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

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

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

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

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) 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

<i>Schistosoma mansoni</i> Infection of Mice, Rats and Humans Elicits a Strong Antibody Response to a Limited Number of Reduction-Sensitive Epitopes on Five Major Tegumental Membrane Proteins

<div><p>Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous <i>Schistosoma mansoni</i> worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by <i>S</i>. <i>mansoni</i> infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human <i>S</i>. <i>mansoni</i> infection sera antibodies. We conclude that, <i>Schistosoma mansoni</i> infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.</p></div

Anti-tegumental rat scFvs recognize distinct juvenile and adult <i>S</i>. <i>mansoni</i> antigens when resolved on non-reducing gels.

<p>Total extracts from juvenile or adult mammalian-stage <i>Schistosoma mansoni</i> worms were resolved by SDS-PAGE under non-reducing conditions and transferred to filters. Filters were probed by one of four different anti-tegumental scFvs (Teg1, S3. Teg4 and Teg5, all at 1 μg/ml) previously obtained from <i>S</i>. <i>mansoni</i> infected rats. Detection was by HRP/anti-E-tag recognition of a peptide E-tag expressed with each scFv. Numbers indicate the positions of migration of molecular mass markers (kDa).</p