Characteristics of Mycobacterium tuberculosis PtpA interaction and activity on the alpha subunit of human mitochondrial trifunctional protein, a key enzyme of lipid metabolism

During Mycobacterium tuberculosis (Mtb) infection, the virulence factor PtpA belonging to the protein tyrosine phosphatase family is delivered into the cytosol of the macrophage. PtpA interacts with numerous eukaryotic proteins modulating phagosome maturation, innate immune response, apoptosis, and potentially host-lipid metabolism, as previously reported by our group. In vitro, the human trifunctional protein enzyme (hTFP) is a bona fide PtpA substrate, a key enzyme of mitochondrial β-oxidation of long-chain fatty acids, containing two alpha and two beta subunits arranged in a tetramer structure. Interestingly, it has been described that the alpha subunit of hTFP (ECHA, hTFPα) is no longer detected in mitochondria during macrophage infection with the virulent Mtb H37Rv. To better understand if PtpA could be the bacterial factor responsible for this effect, in the present work, we studied in-depth the PtpA activity and interaction with hTFPα. With this aim, we performed docking and in vitro dephosphorylation assays defining the P-Tyr-271 as the potential target of mycobacterial PtpA, a residue located in the helix-10 of hTFPα, previously described as relevant for its mitochondrial membrane localization and activity. Phylogenetic analysis showed that Tyr-271 is absent in TFPα of bacteria and is present in more complex eukaryotic organisms. These results suggest that this residue is a specific PtpA target, and its phosphorylation state is a way of regulating its subcellular localization. We also showed that phosphorylation of Tyr-271 can be catalyzed by Jak kinase. In addition, we found by molecular dynamics that PtpA and hTFPα form a stable protein complex through the PtpA active site, and we determined the dissociation equilibrium constant. Finally, a detailed study of PtpA interaction with ubiquitin, a reported PtpA activator, showed that additional factors are required to explain a ubiquitin-mediated activation of PtpA. Altogether, our results provide further evidence supporting that PtpA could be the bacterial factor that dephosphorylates hTFPα during infection, potentially affecting its mitochondrial localization or β-oxidation activity

A Family of Diverse Kunitz Inhibitors from Echinococcus granulosus Potentially Involved in Host-Parasite Cross-Talk

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H+-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 – EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (KI*) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection

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

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

Structural analyses of <i>Eg</i>KU-1/<i>Eg</i>KU-4 and <i>Eg</i>KU-3/<i>Eg</i>KU-8.

<p>(A) Cartoon representation of structural models from the <i>Eg</i>KUs and the crystal structure of α-DTX (1DTX) featuring solvent-accessible (> 40 Ų) aromatic (purple), acid (red) and basic (blue) residues. N and C terminal ends are labeled. Note the presence of patches of basic amino acids with close aromatic residues in the models of α-DTX, <i>Eg</i>KU-1 and <i>Eg</i>KU-4. (B) Molecular surface electrostatic representations of the same proteins in the same orientation, highlighting global differences in charge distribution; scale represents charge from positive blue to negative red. (C) Sequence alignment produced with TEXshade [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006169#ppat.1006169.ref082" target="_blank">82</a>] and hand-edited, featuring aromatic, acid and basic residues; those with solvent-accessibilities < 40 Ų are grey shaded. Note that structurally equivalent positions in the <i>Eg</i>KUs and α-DTX are shifted two residues in the primary sequence. The P1 site of serine peptidase inhibitors, located at the center of the antipeptidase loop, is indicated with arrowheads in (A) and (C).</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

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

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

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