Studies with <i>Eg</i>KU-3 and <i>Eg</i>KU-8 on total K⁺ currents from DRG neurons.

<p>Representative experiments showing that recombinant <i>Eg</i>KU-3 and <i>Eg</i>KU-8 (1 μM) do not block voltage-dependent K⁺ currents elicited by a pulse of -100 to 0 mV during 800 ms (V_h = -60 mV). The superimposed traces correspond to control recordings (black) and records after the perfusion of each <i>Eg</i>KU (red). Positive and negative controls were carried out in parallel, using α-DTX (100 nM) and albumin (15 μM), respectively.</p

Titration assays of recombinant <i>Eg</i>KUs: results for <i>Eg</i>KU-3 and <i>Eg</i>KU-4.

<p>Increasing concentrations of bovine chymotrypsin or trypsin were pre-incubated with fixed amounts of recombinant <i>Eg</i>KU-3 (A) or <i>Eg</i>KU-4 (B), respectively, and mixed with the corresponding enzyme substrate. The plots show the initial steady-state rate of substrate hydrolysis for each enzyme concentration; the activity in the absence of inhibitor is indicated in grey. (A) <i>Eg</i>KU-3 is a high affinity inhibitor of chymotrypsin. Note that the slope at the enzyme concentrations for which activity is detected compares very well with the slope in the absence of inhibitor. The x-intercept of this plot (1.5 nM) represents the enzyme concentration interacting with 1.5 nM of <i>Eg</i>KU-3. Thus, <i>Eg</i>KU-3 inhibits chymotrypsin with a 1:1 stoichiometry. (B) <i>Eg</i>KU-4 is a low affinity inhibitor of trypsin. Note that trypsin activity is detected all over the assayed enzyme range in the presence of an inhibitor concentration 1000-fold higher than the peptidase concentration. Representative results are shown. Experiments with <i>Eg</i>KU-3 and <i>Eg</i>KU-4 were carried out five and two independent times, respectively. Within each experiment, measurements were performed in duplicates.</p

Global inhibition constants (<i>K</i>_I^*) of <i>Eg</i>KU-3, <i>Eg</i>KU-4 and <i>Eg</i>KU-8 acting on pancreatic serine peptidases.

<p>Global inhibition constants (<i>K</i>_I^*) of <i>Eg</i>KU-3, <i>Eg</i>KU-4 and <i>Eg</i>KU-8 acting on pancreatic serine peptidases.</p

Expanded view of the <i>E</i>. <i>granulosus</i> Kunitz family.

<p>Unrooted phylogenetic tree highlighting sequence groupings within the family that roughly correlate with functional features described in the main text. Of outmost notice are sub-clades which include pairs such as the serine peptidase inhibitors <i>Eg</i>KU-3/<i>Eg</i>KU-8 (red clade) and <i>Eg</i>KU-6/<i>Eg</i>KU-7 (green clade); and the channel blockers <i>Eg</i>KU-1/<i>Eg</i>KU-4 (blue clade). Note that the sequences from <i>T</i>. <i>solium</i> pair with their close <i>E</i>. <i>granulosus</i> paralogs. Interestingly, the serine peptidase inhibitors SjKI-1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref037" target="_blank">37</a>], SmKI-1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref038" target="_blank">38</a>] and EGR_07242 (EgKI-2 in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref036" target="_blank">36</a>]) group in the red clade. The sequences from <i>F</i>. <i>hepatica</i> (FhKTM [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref042" target="_blank">42</a>] and FhKT1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref043" target="_blank">43</a>]) define a basal, separate clade that could reflect functional diversity (cysteine peptidase inhibition; [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref043" target="_blank">43</a>]). The long branch of <i>Eg</i>KU-2 (and its putative <i>T</i>. <i>solium</i> ortholog) may reflect either a basal position of the protein (ancient/extreme sequence divergence), an accelerated evolution (<i>e</i>.<i>g</i>. through positive selection) or even relaxed selective pressures resulting in high tolerance to mutation accumulation. Data are insufficient to distinguish between such alternative scenarios. EgrG_001136500, in a black clade to the left, was also found to be a potent serine peptidase inhibitor (EgKI-1 in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref036" target="_blank">36</a>]). The position of the mollusk sequence (Conkunitzin S1), which was characterized as a channel blocker [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref014" target="_blank">14</a>], is probably derived from the fact that, similar to EGR_07242, it lacks Cys14. This artifact is to be expected in short sequences. Bottom scale bar denotes average substitutions per site.</p

Inhibitory kinetics of <i>Eg</i>KU-3 on bovine chymotrypsin A.

<p>Inhibitory kinetics of <i>Eg</i>KU-3 on bovine chymotrypsin A.</p

Detection of <i>Eg</i>KU-3 and <i>Eg</i>KU-8 in parasite secretions.

<p>Analysis by MALDI-TOF MS of hydatid fluid from a bovine cyst (A), as well as of chymotrypsin-affinity purified fractions from the same sample (B) and from the supernatant of cultured immature adults (C). Signals whose m/z values could derive from the <i>Eg</i>KUs are indicated (MH⁺ predicted for mature <i>Eg</i>KU-3 and <i>Eg</i>KU-8 are: 6406.8 and 6520.9, respectively). Note that the signals putatively corresponding to the <i>Eg</i>KUs are significantly enriched in the eluate from the affinity matrix. The identity of <i>Eg</i>KU-3 and <i>Eg</i>KU-8 purified from cyst fluid was subsequently confirmed by peptide mass fingerprinting (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.s001" target="_blank">S1 Dataset</a> and the text for further details), as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref003" target="_blank">3</a>].</p

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels

<div><p>We previously reported a multigene family of monodomain Kunitz proteins from <i>Echinococcus granulosus</i> (<i>Eg</i>KU-1-<i>Eg</i>KU-8), and provided evidence that some <i>Eg</i>KUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the <i>E</i>. <i>granulosus</i> family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the <i>Eg</i>KUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (<i>Eg</i>KU-2-<i>Eg</i>KU-3) or trypsin (<i>Eg</i>KU-5-<i>Eg</i>KU-8). In contrast, the close paralogs <i>Eg</i>KU-1 and <i>Eg</i>KU-4 blocked voltage-dependent potassium channels (K_v); and also pH-dependent sodium channels (ASICs), while showing null (<i>Eg</i>KU-1) or marginal (<i>Eg</i>KU-4) peptidase inhibitory activity. We also confirmed the presence of <i>Eg</i>KUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular <i>Eg</i>KUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.</p></div

Concentration-response analysis of native <i>Eg</i>KU-1 on total K⁺ currents from DRG neurons.

<p>(A) Representative traces showing total K⁺ currents elicited by a voltage pulse of -100 to 0 mV during 1000 ms (as indicated above the current trace) under control conditions, after 1 min perfusion of 200 nM of native <i>Eg</i>KU-1 and after washing. (B) Concentration-response analysis of <i>Eg</i>KU-1 inhibitory effect on K⁺ currents, measured at the end of the voltage pulse, on the steady-state component of the current. The black line shows the best fit to the dose-response equation, from which the IC₅₀ was calculated (216 ± 26 nM). The data correspond to the mean ± standard error (n = 5 in all cases). The asterisks indicate Student’s <i>t</i>-test significance with respect to the effect in the absence of inhibitor (P ≤ 0.05).</p

Concentration-response analysis of native <i>Eg</i>KU-1 on ASIC currents from DRG neurons.

<p>(A) Analysis of native <i>Eg</i>KU-1 inhibitory effect on the ASIC current amplitude (n = 26). The black line shows the best fit to the dose-response equation, from which the IC₅₀ was calculated (7.8 ± 0.7 nM). The data correspond to the mean ± standard error (n ≥ 6 in all cases, except for 1 nM in which n = 4). The asterisks indicate Student’s <i>t</i>-test significance with respect to the effect in the absence of inhibitor (P ≤ 0.05). (B) and (C) correspond to positive and negative controls, respectively. (B) Representative traces showing the acid (pH 6.1, 5 s) activated current under control conditions (left), after sustained (25 s) perfusion of α-DTX (center), and after 1 min washout (right). α-DTX (1 μM; n = 6) significantly decreased the current amplitude (44.5 ± 7.0%; P = 0.045). (C) The application of <i>Eg</i>KU-1 in extracellular solution, without any pH change, had no effect.</p

Inhibition studies with <i>Eg</i>KU-1 and <i>Eg</i>KU-4: results for ASIC currents from DRG neurons.

<p>(A-C) Representative traces showing the acid (pH 6.1, 5 s) activated current under control conditions (left), after sustained (25 s) perfusion of 30 nM of each <i>Eg</i>KU (center) and after 1 min washout of the inhibitors (right). Note that <i>Eg</i>KU-1 and <i>Eg</i>KU-4 reduced the amplitude of the Na⁺ current, that recombinant <i>Eg</i>KU-1 reproduced the effect of the native inhibitor and that the recovery after washout was higher than 90% in all cases. (D-E) Representative traces from analogous assays with 30 nM of <i>Eg</i>KU-3 and <i>Eg</i>KU-8. The slight decrement of the current amplitude induced by <i>Eg</i>KU-3 was significant (see the text for further details); <i>Eg</i>KU-8 had no effect. (F) Albumin (15 μM) was used as negative control. Calibration in each case applies to the control, effect and washout recordings of each panel.</p