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Characteristics of plasminogen binding to Trypanosoma cruzi epimastigotes
Acta Tropica 107 (2008) 54–58
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Characteristics of plasminogen binding to Trypanosoma cruzi epimastigotes ´ b , Elis Aldana c , Luisana Avilan a,∗ Masyelly Rojas a , Indira Labrador a , Juan L. Concepcion a
Laboratorio de Fisiolog´ıa, Facultad de Ciencias, Universidad de Los Andes, M´erida 5101, Venezuela Laboratorio de Enzimolog´ıa de Par´ asitos, Facultad de Ciencias, Universidad de Los Andes, M´erida 5101, Venezuela c Laboratorio Herman Lentz, Facultad de Ciencias, Universidad de Los Andes, M´erida 5101, Venezuela b
a r t i c l e
i n f o
Article history: Received 4 September 2007 Received in revised form 5 April 2008 Accepted 8 April 2008 Available online 22 April 2008 Keywords: Trypanosoma cruzi Plasminogen Kringles
a b s t r a c t The binding constants of the interaction between plasminogen and Trypanosoma cruzi epimastigotes were determined. An indirect method in which the bound plasminogen is detached from the cell by -aminocaproic acid and a direct method through biotinylated plasminogen were used. The analyses revealed a dissociation constant (Kd ) from 0.4 to 1.2 M, these values being compatible with recognition in vivo. Moreover, epimastigotes from the gut of Rhodnius prolixus were able to bind plasminogen from the blood meal. Fragments derived from elastase digestion of plasminogen were tested for their ability to bind T. cruzi cells. The fragment with highest ability to interact with the parasite was miniplasminogen that bound in a concentration-dependent and saturable manner with a Kd similar to that for plasminogen. This binding was inhibited by -aminocaproic acid indicating that the lysine-binding site of kringle 5 may be responsible for the interaction of plasminogen with T. cruzi. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Plasminogen is the zymogenic form of the serine protease plasmin and is found in plasma and interstitial fluids. This protein is well known for its participation in fibrinolysis (Cesarman-Maus and Hajjar, 2005). However, several other functions have been attributed to this molecule such as extracellular matrix degradation and cell migration (Syrovets and Simmet, 2004). Due to its interaction with an increasing number of pathogenic organisms both prokaryotes and eukaryotes, plasminogen has also been suggested to be involved in infectious processes (Boyle and Lottenberg, 1997; ¨ ¨ et al., 2005; Sun, 2005). Coleman and Benach, 1999; Lahteenm aki Indeed, in several pathogens it has been demonstrated that plasminogen bound on their surface facilitates the establishment of infection within the host (Sun et al., 2004; Coleman et al., 1997). Trypanosoma cruzi, that belongs to the family of trypanosomatidae and is the causative agent of Chagas disease, is one of the pathogens capable of binding plasminogen (Almeida et al., 2004). This binding was previously observed for metacyclic trypomastigotes and epimastigotes. Binding to epimastigotes is not well understood since this form is found in the insect vector. However, in the case of Borrelia burdorgferi, it has been demonstrated that binding of plasminogen is required for dissemination in both ticks and mammals
∗ Corresponding author at: Dept. Biolog´ıa, Facultad de Ciencias, Universidad de ´ Los Andes, Merida 5101, Venezuela. Tel.: +58 274 2401307; fax: +58 274 2401286. E-mail address: [email protected] (L. Avilan). 0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.04.013
(Coleman et al., 1997). Thus one may hypothesize that in the case of T. cruzi, plasminogen might be used in the vector. To determine the function of the interaction between T. cruzi and plasminogen, a better characterization of this interaction is needed. Here, we determined the binding constants of this interaction to assess whether it is compatible with recognition in vivo. In addition, we attempted to localize the region in the plasminogen molecule that is recognized by T. cruzi. The plasminogen molecule has five substructures called kringles some of which (K1, K4 and K5) are implicated in the recognition of fibrin, and cell surfaces (Ponting et al., 1992). Plasminogen can be digested by elastase yielding three fragments: K1–K2–K3, K4 and the K5-catalytic site (miniplasminogen). These plasminogen fragments were tested for their capacity to interact with T. cruzi. The study with these fragments is of value in elucidating the mechanism of binding on the parasite. 2. Materials and methods 2.1. Parasites, insects and T. cruzi infection Epimastigotes of the T. cruzi EP strain were used in the present study. The parasites were grown in liver-infusion tryptose (LIT) medium, supplemented with 10% heat-inactivated fetal bovine serum, at 28 ◦ C with agitation. The parasites harvested during the exponential phase were washed three times with phosphatebuffered saline (PBS). Rhodnius prolixus (Heteroptera: Triatominae) were from a colony maintained in the Herman Lentz Laboratory at the Universidad de Los Andes, Venezuela. For infection, a group of
M. Rojas et al. / Acta Tropica 107 (2008) 54–58
insects (3rd to 5th instar nymphs) were fed with LIT medium containing 1 × 108 epimastigotes/ml of T. cruzi strain EP. After infection the insects were maintained in the laboratory by feeding on mice. These insects were used here as adults after a month of starvation. 2.2. Proteins Plasminogen was purified from human blood as reported previously (Deutsch and Mertz, 1970). Polyclonal plasminogen antibodies were prepared in the laboratory as described elsewhere ˜ (Quinones et al., 2007). IgG fractions were purified using ammonium sulfate precipitation and DEAE-chromatography (Harlow and Lane, 1988).
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To determine the specific binding activity of the plasminogen fragments, T. cruzi parasites in PBS, were immobilized overnight in microtitration plates (Maxisorp, Nalge Nunc International, New York, USA). Parasites (50 l, 2 × 107 parasites/ml in PBS) were added to each well and left to dry overnight at room temperature. The plates were blocked with PBS containing 2% BSA. Non-specific binding was determined using BSA-coated wells. The wells were washed with PBS and incubated with the biotinylated ligand (final volume 100 l) in the presence of 0.5 mM phenylmethylsulfonyl fluoride, for 2 h at room temperature. After three washings with PBS, the biotinylated molecules were revealed with streptavidine and biotinylated alkaline phosphatase from the Phototope-star
2.3. Plasminogen binding assay The association of plasminogen to T. cruzi was tested in a final volume of 250 l by incubating motile parasites (2 × 108 cells) with plasminogen in the presence or absence of -aminocaproic acid for 90 min at room temperature in PBS containing 0.1% bovine serum albumin (BSA). After three washings with PBS, the parasite pellet was resuspended in 70 l PBS and incubated with either the plasminogen activator urokinase (75 nM) or streptokinase (20 nM) for a further hour. The plasmin generated was measured using the chromogenic substrate S2251 (0.6 mM) in a final volume of 100 l using an aliquot of the cell suspension. The change in absorbance at 405 nm was determined over time in a microtiter reader. To evaluate the amount of plasminogen bound at different plasminogen concentrations, 100 mM -aminocaproic acid in PBS (final volume 70 l) was added to the pellet after incubation and the washing steps, to remove the plasminogen from the cells. In this case, the incubation step with plasminogen included 2 × 108 cells in a final volume of 0.5 ml. After centrifugation, the supernatant obtained was similarly activated with urokinase (75 nM) or streptokinase (20 nM) and the plasmin activity measured. A calibration curve obtained from different concentrations of plasminogen activated by the appropriate plasminogen activator in the presence of 100 mM -aminocaproic acid was used to estimate the plasminogen bound. A linear relationship between absorbance at 405 nm and plasminogen was obtained making it possible to estimate the amount of ligand in each assay. The amount of ligand per cell was calculated taking into account the number of parasites in the test. 2.4. Binding assay of plasminogen fragments Digestion of plasminogen was performed by incubating plasminogen with porcine pancreatic elastase (Sigma, USA) in a 2:1 (mol:mol) ratio for 8 h at room temperature, in 0.15 M sodium phosphate buffer pH 8 containing 15 mM -aminocaproic acid and 5 mM benzamidine. After digestion, the fragments were loaded onto a Lysine-Sepharose column (18 cm × 2.4 cm) equilibrated with 0.2 M sodium phosphate buffer, pH 8. Miniplasminogen was recovered in the flowthrough volume. K1–K2–K3 and K4 were eluted using a gradient of -aminocaproic acid (0–15 mM). K4 was further purified using a Sephadex G-75 column (75 cm × 1.2 cm). Fragments were precipitated with 80% ammonium sulfate and extensive dialysis was performed before fragment labelling (three dialysis in a 5 l volume each) to assure the elimination of -aminocaproic acid. Fragments were labelled with biotin using the BiotinTag kit (Sigma, USA). Free biotin was separated from biotinylated fragments by PD-10 (Amersham Biosciences, USA) gel filtration chromatography using PBS as equilibration column. The resulting fragments appeared pure as tested by SDS-PAGE (Fig. 2A).
Fig. 1. Plasminogen binding to T. cruzi parasites. (A) Cells were incubated with 2 M plasminogen (Plg) in the absence or presence of 100 mM -aminocaproic acid (EACA). Plasmin activity of an aliquot of cell suspension was detected after several washings and activation with 75 nM urokinase, using the chromogenic substrate S2251. (B) Cells (2 × 108 ) were incubated with different concentrations of plasminogen. After several washings, the plasminogen was detached with 100 mM EACA and activated with 75 nM urokinase. Plasmin activity was measured as above. The plasminogen bound was calculated using a calibration curve. Inset: Scatchard plot. (C) Interaction of biotinylated plasminogen with immobilized T. cruzi cells.
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M. Rojas et al. / Acta Tropica 107 (2008) 54–58
Fig. 2. Binding of plasminogen fragments on T. cruzi cells. (A) SDS-PAGE of the purified fragments derived from elastase digestion of plasminogen. Lane 1: K4; lane 2: K1–K2–K3; lane 3: miniplasminogen. (B) Interaction of the plasminogen fragments: K1–K2–K3 (), K4 () and miniplasminogen (䊉) with immobilized T. cruzi cells. (C) Inhibition of plasminogen binding on immobilized T. cruzi cells by K1–K2–K3 () and miniplasminogen (䊉). The plasminogen concentration was 1 M. (D) Effect of aminocaproic acid on the miniplasminogen binding on immobilized T. cruzi cells. Values are in % of the activity without inhibitor. The concentration of miniplasminogen used was 2 M. (E) Miniplasminogen binding on motile parasites. Cells were incubated with different plasminogen concentration. After several washings, the plasminogen was detached with 100 mM EACA and activated with 75 nM urokinase. Plasmin activity was measured using the chromogenic substrate S2251. Inset: Scatchard plot.
detection kit (New England Biolabs, USA). The substrate p-nitrophenyl phosphate in 50 mM Tris pH 9.5 and 0.5 mM MgCl2 was added and the absorbance at 405 nm was recorded over time. The experimental data were fitted to a hyperbola from which the dissociation constant was calculated. For competitive inhibition assays, the plates were incubated with biotinylated plasminogen at a constant concentration (1 M) in PBS and varying concentrations (0–6 M) of miniplasminogen or K1–K2–K3. Miniplasminogen binding experiments were also performed with living parasites as described above for plasminogen binding experiments. In this case the calibration curve used to estimate the amount of bound ligand was built with miniplasminogen.
2.6. Zymogram analysis Mid gut extract from infected insects either starved or blood fed were submitted to SDS-PAGE under non-reducing conditions. The resolving gel was co-polymerized with 0.4% casein in the presence or absence of 15 g/ml streptokinase. Blood, plasmin and plasminogen were included as controls. Following electrophoresis, the gels were washed for 30 min in 2.5% Triton X-100 and rinsed twice with 50 mM Tris, 10 mM NaCl pH 7.5. Gels were then incubated in the same buffer for 6 h at 37 ◦ C. Proteolytic activity was detected as translucent band in the gel stained with 0.1% Coomassie blue. 3. Results and discussion
2.5. Immunofluorescence experiment Infected insects were fed with citrated human blood in a feeding apparatus. After 2 h, mid guts were dissected and after disrupting the walls, the contents of the stomachs were pooled and washed three times with PBS or PBS containing 100 mM -aminocaproic acid. Epimastigote forms were present in this preparation. A preparation using starved infected insects was served as control. The suspension was then allowed to adhere to poly-(l-lysine)-coated slides and air dried. The suspension was incubated with PBS containing 3% BSA for 30 min at room temperature and washed again with PBS. It was then, incubated with anti-plasminogen antibodies (dilution 1:80) for 1 h, rinsed with PBS and incubated for 1 h with anti-rabbit IgG coupled to FITC (dilution 1:200). The slides were examined using a fluorescence microscope.
In a previous study, we determined by immunostaining, that T. cruzi parasites bind plasminogen (Almeida et al., 2004). This binding was inhibited by -aminocaproic acid. In the present study, we measured the plasminogen binding ability on living cells using a ligand system in which the plasminogen bound is detected by plasmin activity after plasminogen activation by either urokinase or streptokinase. Fig. 1A shows that plasmin activity can be detected in this assay and that -aminocaproic acid inhibits up to 75% of the binding, this value being independent of the plasminogen activator used. The inhibition was calculated taken into account the control value. This result indicates that at least 25% of plasminogen binding is independent of lysine-binding sites in the plasminogen molecule. Since an important part of the binding to T. cruzi is lysine-binding dependent we attempted to determine the dissociation constants and the amount of plasminogen bound on T. cruzi through these
M. Rojas et al. / Acta Tropica 107 (2008) 54–58
lysine-binding sites. For this purpose plasminogen was removed from the cells with -aminocaproic acid and further activated with either urokinase or streptokinase. The plasmin activity was correlated with the amount of plasminogen through a calibration curve. As shown in Fig. 1B, plasminogen bound T. cruzi cells in a concentration-dependent and saturable manner. Similar results were obtained when either streptokinase or urokinase were used as activator. Scatchard analysis of the binding showed a single specific mechanism with a dissociation constant Kd of 0.4 ± 0.1 M (inset in Fig. 1B). The number of binding sites per cell was 5600 ± 900. The Kd value reported here is compatible with in vivo recognition since the plasminogen concentration in the blood is 2 M (Ponting et al., 1992). This Kd corresponds to binding through lysine-binding sites. We also measured plasminogen binding on immobilized parasites on microtiter plates using plasminogen labelled with biotin (Fig. 1C). In this case, in contrast with the method used in which ligand is detached by -aminocaproic acid, lysine-dependent as well as lysine-independent is being measured. The Kd (1.2 ± 0.2 M) estimated by this method was slightly higher. The dissociation constant for plasminogen binding on T. cruzi epimastigote agrees with those from other organisms and cell types. The dissociation constants for plasminogen binding on several human pathogens range from 10−6 to 10−10 M (Boyle and Lottenberg, 1997). In the case of eukaryotic cells the dissociation constants are relatively high, 0.1–2 M (Redlitz and Plow, 1995).
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The number of plasminogen binding sites per T. cruzi cell (5600) differs from that reported for another trypanosomatid, Leishmania mexicana. In this latter case, the number of binding sites per ´ et al., cell (1.6 × 105 ) is two orders of magnitude higher (Avilan 2000). Biotinylated miniplasminogen bound to immobilized cells in a concentration-dependent and saturable manner (Fig. 2B). The binding of biotinylated miniplasminogen to control plates containing BSA was minimal (data not shown). Weakly interaction was observed with K1–K2–K3 and K4, indicating that the main site of interaction with T. cruzi proteins is located on miniplasminogen. The Kd for the miniplasminogen estimated from this curve resulted in a similar value (Kd = 0.8 ± 0.05 M) to that for plasminogen. In competitive binding experiments, the miniplasminogen (6 M) inhibited binding of labelled plasminogen by 72% (Fig. 2C). Inhibition by K1–K2–K3 (6 M) was only 13% (Fig. 2C). These results support the view that among the elastase-derived fragments of plasminogen, miniplasminogen is the most actively recognized fragment. Plasminogen binds to proteins through its kringle domains where lysine-binding sites are located. As expected, miniplasminogen binding was inhibited by -aminocaproic acid (Fig. 2D). The Kd value related to the lysine-dependent sites of miniplasminogen was also measured after removal of the ligand from the cells with -aminocaproic acid and further activation with urokinase to detect miniplasmin activity (Fig. 2E). This value
Fig. 3. Inmunostaining using anti-plasminogen antibodies, of epimastigotes from the midgut of R. prolixus infected with T. cruzi. (A) From insects fed with human blood and washed with PBS. (B) From insects fed with human blood and washed with PBS containing 100 mM -aminocaproic. (C) From starved infected insects. (D and E) Zymogram analyses of the midgut extract electrophoresed under non-reducing conditions, in a 10% polyacrylamide gel containing 0.4% casein without plasminogen activator (D) or with 15 g/ml streptokinase (E). Lane 1: mid gut of starved insect (30 g protein), lane 2: mid gut of blood-fed insect (30 g protein), lane 3: human blood (30 g protein), lane 4: purified plasminogen (0.5 g) and lane 5: plasmin (0.06 g).
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(Kd = 0.7 ± 0.1 M) is of the same order as obtained for plasminogen as is also the number of ligand binding sites (4665 ± 600). From the results reported here, it is clear that the lysine-binding site involved in binding with T. cruzi is mainly that from kringle 5. The lysine-binding site in this kringle is designated AH site since it recognizes the aminohexyl function of the lysine residue without a requirement of free carboxylates (Christensen, 1984; Thewes et al., 1990). Thus we can suggest that internal lysines in T. cruzi proteins might be involved in interaction with plasminogen. Kringle 5 of plasminogen has been involved in the binding of plasminogen to other proteins. In the fibrinolysis process, the binding of plasminogen with fibrin is mainly determined by kringle 5 (Wu et al., 1990). This kringle in particular has also been involved in the binding of plasminogen on the surface of Helicobacter pylori (Pantzar et ´ al., 1998) and with the protein 2-glycoprotein I (Lopez-Lira et al., 2006). From these results we assume that plasminogen arriving in the insect gut through the meal might interact with T. cruzi epimastigotes. To determine whether true epimastigotes have plasminogen binding capacity, we performed an immunofluorescence experiment with epimastigotes obtained from the mid gut of infected insect vectors (R. prolixus) that had been fed with human blood. This insect is the main vector of T. cruzi in many countries of South and Central America. The mid guts of the insects were dissected and after several washings the contents of the stomachs were fixed onto slides and incubated with anti-plasminogen antibodies and FITC-coupled secondary antibodies. As shown in Fig. 3A, parasites from the insect gut of the vector were stained. When the washing step contained -aminocaproic acid, the staining was reduced (Fig. 3B), although staining was higher than that from the control experiment with starved infected insects (Fig. 3C). This result indicates that in the vector binding occurs in part through the lysine-binding sites. We can conclude from these immunofluorescence experiments that inside the gut of the vector, epimastigotes can bind plasminogen from the blood meal. To determine if the bound molecule is plasminogen or plasmin a zymogram was performed in the presence or absence of plasminogen activator. In this zymogram the gut extract from the infected insects (starved and blood fed) as well as the controls blood sample, plasminogen and plasmin were loaded on the gel. As shown in Fig. 3D, no plasmin activity was detected. However, plasminogen is activatable in the gut since plasmin activity is detected when plasminogen activator is present in the gel (Fig. 3E). The physiological significance of plasminogen interaction with epimastigotes remains unknown. The importance of the interaction of plasminogen with cells has been attributed mainly to the plasmin activity that can be generated from plasminogen. Since T. cruzi parasites do not cross tissue barriers in the insect, plasmin activity would be used exclusively inside the gut where the parasites proliferate (Garcia and Azambuja, 1991). Thus, in the case of T. cruzi epimastigotes this acquired proteolytic activity could be used either to degrade fibrin, to digest proteins to obtain amino acids inside the gut or to degrade molecules from the blood meal. Regarding this latter possibility, plasmin can degrade molecules from the blood such as antibodies or complement (Rooijakkers et al., 2005; Lambris et al., 2008). However, we were unable to detect plasmin activity in the insect gut under the conditions tested. Inside the gut, a function independent of plasmin activity cannot be discarded. Plasminogen from the blood could trigger signaling pathways that can be involved in parasite differentiation or metabolism changes. Triggering a signalization pathway and regulation of transcriptional activators by plasminogen binding on cell surface has been described previously (De Sousa et al., 2005). However, to assess
the physiological function of plasminogen binding in epimastigotes more investigations are needed. In conclusion, the estimated dissociation constant for the binding of plasminogen on T. cruzi epimastigotes is biologically relevant at the plasma concentration of plasminogen. In the gut of the insect vector, epimastigotes can interact with plasminogen. This interaction may occur in vivo mainly through the lysine-binding site of kringle 5 in the plasminogen molecule. Acknowledgments This study was supported by CDCHT-ULA, grant C-1321-05-03B. We thank Silverio Diaz for his help in the manipulation of the insects. We wish to thank Dr. Anne-Lise Haenni for the revision of the manuscript. References ´ J.L., Avilan, ´ L., 2004. Plasminogen Almeida, L., Vanegas, G., Calcagno, M., Concepcion, interaction with Trypanosoma cruzi. Mem. Inst. Oswaldo Cruz 99, 63–67. ´ L., Calcagno, M., Figuera, M., Lemus, L., Puig, J., Rodr´ıguez, A.M., 2000. InterAvilan, action of Leishmania mexicana promastigotes with the plasminogen-plasmin system. Mol. Biochem. Parasitol. 110, 183–193. Boyle, M.D.P., Lottenberg, R., 1997. Plasminogen activation by invasive human pathogens. Thromb. Haemost. 77, 1–10. Cesarman-Maus, G., Hajjar, K.A., 2005. Molecular mechanisms of fibrinolysis. Br. J. Haematol. 129, 307–321. Christensen, U., 1984. The AH-site of plasminogen and two C-terminal fragments. A weak lysine-binding site preferring ligands not carrying a free carboxylate function. Biochem. J. 223, 413–421. Coleman, J.L., Gebbia, J.A., Piesman, J., Degen, J.L., Bugge, T.H., Benach, J.L., 1997. Plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochetemia in mice. Cell 89, 1111–1119. Coleman, J.L., Benach, J.L., 1999. Use of the plasminogen activation system by microorganisms. J. Lab. Clin. Med. 134, 567–576. Deutsch, D.G., Mertz, E.T., 1970. Plasminogen: purification from human plasma by affinity chromatography. Science 170, 1095–1096. De Sousa, L.P., Brasil, B.S., Silva, B.M., Freitas, M.H., Nogueira, S.V., Ferreira, P.C., Kroon, E.G., Bonjardim, C.A., 2005. Plasminogen/plasmin regulates c-fos and egr1 expression via the MEK/ERK pathway. Biochem. Biophys. Res. Commun. 329, 237–245. Garcia, E.S., Azambuja, P., 1991. Development and interactions of Trypanosoma cruzi within the insect vector. Parasitol. Today 7, 240–244. Harlow, E., Lane, D., 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Lambris, J.D, Ricklin, D., Geisbrecht, B.V., 2008. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142. ´ ´ L., Mart´ınez, V.M., Ruiz Ordaz, B.H., 2006. The role of Lopez-Lira, F., Rosales-Leon, (-glycoprotein I (2GPI) in the activation of plasminogen. Biochim. Biophys. Acta 1764, 815–823. ¨ ¨ Lahteenm aki, K., Edelman, S., Korhonen, T.K., 2005. Bacterial metastasis: the host plasminogen system in bacterial invasion. Trends Microbiol. 13, 79–85. Ponting, C.P., Marshall, J.M., Cederholm-Williams, S.A., 1992. Plasminogen: a structural review. Blood Coagul. Fibrinolysis 3, 605–614. Pantzar, M., Ljungh, A., Wadstrom, T., 1998. Plasminogen binding and activation at the surface of Helicobacter pylori CCUG 17874. Infect. Immun. 66, 4976–4980. ˜ ˜ P., Domingo-Sananes, M., Caceres, ´ Quinones, W., Pena, A., Michels, P.A., Avilan, L., ´ J.L., 2007. Leishmania mexicana: molecular cloning and characteriConcepcion, zation of enolase. Exp. Parasitol. 116, 241–251. Redlitz, A., Plow, E.F., 1995. Receptors for plasminogen and t-PA: an update. Baillieres Clin. Haematol. 8, 313–327. Rooijakkers, S.H., van Wamel, W.J., Ruyken, M., van Kessel, K.P., van Strijp, J.A., 2005. Anti-opsonic properties of staphylokinase. Microbes Infect. 7, 476–484. Syrovets, T., Simmet, T., 2004. Novel aspects and new roles for the serine protease plasmin. Cell Mol. Life Sci. 61, 873–885. Sun, H., 2005. The interaction between pathogens and the host coagulation system. Physiology 21, 281–288. Sun, H., Ringdahl, U., Homeister, J.W., Fay, W.P., Engleberg, N.C., Yang, A.Y., Rozek, L.S., Wang, X., Sjobring, U., Ginsburg, D., 2004. Plasminogen is a critical host pathogenicity factor for group A streptococcal infection. Science 305, 1283–1286. Thewes, T., Constantine, K., Byeon, I.J., Llinas, M., 1990. Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy. J. Biol. Chem. 265, 3906–3915. Wu, H.L., Chang, B.I., Wu, D.H., Chang, L.C., Gong, C.C., Lou, K.L., Shi, G.Y., 1990. Interaction of plasminogen and fibrin in plasminogen activation. J. Biol. Chem. 265, 19658–19664.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
Characteristics of plasminogen binding to Trypanosoma cruzi epimastigotes ´ b , Elis Aldana c , Luisana Avilan a,∗ Masyelly Rojas a , Indira Labrador a , Juan L. Concepcion a
Laboratorio de Fisiolog´ıa, Facultad de Ciencias, Universidad de Los Andes, M´erida 5101, Venezuela Laboratorio de Enzimolog´ıa de Par´ asitos, Facultad de Ciencias, Universidad de Los Andes, M´erida 5101, Venezuela c Laboratorio Herman Lentz, Facultad de Ciencias, Universidad de Los Andes, M´erida 5101, Venezuela b
a r t i c l e
i n f o
Article history: Received 4 September 2007 Received in revised form 5 April 2008 Accepted 8 April 2008 Available online 22 April 2008 Keywords: Trypanosoma cruzi Plasminogen Kringles
a b s t r a c t The binding constants of the interaction between plasminogen and Trypanosoma cruzi epimastigotes were determined. An indirect method in which the bound plasminogen is detached from the cell by -aminocaproic acid and a direct method through biotinylated plasminogen were used. The analyses revealed a dissociation constant (Kd ) from 0.4 to 1.2 M, these values being compatible with recognition in vivo. Moreover, epimastigotes from the gut of Rhodnius prolixus were able to bind plasminogen from the blood meal. Fragments derived from elastase digestion of plasminogen were tested for their ability to bind T. cruzi cells. The fragment with highest ability to interact with the parasite was miniplasminogen that bound in a concentration-dependent and saturable manner with a Kd similar to that for plasminogen. This binding was inhibited by -aminocaproic acid indicating that the lysine-binding site of kringle 5 may be responsible for the interaction of plasminogen with T. cruzi. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Plasminogen is the zymogenic form of the serine protease plasmin and is found in plasma and interstitial fluids. This protein is well known for its participation in fibrinolysis (Cesarman-Maus and Hajjar, 2005). However, several other functions have been attributed to this molecule such as extracellular matrix degradation and cell migration (Syrovets and Simmet, 2004). Due to its interaction with an increasing number of pathogenic organisms both prokaryotes and eukaryotes, plasminogen has also been suggested to be involved in infectious processes (Boyle and Lottenberg, 1997; ¨ ¨ et al., 2005; Sun, 2005). Coleman and Benach, 1999; Lahteenm aki Indeed, in several pathogens it has been demonstrated that plasminogen bound on their surface facilitates the establishment of infection within the host (Sun et al., 2004; Coleman et al., 1997). Trypanosoma cruzi, that belongs to the family of trypanosomatidae and is the causative agent of Chagas disease, is one of the pathogens capable of binding plasminogen (Almeida et al., 2004). This binding was previously observed for metacyclic trypomastigotes and epimastigotes. Binding to epimastigotes is not well understood since this form is found in the insect vector. However, in the case of Borrelia burdorgferi, it has been demonstrated that binding of plasminogen is required for dissemination in both ticks and mammals
∗ Corresponding author at: Dept. Biolog´ıa, Facultad de Ciencias, Universidad de ´ Los Andes, Merida 5101, Venezuela. Tel.: +58 274 2401307; fax: +58 274 2401286. E-mail address: [email protected] (L. Avilan). 0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2008.04.013
(Coleman et al., 1997). Thus one may hypothesize that in the case of T. cruzi, plasminogen might be used in the vector. To determine the function of the interaction between T. cruzi and plasminogen, a better characterization of this interaction is needed. Here, we determined the binding constants of this interaction to assess whether it is compatible with recognition in vivo. In addition, we attempted to localize the region in the plasminogen molecule that is recognized by T. cruzi. The plasminogen molecule has five substructures called kringles some of which (K1, K4 and K5) are implicated in the recognition of fibrin, and cell surfaces (Ponting et al., 1992). Plasminogen can be digested by elastase yielding three fragments: K1–K2–K3, K4 and the K5-catalytic site (miniplasminogen). These plasminogen fragments were tested for their capacity to interact with T. cruzi. The study with these fragments is of value in elucidating the mechanism of binding on the parasite. 2. Materials and methods 2.1. Parasites, insects and T. cruzi infection Epimastigotes of the T. cruzi EP strain were used in the present study. The parasites were grown in liver-infusion tryptose (LIT) medium, supplemented with 10% heat-inactivated fetal bovine serum, at 28 ◦ C with agitation. The parasites harvested during the exponential phase were washed three times with phosphatebuffered saline (PBS). Rhodnius prolixus (Heteroptera: Triatominae) were from a colony maintained in the Herman Lentz Laboratory at the Universidad de Los Andes, Venezuela. For infection, a group of
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insects (3rd to 5th instar nymphs) were fed with LIT medium containing 1 × 108 epimastigotes/ml of T. cruzi strain EP. After infection the insects were maintained in the laboratory by feeding on mice. These insects were used here as adults after a month of starvation. 2.2. Proteins Plasminogen was purified from human blood as reported previously (Deutsch and Mertz, 1970). Polyclonal plasminogen antibodies were prepared in the laboratory as described elsewhere ˜ (Quinones et al., 2007). IgG fractions were purified using ammonium sulfate precipitation and DEAE-chromatography (Harlow and Lane, 1988).
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To determine the specific binding activity of the plasminogen fragments, T. cruzi parasites in PBS, were immobilized overnight in microtitration plates (Maxisorp, Nalge Nunc International, New York, USA). Parasites (50 l, 2 × 107 parasites/ml in PBS) were added to each well and left to dry overnight at room temperature. The plates were blocked with PBS containing 2% BSA. Non-specific binding was determined using BSA-coated wells. The wells were washed with PBS and incubated with the biotinylated ligand (final volume 100 l) in the presence of 0.5 mM phenylmethylsulfonyl fluoride, for 2 h at room temperature. After three washings with PBS, the biotinylated molecules were revealed with streptavidine and biotinylated alkaline phosphatase from the Phototope-star
2.3. Plasminogen binding assay The association of plasminogen to T. cruzi was tested in a final volume of 250 l by incubating motile parasites (2 × 108 cells) with plasminogen in the presence or absence of -aminocaproic acid for 90 min at room temperature in PBS containing 0.1% bovine serum albumin (BSA). After three washings with PBS, the parasite pellet was resuspended in 70 l PBS and incubated with either the plasminogen activator urokinase (75 nM) or streptokinase (20 nM) for a further hour. The plasmin generated was measured using the chromogenic substrate S2251 (0.6 mM) in a final volume of 100 l using an aliquot of the cell suspension. The change in absorbance at 405 nm was determined over time in a microtiter reader. To evaluate the amount of plasminogen bound at different plasminogen concentrations, 100 mM -aminocaproic acid in PBS (final volume 70 l) was added to the pellet after incubation and the washing steps, to remove the plasminogen from the cells. In this case, the incubation step with plasminogen included 2 × 108 cells in a final volume of 0.5 ml. After centrifugation, the supernatant obtained was similarly activated with urokinase (75 nM) or streptokinase (20 nM) and the plasmin activity measured. A calibration curve obtained from different concentrations of plasminogen activated by the appropriate plasminogen activator in the presence of 100 mM -aminocaproic acid was used to estimate the plasminogen bound. A linear relationship between absorbance at 405 nm and plasminogen was obtained making it possible to estimate the amount of ligand in each assay. The amount of ligand per cell was calculated taking into account the number of parasites in the test. 2.4. Binding assay of plasminogen fragments Digestion of plasminogen was performed by incubating plasminogen with porcine pancreatic elastase (Sigma, USA) in a 2:1 (mol:mol) ratio for 8 h at room temperature, in 0.15 M sodium phosphate buffer pH 8 containing 15 mM -aminocaproic acid and 5 mM benzamidine. After digestion, the fragments were loaded onto a Lysine-Sepharose column (18 cm × 2.4 cm) equilibrated with 0.2 M sodium phosphate buffer, pH 8. Miniplasminogen was recovered in the flowthrough volume. K1–K2–K3 and K4 were eluted using a gradient of -aminocaproic acid (0–15 mM). K4 was further purified using a Sephadex G-75 column (75 cm × 1.2 cm). Fragments were precipitated with 80% ammonium sulfate and extensive dialysis was performed before fragment labelling (three dialysis in a 5 l volume each) to assure the elimination of -aminocaproic acid. Fragments were labelled with biotin using the BiotinTag kit (Sigma, USA). Free biotin was separated from biotinylated fragments by PD-10 (Amersham Biosciences, USA) gel filtration chromatography using PBS as equilibration column. The resulting fragments appeared pure as tested by SDS-PAGE (Fig. 2A).
Fig. 1. Plasminogen binding to T. cruzi parasites. (A) Cells were incubated with 2 M plasminogen (Plg) in the absence or presence of 100 mM -aminocaproic acid (EACA). Plasmin activity of an aliquot of cell suspension was detected after several washings and activation with 75 nM urokinase, using the chromogenic substrate S2251. (B) Cells (2 × 108 ) were incubated with different concentrations of plasminogen. After several washings, the plasminogen was detached with 100 mM EACA and activated with 75 nM urokinase. Plasmin activity was measured as above. The plasminogen bound was calculated using a calibration curve. Inset: Scatchard plot. (C) Interaction of biotinylated plasminogen with immobilized T. cruzi cells.
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Fig. 2. Binding of plasminogen fragments on T. cruzi cells. (A) SDS-PAGE of the purified fragments derived from elastase digestion of plasminogen. Lane 1: K4; lane 2: K1–K2–K3; lane 3: miniplasminogen. (B) Interaction of the plasminogen fragments: K1–K2–K3 (), K4 () and miniplasminogen (䊉) with immobilized T. cruzi cells. (C) Inhibition of plasminogen binding on immobilized T. cruzi cells by K1–K2–K3 () and miniplasminogen (䊉). The plasminogen concentration was 1 M. (D) Effect of aminocaproic acid on the miniplasminogen binding on immobilized T. cruzi cells. Values are in % of the activity without inhibitor. The concentration of miniplasminogen used was 2 M. (E) Miniplasminogen binding on motile parasites. Cells were incubated with different plasminogen concentration. After several washings, the plasminogen was detached with 100 mM EACA and activated with 75 nM urokinase. Plasmin activity was measured using the chromogenic substrate S2251. Inset: Scatchard plot.
detection kit (New England Biolabs, USA). The substrate p-nitrophenyl phosphate in 50 mM Tris pH 9.5 and 0.5 mM MgCl2 was added and the absorbance at 405 nm was recorded over time. The experimental data were fitted to a hyperbola from which the dissociation constant was calculated. For competitive inhibition assays, the plates were incubated with biotinylated plasminogen at a constant concentration (1 M) in PBS and varying concentrations (0–6 M) of miniplasminogen or K1–K2–K3. Miniplasminogen binding experiments were also performed with living parasites as described above for plasminogen binding experiments. In this case the calibration curve used to estimate the amount of bound ligand was built with miniplasminogen.
2.6. Zymogram analysis Mid gut extract from infected insects either starved or blood fed were submitted to SDS-PAGE under non-reducing conditions. The resolving gel was co-polymerized with 0.4% casein in the presence or absence of 15 g/ml streptokinase. Blood, plasmin and plasminogen were included as controls. Following electrophoresis, the gels were washed for 30 min in 2.5% Triton X-100 and rinsed twice with 50 mM Tris, 10 mM NaCl pH 7.5. Gels were then incubated in the same buffer for 6 h at 37 ◦ C. Proteolytic activity was detected as translucent band in the gel stained with 0.1% Coomassie blue. 3. Results and discussion
2.5. Immunofluorescence experiment Infected insects were fed with citrated human blood in a feeding apparatus. After 2 h, mid guts were dissected and after disrupting the walls, the contents of the stomachs were pooled and washed three times with PBS or PBS containing 100 mM -aminocaproic acid. Epimastigote forms were present in this preparation. A preparation using starved infected insects was served as control. The suspension was then allowed to adhere to poly-(l-lysine)-coated slides and air dried. The suspension was incubated with PBS containing 3% BSA for 30 min at room temperature and washed again with PBS. It was then, incubated with anti-plasminogen antibodies (dilution 1:80) for 1 h, rinsed with PBS and incubated for 1 h with anti-rabbit IgG coupled to FITC (dilution 1:200). The slides were examined using a fluorescence microscope.
In a previous study, we determined by immunostaining, that T. cruzi parasites bind plasminogen (Almeida et al., 2004). This binding was inhibited by -aminocaproic acid. In the present study, we measured the plasminogen binding ability on living cells using a ligand system in which the plasminogen bound is detected by plasmin activity after plasminogen activation by either urokinase or streptokinase. Fig. 1A shows that plasmin activity can be detected in this assay and that -aminocaproic acid inhibits up to 75% of the binding, this value being independent of the plasminogen activator used. The inhibition was calculated taken into account the control value. This result indicates that at least 25% of plasminogen binding is independent of lysine-binding sites in the plasminogen molecule. Since an important part of the binding to T. cruzi is lysine-binding dependent we attempted to determine the dissociation constants and the amount of plasminogen bound on T. cruzi through these
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lysine-binding sites. For this purpose plasminogen was removed from the cells with -aminocaproic acid and further activated with either urokinase or streptokinase. The plasmin activity was correlated with the amount of plasminogen through a calibration curve. As shown in Fig. 1B, plasminogen bound T. cruzi cells in a concentration-dependent and saturable manner. Similar results were obtained when either streptokinase or urokinase were used as activator. Scatchard analysis of the binding showed a single specific mechanism with a dissociation constant Kd of 0.4 ± 0.1 M (inset in Fig. 1B). The number of binding sites per cell was 5600 ± 900. The Kd value reported here is compatible with in vivo recognition since the plasminogen concentration in the blood is 2 M (Ponting et al., 1992). This Kd corresponds to binding through lysine-binding sites. We also measured plasminogen binding on immobilized parasites on microtiter plates using plasminogen labelled with biotin (Fig. 1C). In this case, in contrast with the method used in which ligand is detached by -aminocaproic acid, lysine-dependent as well as lysine-independent is being measured. The Kd (1.2 ± 0.2 M) estimated by this method was slightly higher. The dissociation constant for plasminogen binding on T. cruzi epimastigote agrees with those from other organisms and cell types. The dissociation constants for plasminogen binding on several human pathogens range from 10−6 to 10−10 M (Boyle and Lottenberg, 1997). In the case of eukaryotic cells the dissociation constants are relatively high, 0.1–2 M (Redlitz and Plow, 1995).
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The number of plasminogen binding sites per T. cruzi cell (5600) differs from that reported for another trypanosomatid, Leishmania mexicana. In this latter case, the number of binding sites per ´ et al., cell (1.6 × 105 ) is two orders of magnitude higher (Avilan 2000). Biotinylated miniplasminogen bound to immobilized cells in a concentration-dependent and saturable manner (Fig. 2B). The binding of biotinylated miniplasminogen to control plates containing BSA was minimal (data not shown). Weakly interaction was observed with K1–K2–K3 and K4, indicating that the main site of interaction with T. cruzi proteins is located on miniplasminogen. The Kd for the miniplasminogen estimated from this curve resulted in a similar value (Kd = 0.8 ± 0.05 M) to that for plasminogen. In competitive binding experiments, the miniplasminogen (6 M) inhibited binding of labelled plasminogen by 72% (Fig. 2C). Inhibition by K1–K2–K3 (6 M) was only 13% (Fig. 2C). These results support the view that among the elastase-derived fragments of plasminogen, miniplasminogen is the most actively recognized fragment. Plasminogen binds to proteins through its kringle domains where lysine-binding sites are located. As expected, miniplasminogen binding was inhibited by -aminocaproic acid (Fig. 2D). The Kd value related to the lysine-dependent sites of miniplasminogen was also measured after removal of the ligand from the cells with -aminocaproic acid and further activation with urokinase to detect miniplasmin activity (Fig. 2E). This value
Fig. 3. Inmunostaining using anti-plasminogen antibodies, of epimastigotes from the midgut of R. prolixus infected with T. cruzi. (A) From insects fed with human blood and washed with PBS. (B) From insects fed with human blood and washed with PBS containing 100 mM -aminocaproic. (C) From starved infected insects. (D and E) Zymogram analyses of the midgut extract electrophoresed under non-reducing conditions, in a 10% polyacrylamide gel containing 0.4% casein without plasminogen activator (D) or with 15 g/ml streptokinase (E). Lane 1: mid gut of starved insect (30 g protein), lane 2: mid gut of blood-fed insect (30 g protein), lane 3: human blood (30 g protein), lane 4: purified plasminogen (0.5 g) and lane 5: plasmin (0.06 g).
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(Kd = 0.7 ± 0.1 M) is of the same order as obtained for plasminogen as is also the number of ligand binding sites (4665 ± 600). From the results reported here, it is clear that the lysine-binding site involved in binding with T. cruzi is mainly that from kringle 5. The lysine-binding site in this kringle is designated AH site since it recognizes the aminohexyl function of the lysine residue without a requirement of free carboxylates (Christensen, 1984; Thewes et al., 1990). Thus we can suggest that internal lysines in T. cruzi proteins might be involved in interaction with plasminogen. Kringle 5 of plasminogen has been involved in the binding of plasminogen to other proteins. In the fibrinolysis process, the binding of plasminogen with fibrin is mainly determined by kringle 5 (Wu et al., 1990). This kringle in particular has also been involved in the binding of plasminogen on the surface of Helicobacter pylori (Pantzar et ´ al., 1998) and with the protein 2-glycoprotein I (Lopez-Lira et al., 2006). From these results we assume that plasminogen arriving in the insect gut through the meal might interact with T. cruzi epimastigotes. To determine whether true epimastigotes have plasminogen binding capacity, we performed an immunofluorescence experiment with epimastigotes obtained from the mid gut of infected insect vectors (R. prolixus) that had been fed with human blood. This insect is the main vector of T. cruzi in many countries of South and Central America. The mid guts of the insects were dissected and after several washings the contents of the stomachs were fixed onto slides and incubated with anti-plasminogen antibodies and FITC-coupled secondary antibodies. As shown in Fig. 3A, parasites from the insect gut of the vector were stained. When the washing step contained -aminocaproic acid, the staining was reduced (Fig. 3B), although staining was higher than that from the control experiment with starved infected insects (Fig. 3C). This result indicates that in the vector binding occurs in part through the lysine-binding sites. We can conclude from these immunofluorescence experiments that inside the gut of the vector, epimastigotes can bind plasminogen from the blood meal. To determine if the bound molecule is plasminogen or plasmin a zymogram was performed in the presence or absence of plasminogen activator. In this zymogram the gut extract from the infected insects (starved and blood fed) as well as the controls blood sample, plasminogen and plasmin were loaded on the gel. As shown in Fig. 3D, no plasmin activity was detected. However, plasminogen is activatable in the gut since plasmin activity is detected when plasminogen activator is present in the gel (Fig. 3E). The physiological significance of plasminogen interaction with epimastigotes remains unknown. The importance of the interaction of plasminogen with cells has been attributed mainly to the plasmin activity that can be generated from plasminogen. Since T. cruzi parasites do not cross tissue barriers in the insect, plasmin activity would be used exclusively inside the gut where the parasites proliferate (Garcia and Azambuja, 1991). Thus, in the case of T. cruzi epimastigotes this acquired proteolytic activity could be used either to degrade fibrin, to digest proteins to obtain amino acids inside the gut or to degrade molecules from the blood meal. Regarding this latter possibility, plasmin can degrade molecules from the blood such as antibodies or complement (Rooijakkers et al., 2005; Lambris et al., 2008). However, we were unable to detect plasmin activity in the insect gut under the conditions tested. Inside the gut, a function independent of plasmin activity cannot be discarded. Plasminogen from the blood could trigger signaling pathways that can be involved in parasite differentiation or metabolism changes. Triggering a signalization pathway and regulation of transcriptional activators by plasminogen binding on cell surface has been described previously (De Sousa et al., 2005). However, to assess
the physiological function of plasminogen binding in epimastigotes more investigations are needed. In conclusion, the estimated dissociation constant for the binding of plasminogen on T. cruzi epimastigotes is biologically relevant at the plasma concentration of plasminogen. In the gut of the insect vector, epimastigotes can interact with plasminogen. This interaction may occur in vivo mainly through the lysine-binding site of kringle 5 in the plasminogen molecule. Acknowledgments This study was supported by CDCHT-ULA, grant C-1321-05-03B. We thank Silverio Diaz for his help in the manipulation of the insects. We wish to thank Dr. Anne-Lise Haenni for the revision of the manuscript. References ´ J.L., Avilan, ´ L., 2004. Plasminogen Almeida, L., Vanegas, G., Calcagno, M., Concepcion, interaction with Trypanosoma cruzi. Mem. Inst. Oswaldo Cruz 99, 63–67. ´ L., Calcagno, M., Figuera, M., Lemus, L., Puig, J., Rodr´ıguez, A.M., 2000. InterAvilan, action of Leishmania mexicana promastigotes with the plasminogen-plasmin system. Mol. Biochem. Parasitol. 110, 183–193. Boyle, M.D.P., Lottenberg, R., 1997. Plasminogen activation by invasive human pathogens. Thromb. Haemost. 77, 1–10. Cesarman-Maus, G., Hajjar, K.A., 2005. Molecular mechanisms of fibrinolysis. Br. J. Haematol. 129, 307–321. Christensen, U., 1984. The AH-site of plasminogen and two C-terminal fragments. A weak lysine-binding site preferring ligands not carrying a free carboxylate function. Biochem. J. 223, 413–421. Coleman, J.L., Gebbia, J.A., Piesman, J., Degen, J.L., Bugge, T.H., Benach, J.L., 1997. Plasminogen is required for efficient dissemination of B. burgdorferi in ticks and for enhancement of spirochetemia in mice. Cell 89, 1111–1119. Coleman, J.L., Benach, J.L., 1999. Use of the plasminogen activation system by microorganisms. J. Lab. Clin. Med. 134, 567–576. Deutsch, D.G., Mertz, E.T., 1970. Plasminogen: purification from human plasma by affinity chromatography. Science 170, 1095–1096. De Sousa, L.P., Brasil, B.S., Silva, B.M., Freitas, M.H., Nogueira, S.V., Ferreira, P.C., Kroon, E.G., Bonjardim, C.A., 2005. Plasminogen/plasmin regulates c-fos and egr1 expression via the MEK/ERK pathway. Biochem. Biophys. Res. Commun. 329, 237–245. Garcia, E.S., Azambuja, P., 1991. Development and interactions of Trypanosoma cruzi within the insect vector. Parasitol. Today 7, 240–244. Harlow, E., Lane, D., 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Lambris, J.D, Ricklin, D., Geisbrecht, B.V., 2008. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142. ´ ´ L., Mart´ınez, V.M., Ruiz Ordaz, B.H., 2006. The role of Lopez-Lira, F., Rosales-Leon, (-glycoprotein I (2GPI) in the activation of plasminogen. Biochim. Biophys. Acta 1764, 815–823. ¨ ¨ Lahteenm aki, K., Edelman, S., Korhonen, T.K., 2005. Bacterial metastasis: the host plasminogen system in bacterial invasion. Trends Microbiol. 13, 79–85. Ponting, C.P., Marshall, J.M., Cederholm-Williams, S.A., 1992. Plasminogen: a structural review. Blood Coagul. Fibrinolysis 3, 605–614. Pantzar, M., Ljungh, A., Wadstrom, T., 1998. Plasminogen binding and activation at the surface of Helicobacter pylori CCUG 17874. Infect. Immun. 66, 4976–4980. ˜ ˜ P., Domingo-Sananes, M., Caceres, ´ Quinones, W., Pena, A., Michels, P.A., Avilan, L., ´ J.L., 2007. Leishmania mexicana: molecular cloning and characteriConcepcion, zation of enolase. Exp. Parasitol. 116, 241–251. Redlitz, A., Plow, E.F., 1995. Receptors for plasminogen and t-PA: an update. Baillieres Clin. Haematol. 8, 313–327. Rooijakkers, S.H., van Wamel, W.J., Ruyken, M., van Kessel, K.P., van Strijp, J.A., 2005. Anti-opsonic properties of staphylokinase. Microbes Infect. 7, 476–484. Syrovets, T., Simmet, T., 2004. Novel aspects and new roles for the serine protease plasmin. Cell Mol. Life Sci. 61, 873–885. Sun, H., 2005. The interaction between pathogens and the host coagulation system. Physiology 21, 281–288. Sun, H., Ringdahl, U., Homeister, J.W., Fay, W.P., Engleberg, N.C., Yang, A.Y., Rozek, L.S., Wang, X., Sjobring, U., Ginsburg, D., 2004. Plasminogen is a critical host pathogenicity factor for group A streptococcal infection. Science 305, 1283–1286. Thewes, T., Constantine, K., Byeon, I.J., Llinas, M., 1990. Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy. J. Biol. Chem. 265, 3906–3915. Wu, H.L., Chang, B.I., Wu, D.H., Chang, L.C., Gong, C.C., Lou, K.L., Shi, G.Y., 1990. Interaction of plasminogen and fibrin in plasminogen activation. J. Biol. Chem. 265, 19658–19664.