onto a short esophagus (es, arrows) that empties into an initial transverse region of gut (tg, arrowhead). This bifurcates to pass round the testes (te) before reuniting approximately halfway down the body to continue to the extreme posterior where it ends blindly. <b>B</b>. Female <i>S. japonicum</i> from a rabbit with the gut lumen almost completely filled with dark hemozoin pigment. The layout is the same as for the male but with the bifurcated caeca (c) passing first around the egg-filled uterus (ut, arrows) and ovary (o), before uniting to form a single tube completely surrounded by vitelline follicles (inset, higher magnification, esophageal region). <b>C</b>. Confocal image of the anterior of a male <i>S. japonicum</i> from a rabbit host highlighting the esophageal gland (green), revealed by detection of esophageal-specific protein SjMEG-4.1, and the nuclei (false-colored orange) stained by DAPI. The short esophagus is lined with atypical tegument syncytium, the surface of the anterior compartment (a) being corrugated while that in the posterior (p), coincident with the gland, is extended ∼50-fold by thin plate-like extensions. Aggregates of host leucocytes (leu, arrow) are evident in the esophageal lumen. Scale bars: A, 0.75 mm mm; B, 0.5 mm (inset, 0.2 mm); C, 0.1 mm.</p

Pathways for diffusion of nutrients in male and female schistosomes.

<p>Confocal images of stained adult male (left) and female (right) schistosome cross sections at the same magnification (yellow, phalloidin for actin; blue, DAPI for nuclei; green, tegument and gut). The parameter values are the mean of 12 females and 11 males in near perfect cross section. Areas and distances were measured using the polygon line and path tools in the Analyzing Digital Images package from the Lawrence Hall of Science, Berkeley, California (<a href="http://www.globalsystemsscience.org/software/download" target="_blank">http://www.globalsystemsscience.org/software/download</a>). Solid white arrows show diffusion distances from the tegument surface to the midpoint, dotted white arrows those from the gut lumen to the furthest extremity. The gut lumen of the female occupies 9.7% of the cross section, that of the male only 1.9%. Nutrients diffusing from the male gut would have to travel >400 µm to reach the furthest tissues whereas the maximum distance from the female gut is only 85 µm. The distance a nutrient has to diffuse from the tegument surface to the tissue midpoint in both male and female is very similar at ∼40 µm. Central boxes list metabolite classes that are transported across the tegument (upper box, yellow arrows) or via the gastrointestinal tract (lower box, yellow arrows). Metabolites that have been shown experimentally to be transported are listed in the upper group in each box. Those that have been inferred to be transported from proteomic, transcriptomic, or other work are indicated by italics.</p