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Trypanosoma cruzi Prolyl Oligopeptidase Tc80 Is Involved in Nonphagocytic Mammalian Cell Invasion by Trypomastigotes*

Open AccessPublished:December 14, 2001DOI:https://doi.org/10.1074/jbc.M106017200
      Trypanosoma cruzi is an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion.
      ECM
      extracellular matrix
      AMC
      7-amido-4-methylcoumarin
      Boc
      N-tert-butoxycarbonyl
      DAPI
      4′,6-diamidino-2-phenylindol
      E-64
      trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane
      IC50
      concentration inhibiting 50%
      IFA
      immunofluorescence assay(s)
      PBS
      phosphate-buffered saline
      POP
      prolyl oligopeptidase
      Suc
      succinyl
      Z
      benzyloxycarbonyl
      Tic
      tetrahydroisoquinoline carboxylic acid
      TLCK
      N α-p-tosyl-l-lysine chloromethylketone
      Trypanosoma cruzi, a flagellated protozoan, is the etiologic agent of Chagas' disease, a chronic incurable illness prevalent in Latin America (
      • Brener Z.
      ). In vertebrate hosts, T. cruzi transmission is carried out through reduviid bug feces contaminated with metacyclic trypomastigotes that infect cells via mucosa or skin wounds. The parasite can infect and multiply inside a broad range of mammalian tissues or cells. Trypomastigotes penetrate nonphagocytic cells by a nonconventional mechanism involving host cell lysosome recruitment and their fusion at the parasite attachment site to form a parasitophorous vacuole (
      • Tardieux I.
      • Webster P.
      • Ravesloot J.
      • Boron W.
      • Lunn J.A.
      • Heuser J.E.
      • Andrews N.W.
      ). An acid-active membrane pore-forming protein and a neuraminidase activity synergistically promote the escape from the parasitophorous vacuole to the host cell cytoplasm, where it differentiates into the dividing amastigote form (
      • Burleigh B.A.
      • Andrews N.W.
      ). After multiplying, the amastigotes differentiate into trypomastigotes, which are released into the extracellular medium. To have access to different cell types, the trypomastigote must cross the basal laminae (the barrier surrounding organs and blood vessels) and ECM.1 Specific interactions between trypomastigote surface molecules and basal laminae/ECM components, such as collagen (
      • Velge P.
      • Ouaissi M.A.
      • Cornette J.
      • Afchain D.
      • Capron A.
      ,
      • Ortega-Barria E.
      • Pereira M.E.
      ), fibronectin (
      • Ouaissi M.A.
      • Cornette J.
      • Afchain D.
      • Capron A.
      • Gras-Masse H.
      • Tartar A.
      ), and laminin (
      • Giordano R.
      • Fouts D.L.
      • Tewari D.
      • Colli W.
      • Manning J.E.
      • Alves M.J.
      ), have been described, which may facilitate the trypomastigote migration through ECM or its interaction with the host cell membrane. T. cruzi proteases play important roles in the host-parasite relationship. They have been involved in nutritional functions, metacyclogenesis, and host cell invasion (
      • Avila J.L.
      • Bretana A.
      • Casanova M.A.
      • Avila A.
      • Rodriguez F.
      ,
      • Bonaldo M.C.
      • d'Escoffier L.N.
      • Sales J.M.
      • Goldenberg S.
      ,
      • Meirelles M.N.
      • Juliano L.
      • Carmona E.
      • Silva S.G.
      • Costa E.M.
      • Murta A.C.
      • Scharfstein J.
      ,
      • Harth G.
      • Andrews N.W.
      • Mills A.A.
      • Engel J.C.
      • Smith R.
      • McKerrow J.H.
      ). A serine proteinase, called oligopeptidase B, plays a central role in host cell invasion by generating a Ca2+-signaling factor required for the host lysosome recruitment at the invasion site (
      • Burleigh B.A.
      • Andrews N.W.
      ,
      • Burleigh B.A.
      • Caler E.V.
      • Webster P.
      • Andrews N.W.
      ,
      • Caler E.V.
      • Vaena de Avalos S.
      • Haynes P.A.
      • Andrews N.W.
      • Burleigh B.A.
      ). Development of specific inhibitors of T. cruziproteases seems to be a promising approach to novel chemotherapy for Chagas' disease (
      • Engel J.C.
      • Doyle P.S.
      • Hsieh I.
      • McKerrow J.H.
      ).
      We characterized a T. cruzi 80-kDa proteinase (Tc80), secreted by the infective trypomastigote form, that specifically hydrolyzes purified human collagens types I and IV and native collagen in rat mesentery at neutral pH (
      • Santana J.M.
      • Grellier P.
      • Schrével J.
      • Teixeira A.R.L.
      ). By its property of cleaving peptide bonds at the carboxyl side of proline residues, Tc80 proteinase could be classified as prolyl oligopeptidase (POP), a representative of a new serine peptidase family (EC 3.4.21.26) (
      • Barrett A.J.
      ). As POPs, Tc80 proteinase has an unusual inhibitor profile characterized by its inactivation by diisopropyl fluorophosphate but not by the serine protease inhibitor phenylmethylsulfonyl fluoride and its susceptibility to p-chloromercuribenzoate, a cysteine protease inhibitor, indicating the presence of a cysteine residue at or near the active site (
      • Joyeau R.
      • Maoulida C.
      • Guillet C.
      • Frappier F.
      • Teixeira A.R.L.
      • Schrével J.
      • Santana J.
      • Grellier P.
      ). Tc80 proteinase secretion by the trypomastigotes suggests that this enzyme could be involved in the host cell infection by facilitating the parasite migration through ECM or the host cell invasion per se (e.g. by cleaving collagens interacting with integrin receptors). Reversible inhibitors of Tc80 proteinase have been obtained based on 1) the substrate recognition sequence (Leu-Gly-Pro), whose C terminus was modified with the functional groups vinyl sulfone, 2-ketobenzothiazole, or nitrile likely to interact with the active site (
      • Joyeau R.
      • Maoulida C.
      • Guillet C.
      • Frappier F.
      • Teixeira A.R.L.
      • Schrével J.
      • Santana J.
      • Grellier P.
      ); 2) the screening of combinatorial peptide libraries with phenylpropylcarbonyl-l-tetrahydroisoquinoline(Tic)-pyrrolidine (inhibitor 1; see Table II), which was highly efficient in inhibiting the enzyme with an IC50 of 7 nm(
      • Vendeville S.
      • Buisine E.
      • Williard X.
      • Schrével J.
      • Grellier P.
      • Santana J.
      • Sergheraert C.
      ,
      • Vendeville S.
      • Bourel L.
      • Davioud-Charvet E.
      • Grellier P.
      • Deprez B.
      • Sergheraert C.
      ). In this study, we evaluated the specificity of these inhibitors toward Tc80 proteinase and other known T. cruziproteases as well as mammalian POP. Furthermore, irreversible inhibitors were synthesized based on the dipeptide 1, possessing an electrophilic group on pyrrolidine in the P1 position and a proline or a proline mimic (Tic) in the P2 position. The importance of Tc80 proteinase in host cell invasion by the trypomastigotes was investigated by using these inhibitors, and its biological role during this essential step of T. cruzi life cycle is discussed.
      Table IISummary of the inhibition data of POP Tc80 by the irreversible inhibitors 1–4
      Figure thumbnail fx1
      2-a In the presence of 11 μm Suc-GPLGP-AMC as substrate. nd, not determined. The reversible inhibitor 1 was used as the negative control in the inactivation experiments.

      DISCUSSION

      By its biochemical properties (
      • Santana J.M.
      • Grellier P.
      • Schrével J.
      • Teixeira A.R.L.
      ,
      • Joyeau R.
      • Maoulida C.
      • Guillet C.
      • Frappier F.
      • Teixeira A.R.L.
      • Schrével J.
      • Santana J.
      • Grellier P.
      ) and the high similarity of an internal amino acid sequence to sequences containing the Asp residue of the catalytic site of the POP active site (Fig. 1), the Tc80 proteinase of T. cruzi can be classified as a member of the prolyl oligopeptidase family of serine peptidases. POPs are ubiquitous proteases found in various organisms including prokaryotes (
      • Kanatani A.
      • Yoshimoto T.
      • Kitazono A.
      • Kokubo T.
      • Tsuru D.
      ), yeast (
      • Roberts C.J.
      • Pohlig G.
      • Rothman J.H.
      • Stevens T.H.
      ), higher eukaryotes (
      • Rennex D.
      • Hemmings B.A.
      • Hofsteenge J.
      • Stone S.R.
      ), and plants (
      • Yoshimoto T.
      • Sattar A.K.M.A.
      • Hirose W.
      • Tsuru D.
      ). Their biological roles are not well known. POPs seem to be unable to hydrolyze peptides longer than 30 residues (
      • Moriyama A.
      • Nakanishi M.
      • Sasaki M.
      ). Access to its catalytic site is impeded by a tunnel of an unusual β propeller excluding large structured peptides from the active site (
      • Fülöp V.
      • Böcskei Z.
      • Polgàr L.
      ). POPs were proposed to be involved in the metabolism of proline-containing neuropeptides or hormones (
      • Mentlein R.
      ,
      • Yoshimoto T.
      • Kado A.
      • Matsubara F.
      • Koriyama N.
      • Kaneto H.
      • Tsuru D.
      ,
      • Welches W.R.
      • Brosnihan K.B.
      • Ferrario C.M.
      ). However, the substrates of POPs in vivo remain to be identified. In contrast, POP Tc80 specifically hydrolyzes collagens and fibronectin but not other large proteins such as albumin or laminin, or small proteins (insulin or cytochrome c) (Ref.
      • Santana J.M.
      • Grellier P.
      • Schrével J.
      • Teixeira A.R.L.
      ; this study). The ability to hydrolyze specific large molecules suggests that POP Tc80 might show divergence with other POPs.
      POP Tc80 selectivity for specific ECM components and its secretion by the infective parasite form, the trypomastigote, led us to hypothesize that POP Tc80 might be involved in the host cell invasion either by facilitating the parasite progression through ECM or the invasionper se by modifying interactions of ECM components with the host cells. Indeed, T. cruzi interaction with ECM is essential for a successful infection. The parasite must cross the endothelium of blood vessels, the basal laminae, and the extracellular connective medium before invading targeted cells. Receptors for basal laminae or ECM components on the surface of trypomastigotes have been identified and are involved in parasite migration through ECM or parasite attachment to host cells (
      • Velge P.
      • Ouaissi M.A.
      • Cornette J.
      • Afchain D.
      • Capron A.
      ,
      • Ortega-Barria E.
      • Pereira M.E.
      ,
      • Ouaissi M.A.
      • Cornette J.
      • Afchain D.
      • Capron A.
      • Gras-Masse H.
      • Tartar A.
      ,
      • Giordano R.
      • Fouts D.L.
      • Tewari D.
      • Colli W.
      • Manning J.E.
      • Alves M.J.
      ). Proteases secreted by the infective parasite and active on basal laminae or ECM components could then be important during this step of the T. cruzi life cycle. POP Tc80, by its ability to hydrolyze collagen type IV, a basal laminae component, collagen type I, and fibronectin, ECM components, represents a putative candidate for such a purpose.
      To investigate the biological role of POP Tc80, we looked for specific inhibitors (
      • Joyeau R.
      • Maoulida C.
      • Guillet C.
      • Frappier F.
      • Teixeira A.R.L.
      • Schrével J.
      • Santana J.
      • Grellier P.
      ,
      • Vendeville S.
      • Buisine E.
      • Williard X.
      • Schrével J.
      • Grellier P.
      • Santana J.
      • Sergheraert C.
      ,
      • Vendeville S.
      • Bourel L.
      • Davioud-Charvet E.
      • Grellier P.
      • Deprez B.
      • Sergheraert C.
      ). Their selectivity for POP Tc80 was evaluated by comparison with the inhibition of well characterized T. cruzi serine and cysteine proteases (Table I) and the rodent L-6 cell POP. Surprisingly, a moderate selectivity with T. cruzicysteine proteases was measured for the commercial POP inhibitors, Z-P-prolinal dimethylacetal and Boc-NFP-aldehyde (the lowest factors of selectivity were of 28 and 59 for cruzipain, respectively; Table I). The vinyl sulfone derivative based on the POP Tc80 recognition sequence Leu-Gly-Pro was synthesized to react with the cysteine residue in close proximity to the catalytic site as a Michael acceptor (e.g.Cys255 for porcine muscle POP (
      • Fülöp V.
      • Böcskei Z.
      • Polgàr L.
      )). Covalent attachment to this cysteine residue is predicted to inactivate POPs by steric hindrance (
      • Fülöp V.
      • Böcskei Z.
      • Polgàr L.
      ). However, it has a weak inhibition activity on POP Tc80, and no selectivity or a weak selectivity was observed between the serine and cysteine proteases tested. It was, however, the only inhibitor to inhibit significantly the serine oligopeptidase B. This suggests the presence of a crucial cysteine residue close to the catalytic site in the folded enzyme, as observed for POPs. However, no equivalent cysteine residue to Cys255 of porcine POP was noted in T. cruzi oligopeptidase B (
      • Burleigh B.A.
      • Caler E.V.
      • Webster P.
      • Andrews N.W.
      ), in contrast to itsT. brucei homologue (Cys256 (
      • Morty R.E.
      • Lonsdale-Eccles J.D.
      • Morehead J.
      • Caler E.V.
      • Mentele R.
      • Auerswald E.A.
      • Coetzer T.H.
      • Andrews N.W.
      • Burleigh B.A.
      )). Peptidyl α-keto heterocycles were designed to act as mechanism-based inhibitors of serine-proteases, including POPs, by interacting with both the serine hydroxyl group and the histidine imidazole ring of the catalytic triad (
      • Edwards P.D.
      • Meyer E.F.
      • Vijayalakshmi J.
      • Tuthill P.A.
      • Andisik D.A.
      • Gomes B.
      • Strimpler A.
      ). Nitrile derivatives have been reported to exert potent inhibitory effects toward POP (
      • Li J.
      • Wilk E.
      • Wilk S.
      ). By associating both reactive groups with the substrate recognition sequence, highly selective inhibitors for POP Tc80 were obtained (Table I, selectivity ratio >3000). However, no selectivity toward the L-6 cell POP was recorded. In contrast, inhibitor 1 inhibits more efficiently POP Tc80 than the L-6 POP in addition to its strict selectivity for POP Tc80 when compared with the other T. cruzi proteases. Inhibitor1 differs from the classical inhibitors of POP by introduction of nonnatural amino acid Tic instead of proline residue, which confers a high hydrophobicity to the resulting inhibitor (
      • Vendeville S.
      • Bourel L.
      • Davioud-Charvet E.
      • Grellier P.
      • Deprez B.
      • Sergheraert C.
      ). The hydrophobic character could constitute a criterion of specificity between POPs and POP Tc80. Screening of a focused Tic-based library showed, furthermore, a high discrepancy of inhibition of POP Tc80 relative to human POP according to the residue at the subsite P3, supporting divergence between POP Tc80 and POPs.
      S. Vendeville, personal communication.
      Since entry of T. cruzi into a wide variety of cell types is facilitated by the parasite's properties to bind ubiquitous tissue components such as those found in ECM, we looked for the inhibitory effect of POP Tc80 inhibitors on the invasion of adherent nonphagocytic cells. We found that they blocked the invasion process in a dose-dependent manner with an efficiency that reflects their inhibitory effects on the POP Tc80 activity. Preincubation of trypomastigotes with irreversible inhibitors of POP Tc80 also blocked the invasion, indicating that the inhibitors act on parasite components rather than on host cell components. Furthermore, the selective inhibition of the POP Tc80 activity in trypomastigotes pretreated with the irreversible inhibitor (Fig. 8) strongly suggests that the inhibition of the invasion results from an inhibition of the POP Tc80 activity rather from an interaction with other parasite components. However, this remains to be confirmed. Several attempts to modify inhibitors with specific chemical groups (e.g. biotin) that would allow one to identify parasite proteins interacting with the inhibitor were unsuccessful. Such modifications resulted in a dramatic loss of affinity for purified POP Tc80.
      Invasion is an active and complex process whose molecular partners are beginning to be characterized, involving, in particular, heparin-binding protein, fibronectin-binding protein, members of large multigene families includingtrans-sialidase/sialidases and mucins of the parasite, and ECM components, integrins, and cell surface carbohydrates of the mammalian cells (
      • Burleigh B.A.
      • Andrews N.W.
      ). Recent evidence indicates that the invasion process requires early signal transduction events triggered in the host cells as well as in the parasite by parasite-host cell interactions. An increase of intracellular free calcium (
      • Tardieux I.
      • Webster P.
      • Ravesloot J.
      • Boron W.
      • Lunn J.A.
      • Heuser J.E.
      • Andrews N.W.
      ,
      • Moreno S.N.
      • Silva J.
      • Vercesi A.E.
      • Docampo R.
      ) and cyclic AMP (
      • Caler E.V.
      • Morty R.E.
      • Burleigh B.A.
      • Andrews N.W.
      ), tyrosine phosphorylation (
      • Villalta F.
      • Zhang Y.
      • Bibb K.E.
      • Burns J.M.
      • Lima M.F.
      ,
      • Favoreto S.
      • Dorta M.L.
      • Yoshida N.
      ), mitogen-activated protein kinase activation (
      • Villalta F.
      • Zhang Y.
      • Bibb K.E.
      • Burns J.M.
      • Lima M.F.
      ), and transforming growth factor β receptor (
      • Ming M.
      • Ewen M.E.
      • Peireira M.E.A.
      ) are all important in invasion. T. cruzi oligopeptidase B is a key component in the cascade for the generation of a Ca2+-signaling agonist for nonphagocytic mammalian cells required for the recruitment and fusion of host lysosomes that occurs at the parasite attachment site (
      • Caler E.V.
      • Vaena de Avalos S.
      • Haynes P.A.
      • Andrews N.W.
      • Burleigh B.A.
      ). Since POPs are mainly identified as prohormone-processing enzymes, it could be considered that POP Tc80 might be involved in this complex processing cascade. However, trypomastigote extracts treated with 10 μm inhibitor1 or inhibitor 4a, a concentration that totally inhibits POP Tc80 activity, did not inhibit the generation of the transient increase of intracellular free calcium in NRK fibroblasts.
      R. Morty and N. Andrews, personal communication.
      These data rule out POP Tc80 for playing a role in the generation of Ca2+-signaling agonist.
      POP Tc80 localization inside a vesicular compartment surrounding the flagellar pocket, a specialized region of the plasma membrane involved in endocytosis and exocytosis in kinetoplastids, and the fact that POP Tc80 can be released into the extracellular medium by trypomastigotes (
      • Santana J.M.
      • Grellier P.
      • Schrével J.
      • Teixeira A.R.L.
      ) strongly suggest that this vesicular compartment is a part of the secretory pathway utilized by trypanosomes. This transport pathway results in the export of parasite proteins into the flagellar pocket and then into the extracellular medium through its opening. Trypomastigote invasion of nonphagocytic cells is an oriented process (
      • Burleigh B.A.
      • Andrews N.W.
      ); trypomastigotes initiate invasion at their posterior end, the extremity where the flagellar pocket is located. Released POP Tc80 could then have a local action on host cell membranes and ECM components involved in invasion. What could be the POP Tc80 target(s)? According to the specific hydrolysis of collagen and fibronectin by POP Tc80, these ECM components constitute good candidates. A local hydrolysis of these components at the invasion site may be required for the parasite entry. Such hydrolysis could allow a disconnection of the trypomastigote surface proteins from ECM components necessary for penetration. Furthermore, ECM components are connected to the host cell membranes by integrins, and their local hydrolysis by POP Tc80 may trigger a signal that prepares cells for parasite entry (e.g. tyrosine phosphorylation (
      • Villalta F.
      • Zhang Y.
      • Bibb K.E.
      • Burns J.M.
      • Lima M.F.
      ) and cytoskeletal rearrangements (
      • Tardieux I.
      • Webster P.
      • Ravesloot J.
      • Boron W.
      • Lunn J.A.
      • Heuser J.E.
      • Andrews N.W.
      ) that are associated with invasion). However, POP Tc80 action on parasite components cannot be excluded, particularly if taking into account its localization in a vesicular compartment that could be involved in protein export. Since POPs are mainly known as prohormone-processing enzymes, localization in such a compartment is the ideal place for a POP Tc80 involvement in maturation of parasite factors essential for invasion. Further work will be required to investigate the biological role of POP Tc80 during trypomastigote invasion. The specific inhibitors we have developed will be useful tools for that purpose.

      Acknowledgments

      We thank W. Van Voorhis for the generous gift of the β-galactosidase-expressing Tulahuen strain of T. cruzi. We thank M. Dellinger and M. Gèze for assistance and helpful discussion in image acquisition and processing. V. Landry and A. Lemaire are acknowledged for excellent technical assistance. We also thank B. Odaert for considerable help in resolving equations with the use of the nonlinear regression analysis software and fruitful discussion about enzymology. We are very grateful to R. Morty and J. Blum for careful reading of the manuscript.

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