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Infection and Immunity, May 2009, p. 2193-2200, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01542-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

Manjusha M. Kulkarni,¹ Cheryl L. Olson,² David M. Engman,² and Bradford S. McGwire^1*

Division of Infectious Diseases and Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio,¹ Departments of Pathology and Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois²

Received 19 December 2008/ Returned for modification 29 January 2009/ Accepted 25 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Infection by the protozoan Trypanosoma cruzi is a cause of significant morbidity and mortality in Central and South America (15, 34). Mammalian infection is initiated during feeding of the triatomine vector and contamination of the bite wound with infective metacyclic trypomastigotes present within the insect excreta. Once introduced, parasites bind to and invade host cells, where they differentiate and replicate as intracellular amastigotes. Replicating intracellular amastigotes differentiate into trypomastigotes, burst out of their host cells, and invade uninfected host cells. The clinical sequelae of T. cruzi infection include myocarditis, known as Chagas' heart disease, which is a leading cause of cardiac disease in Latin and South America; megaorgan syndromes; and central nervous system disease in immunosuppressed or pediatric patients.

A variety of parasite molecules are known to facilitate the binding of T. cruzi to host cells (4). Soluble and membrane-bound trans-sialidases (8) transfer host sialic acid to parasite surface ligands, including mucin-like molecules (30). Other parasite surface proteins have also been implicated, either positively (29) or negatively (19), in host infection. Invasion of nonphagocytic host cells appears to be an active process involving lysosome recruitment (27), requiring intracellular signaling involving Ca²⁺ mobilization (4, 35). A family of cell surface-localized, zinc-dependent metalloproteases (also known as GP63 proteins, major surface proteases, or leishmanolysins) are expressed by trypanosomes and Leishmania species. In Leishmania, they serve as ligands for host cell attachment and protect the parasite from intraphagolysosomal degradation (6, 20). In the bloodstream form of the African trypanosome Trypanosoma brucei, they function to release variant surface glycoproteins from the cell surface during antigenic variation (17), but they do not function this way in the insect form (12). T. cruzi genes encoding GP63 homologues (TcGP63) are differentially expressed stage specifically, being more abundant in amastigotes than in epimastigotes or trypomastigotes (14). Anti-peptide antibodies against an epitope present in a subset of TcGP63 proteins (termed TcGP63 I) recognized the protein at all life stages and were shown to inhibit trypomastigote infection of host cells (9). Complete genome and proteome analyses of T. cruzi have revealed a vast array of TcGP63 genes (174 "true" copies), encoding at least 29 different proteins. Many of these are expressed at multiple life stages, while some are stage specific (1).

To further understand the pattern of expression and functional role of GP63 proteins in T. cruzi, we developed a polyclonal antiserum against recombinant TcGP63 and used this reagent to study the differential expression, subcellular locations, and posttranslational modifications of TcGP63 in both the mammalian and insect forms of the parasite. This antiserum recognizes a different subset of TcGP63 than an anti-peptide antiserum described previously, and comparison of these two antisera demonstrates that different TcGP63 proteins undergo differential stage-specific posttranslational processing. Infection inhibition studies using both antisera show that each independently contributes to host cell infection. Our characterization of GP63 proteins of T. cruzi paves the way for further biochemical and functional analysis of this family of metalloproteases.

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Parasite cultivation and purification. The Brazil strain, several of its clones, and an uncloned Y strain of T. cruzi were used in this study. Routine cultivation of epimastigotes was performed using LDNT (16) medium supplemented with 10% heat-inactivated fetal bovine serum and 20 µg ml^–1 hemin. Metacyclic trypomastigotes appearing in late-stationary-phase cultures were purified by density gradient centrifugation in 10 to 15% Nycodenz step gradients for 20 min at 900 x g essentially as described previously (10). Amastigotes and tissue culture-derived trypomastigotes (TCTs) were propagated in the H9C2 line of rat heart myoblasts. Myoblasts were routinely grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum. Isolation and purification of emerging trypomastigotes were performed as described above for metacyclic trypomastigotes. Amastigotes were prepared by physical disruption of infected myoblasts by vortexing in phosphate-buffered saline (PBS), followed by purification by density centrifugation in 15 to 17 to 21% Nycodenz step gradients (10). L. amazonensis expressing the flagellar calcium binding protein (FCaBP) of T. cruzi was also used in these studies and will be reported in detail elsewhere. Briefly, the trypanosome expression vector pTEX, containing the gene encoding T. cruzi FCaBP (13), was used to stably transfect L. amazonensis promastigotes (21). Expression and localization of FCaBP were confirmed by Western blotting and immunofluorescence microscopy as described below.

Generation of recombinant T. cruzi GP63 fusion protein and antiserum. A T. cruzi GP63 fusion protein was created using a previously described expressed sequence tag (EST), designated 21n6 (ELEDEGGKGTASSHWERRNAKDELMAGISGIGYYTSLTMAALEDTGFYKANWGMEEPMSWGNNSGCALLTEKCLINGVTQYPEMFCTAETGLISCTSDRLALGYCTIHLYKAELPPQYQYFSNLKLGGSASSLMDLCPYVQPYSNTRCSNGEASVMHGSRVGPRSMCLKGDGLVDFMGPVGDVCAEVSCEKGEVSVRYLGDDTWRQCPEGSSITPTGLFTGGCKILCPKYDDVCIIFDPLRGGGDVSSLLSVFPSISVILLVLIFIS) (26), fused in frame with the glutathione S-transferase (GST) gene at the EcoRI site of pGEX-3X (Amersham Biosciences, Piscataway, NJ). Recombinant protein was purified from Escherichia coli by using glutathione-coupled agarose and used to immunize mice by subcutaneous injection after antigen emulsification with Freund's complete adjuvant. Sera from mice were tested 2 weeks after the second immunization for reactivity to fusion protein and T. cruzi lysates, using preimmune sera as negative controls.

Western blotting and immunoprecipitation. Parasite protein lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Resolved proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) for Western blot analysis using appropriate antisera. The antisera used in these studies were mouse polyclonal anti-TcGP63 (see above), rabbit polyclonal anti-Leishmania amazonensis GP63 (anti-LaGP63) raised against denatured and deglycosylated GP63 purified by monoclonal affinity chromatography (21), rabbit polyclonal anti-P36 raised against the stable cytoplasmic oxidoreductase of L. amazonensis (18), mouse polyclonal anti-green fluorescent protein (anti-GFP; Caltag, Burlingame, CA), and mouse polyclonal anti-FCaBP generated against T. cruzi FCaBP (13). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated or alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G (IgG). Blots were developed with enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ) or nitroblue tetrazolium-BCIP (5-bromo-4-chloro-3-indolylphosphate) (Invitrogen, Carlsbad, CA) reagents, respectively.

Surface biotinylation. Surface proteins on the parasites were labeled with biotin conjugate (EZ-link; Pierce, Rockford, IL) by washing cells thrice in PBS and then incubating them with 0.5 mg/ml biotin reagent in PBS for 20 min on ice. Cells were then pelleted and incubated for another 20 min with fresh biotin stock solution. Quenching of the reactions was performed by washing cells with medium M199 (Invitrogen, Carlsbad, CA) twice.

Immunofluorescence microscopy. Parasites were washed thrice in PBS and then fixed with anhydrous methanol at –20°C for 10 min. Following fixation, cells were again extensively washed in PBS and allowed to adhere to a glass slide. Preparations were then incubated with blocking solution (1% normal goat serum, 1% bovine serum albumin in PBS), followed by 1:500 dilutions of TcGP63 or LaGP63 antisera in blocking buffer. After being washed with PBS, the slides were stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (for anti-TcGP63) or rhodamine-conjugated goat anti-rabbit IgG (for anti-LaGP63) diluted 1:1,000 in blocking buffer and then with 4', 6-diamidino-2-phenylindole (DAPI) for visualization of DNA. Slides were mounted with Permafluor (Immunotech, Marseille, France) and visualized using fluorescence microscopy.

Triton X-100 partitioning analysis. Parasites were subjected to temperature-dependent Triton X-100 phase-partitioning analysis by incubation of equal numbers of cells in PBS containing 0.1% Triton X-100 at 4°C or 37°C or 30 min, followed by removal of insoluble material by centrifugation at 14,000 x g for 10 min (22). Laemmli sample buffer was added to pellets and supernatants in equal proportions, boiled, and analyzed by SDS-PAGE and Western blotting.

Purification of TcGP63 and its fragments. Detergent lysates of T. cruzi epimastigotes were subjected to anion-exchange chromatography using a Hi-Trap QFF column (Amersham Biosciences, Inc., Piscataway, NJ) with a continuous salt gradient of 0 to 1 M NaCl. Fractions were analyzed by Western blotting using the TcGP63 antiserum. Positive fractions were further purified using hydrophobic interaction chromatography with a continuous ammonium sulfate gradient. TcGP63-positive fractions were extensively dialyzed prior to further analysis.

T. cruzi myoblast infections. Purified TCTs were used to infect myoblasts pregrown on coverslips at a multiplicity of infection of 5:1 at 37°C. Prior to infection, parasites were pretreated with either TcGP63 antiserum or the corresponding preimmune serum for 30 min on ice, followed by extensive washing in serum-free medium. At various times postinfection, coverslips were rinsed thrice in fresh medium, fixed in methanol, and stained with DAPI. The percentage of infected myoblasts and number of associated parasites per infected cell were enumerated upon visualization by fluorescence microscopy. For radioisotopic analysis, 5 x 10⁶ TCTs were labeled in Hanks balanced salt solution containing 50 µCi of [³⁵S]Met-[³⁵S]Cys labeling mixture (NEN, Boston, MA) for 1 h at 37°C. Washed parasites were applied to myoblasts pregrown on coverslips. At the indicated times postinfection, the coverslips were withdrawn, rinsed of unbound parasites, dried, and solubilized in 0.4 N NaOH, 0.1% SDS. Scintillation counting was performed on triplicate samples. Myoblasts were preincubated with the indicated preparations at 37°C for 1 h prior to application of labeled parasites.

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Generation of T. cruzi GP63 (TcGP63) antiserum. To begin the study of T. cruzi GP63, we generated an antiserum against the recombinant protein. As a source of the TcGP63-coding sequence, we used an EST clone, designated 21n6, generated from a cDNA library of T. cruzi epimastigotes (26). The EST clone encompasses the C-terminal 40% of the TcGP63 open reading frame. The insert was cloned in-frame with GST in the pGEX-5X-3 plasmid and expressed in E. coli. The expressed protein was approximately 55 kDa in size, composed of the 26-kDa GST protein and the 25-kDa C-terminal fragment of TcGP63. This segment of the protein includes a C-terminal hydrophobic tail thought to serve as a transmembrane domain. The fusion protein was purified using immobilized glutathione and used to immunize mice for generation of TcGP63 antisera. Western blotting of L. amazonensis promastigote and T. cruzi epimastigote lysates was performed with the antiserum (Fig. 1A, left). The antisera reacted specifically with a protein of approximately 61 kDa in T. cruzi and the 63-kDa GP63 protein in L. amazonensis. To verify that the antiserum was reacting to GP63 and not to another comigrating protein, Western blot analysis with the antiserum was done to probe purified GP63 from L. amazonensis (Fig. 1A, right). The TcGP63 antiserum recognized the purified protein as well as the same protein in the total Leishmania lysates (Fig. 1A, lane "Cell") and did not recognize any other parasite protein. Variations in the expression levels of GP63 in different Leishmania strains and clones have been noted (23). To assess expression of TcGP63 in different T. cruzi isolates, we analyzed epimastigote lysates by Western blotting using the TcGP63 antiserum (Fig. 1B). The Y and Brazil strains were tested, as were a number of clones derived from the Brazil strain by limiting dilution. Epimastigotes of all strains and clones expressed the same level of a 61-kDa protein. These results indicate that the TcGP63 protein recognized by this antiserum is expressed at approximately equal levels in multiple T. cruzi strains and clones and that this TcGP63 protein and Leishmania GP63 share antigenic similarities.

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FIG. 1. Characterization of the TcGP63 antiserum and conservation of TcGP63 expression among T. cruzi strains and clones. (A) Protein lysates of L. amazonensis (La) promastigotes and T. cruzi (Tc) epimastigotes (left) were examined by Western blotting using preimmune (PI) or immune (I) sera of mice that had been immunized with a GST-TcGP63 fusion protein. TcGP63 antiserum (right) was used to probe purified LaGP63 (LaGP63) or a control Leishmania cell lysate (Cell). (B) Protein lysates of the Y and Brazil strains of T. cruzi were examined by Western blotting using the TcGP63 antiserum. The uncloned parental Brazil strain (S) and eight independently derived clones (lanes 1 to 4 and A to D) were tested, as was an L. amazonensis promastigote control (La). LaGP63 and TcGP63s are 63 and 61 kDa in size, respectively, as indicated in panel A. (C) Protein lysates of the different life cycle stages of T. cruzi were examined by Western blotting with anti-TcGP63 and anti-tubulin as indicated. The cells examined were epimastigotes (E), metacyclic trypomastigotes (MT), amastigotes (A), and TCTs of the Brazil strain of T. cruzi. The molecular masses of the proteins are as indicated in panels A and C.

Metacyclic trypomastigote GP63 is a nonglycosylated, intracellular protein. We compared the relative levels and molecular masses of TcGP63 at all life stages of T. cruzi by using SDS-PAGE and Western blotting (Fig. 1C). The 61-kDa TcGP63 protein was expressed at approximately equal levels in epimastigotes, amastigotes, and TCTs. Interestingly, the TcGP63 form expressed in metacyclic trypomastigotes was approximately 55 kDa in size and reacted somewhat more weakly with anti-TcGP63 than did the 61-kDa proteins expressed at the other life cycle stages (Fig. 1C). Each sample was loaded with approximately equal amounts of protein, as evidenced by the reactivity of the tubulin-specific antiserum (Fig. 1C, lower). We compared the reactivities of the various TcGP63 proteins with a polyclonal antiserum against purified, deglycosylated GP63 from L. amazonensis promastigotes (20) and found that only the 55-kDa TcGP63 protein of metacyclic trypomastigotes was identified by this antiserum (Fig. 2). Taken together, these results indicate that at least two forms of this TcGP63 protein exist: a 55-kDa form, present in metacyclic trypomastigotes, and a 61-kDa form, present at the other life cycle stages. To investigate the biochemical basis for the smaller size of the metacyclic trypomastigote GP63 protein, epimastigotes and metacyclic trypomastigotes were surface biotinylated, lysed, and immunoprecipitated with the TcGP63 antiserum. The purified proteins were then incubated with endoglycosidase H and analyzed by SDS-PAGE and blotting with either HRP-coupled streptavidin (SA-HRP) or anti-LaGP63 to detect metacyclic GP63 (Fig. 2). The metacyclic GP63 protein was detected by anti-LaGP63 Western blotting but not by SA-HRP, indicating that it is not expressed on the cell surface. In contrast, GP63 of the epimastigote form was detected by SA-HRP, confirming the surface localization of the protein in this life cycle stage. Comparison of the electrophoretic mobilities of GP63 proteins from these two cell types both before (Fig. 2, lane UT) and after (lane EH) endoglycosidase H treatment showed that epimastigote TcGP63 is glycosylated but that metacyclic TcGP63 is not. The sizes of the deglycosylated epimastigote GP63 protein and both the treated and the untreated metacyclic GP63 proteins were the same, suggesting that the lower molecular weight of the metacyclic form may in part be due to lack of N glycosylation. These results indicate that the epimastigote-specific TcGP63 isoform is N glycosylated and found on the surface and that the metacyclic isoform is nonglycosylated and found inside the cell. This conclusion is based upon the inability to detect biotinylated metacyclic TcGP63 but could also be due to the complete absence of basic amino acids in this isoform for the covalent attachment of activated biotin. Although highly unlikely, this possibility cannot be excluded until this isoform is molecularly cloned and sequenced.

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FIG. 2. TcGP63 is glycosylated and located on the surfaces of epimastigotes and nonglycosylated and intracellular in metacyclic trypomastigotes. Epimastigotes (E) and metacyclic trypomastigotes (MT) of T. cruzi were purified and surface biotinylated. Detergent lysates of these treated cells were immunoprecipitated with anti-TcGP63, treated with endoglycosidase H (EH) or left untreated (UT), and analyzed by SDS-PAGE and blotting with LaGP63-specific antiserum or SA-HRP. "g" and "ng" refer to glycosylated and nonglycosylated TcGP63 forms, respectively. The approximate molecular masses of the proteins are indicated.

Since Leishmania GP63 is predominantly localized on the cell surface (6, 7), we tested whether TcGP63 is also expressed on the surface at different life cycle stages of T. cruzi. For this, we performed immunofluorescence microscopy on methanol-fixed epimastigotes and metacyclic trypomastigotes by using the TcGP63 and LaGP63 antisera. This analysis suggested that the TcGP63 isoform identified by anti-TcGP63 is expressed irregularly on the surface of the cell body and flagellum of the epimastigote but is concentrated intracellularly near the kinetoplast DNA and what appears to be the flagellar pocket of the metacyclic trypomastigote (Fig. 3). The isoform identified by anti-LaGP63 was expressed at very low levels within the epimastigote and at somewhat higher levels in the metacyclic trypomastigote. Preimmune sera showed minimal background reaction with the parasites (not shown). Considered together with the surface biotinylation results shown in Fig. 2, these results suggest that TcGP63 is distributed on the membrane of the epimastigote but localized intracellularly in the metacyclic trypomastigote in a structure near the kinetoplast DNA and flagellar pocket.

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FIG. 3. Localization of TcGP63 in epimastigotes and metacyclic trypomastigotes of T. cruzi. Immunofluorescence microscopy was performed on methanol-fixed epimastigotes and metacyclic trypomastigotes by using TcGP63- or LaGP63-specific antisera. Preimmune sera gave minimal background staining (not shown). 4',6-Diamidino-2-phenylindole (DAPI)-stained (blue) kinetoplast DNA is seen in each panel.

Epimastigote TcGP63 is not associated with lipid rafts. The GP63 protein of L. major has recently been shown to be associated with lipid rafts by virtue of insolubility in cold Triton X-100 and characteristic migration at low density upon sucrose gradient centrifugation (11). The ability to partition with the detergent-resistant fraction is thought to be directly related to the presence of the glycosylphosphatidylinositol (GPI) moiety present in Leishmania GP63. We tested the detergent solubility of this TcGP63 protein (Fig. 4) by using transgenic lines of T. cruzi and L. amazonensis which provide negative and positive controls for raft association: T. cruzi expressing GFP, which is cytoplasmic, and L. amazonensis expressing T. cruzi FCaBP, which is raft associated in both Leishmania and T. cruzi (D. Maric and D. Engman, submitted for publication). Triton X-100-soluble and -insoluble fractions were prepared at 4°C and 37°C and compared by SDS-PAGE and Western blotting with antisera specific for the marker proteins (Fig. 4). The detergent-soluble (Fig. 4, lanes S) and -insoluble (lanes P) fractions of LaGP63 were distributed equally at 4°C, and LaGP63 was completely soluble at 37°C. This is consistent with what others have observed (11). The FCaBP trans protein behaved similarly. P36, a cytoplasmic protein, was found only in the soluble phase, regardless of temperature, as expected. Surprisingly, TcGP63 was present only in the soluble phase at both 4°C and 37°C, as was GFP. FCaBP behaved the same whether expressed in T. cruzi or in L. amazonensis. These results suggest that TcGP63 is not raft associated. Western blot analysis of sucrose density gradient-fractionated, detergent-insoluble membranes also supported these results (not shown).

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FIG. 4. Differential Triton X-100 solubilities of T. cruzi and Leishmania GP63 proteins. L. amazonensis expressing T. cruzi FCaBP and T. cruzi expressing GFP and FCaBP were solubilized in 1% Triton X-100 at 4°C or 37°C, separated into soluble (S) and insoluble (P) fractions, and analyzed by SDS-PAGE and Western blotting with the indicated antisera.

Differential posttranslational modification and proteolytic activity of TcGP63 proteins. Genome and proteome analyses of T. cruzi have identified numerous TcGP63 proteins. Since expression of TcGP63 proteins by use of an anti-peptide (PEGSSITPTGLFT) antiserum (11) has been done previously, we compared our antiserum with this to test whether they identify distinct TcGP63 proteins. Western blotting of whole-cell lysates of epimastigotes of four different T. cruzi strains with each antiserum showed distinct banding patterns. Our antiserum (Ab-2) recognized a single, 61-kDa band in all strains, whereas the previously described antiserum (termed Ab-1) recognized several proteins of variable intensities, depending on the strain (Fig. 5A). These data indicate that the antisera indeed recognize different TcGP63 proteins and that Ab-1 recognizes several proteins containing the same peptide epitope but that Ab-2 is more specific to one TcGP63 protein in epimastigotes. We tested whether the TcGP63 proteins recognized by these different antisera have gelatinolytic activities by first immunoprecipitating the TcGP63 protein, followed by zymographic analysis with SDS-PAGE gel containing gelatin with or without Zn, a necessary cofactor for proteolytic activity. Interestingly, only TcGP63 recognized by Ab-2 had detectable Zn-dependent activity, demonstrating differences in the proteolytic activities of these different proteins (Fig. 5B). We also compared these TcGP63 proteins for association with lipid rafts and for membrane release by using the enzyme GPI-phospholipase C (PLC) at different parasite stages. TcGP63 recognized by Ab-1 has been shown to contain a GPI anchor by its ability to be released from the membrane by GPI-PLC (9). We compared the abilities of TcGP63 recognized by Ab-1 and -2 to be released from membrane fractions of TCTs (Fig. 5C). As seen previously, GPI-PLC released immunodetectable TcGP63 recognized by Ab-1 into supernatant fractions whereas TcGP63 recognized by Ab-2 remained in the pellet fractions. This suggests that these distinct TcGP63 proteins have different modes of membrane association and that only the TcGP63 protein recognized by Ab-1 is linked via a GPI anchor. This may be specific to TCTs since we have not found differential release of these TcGP63 proteins at other life stages (data not shown).

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FIG. 5. Distinct biochemical properties of TcGP63 proteins. (A) Comparison of different TcGP63 antisera by Western blot analysis of epimastigotes of different T. cruzi strains. (B) Gelatinolytic zymographic analysis of TcGP63 immunoprecipitated with different anti-TcGP63 antisera in Zn-containing and Zn-depleted conditions (as indicated). (C) Membrane fractions of isolated TCTs were incubated in PBS containing 10 units of GPL-PLC for 1 h at 37°C. Supernatant (S) and pellet (P) fractions were subjected to SDS-PAGE and Western blot analysis with different TcGP63 antisera. The results were compared to those for controls of L. amazonensis treated in the same way and probed with L. amazonensis anti-GP63 (-LaGP63). Ab-1, anti-TcGP63 described by Cuevas et al. (9); Ab-2, anti-TcGP63 described in this paper.

TcGP63 proteins are involved in infection of mammalian cells. Since we have found that distinct TcGP63 proteins are recognized by Ab-1 and -2 and that the TcGP63 protein recognized by Ab-1 has previously been shown to contribute to host cell infection, we hypothesized that both TcGP63 proteins may independently contribute to infection. First, we attempted to purify the unique TcGP63 protein recognized by Ab-2. During the purification of TcGP63 from epimastigotes, we repeatedly observed that a significant proportion of the protein was cleaved into fragments of 34 kDa and 29 kDa (Fig. 6A). Each was reactive with the TcGP63 antiserum, indicating that they both contain epitopes present in the fusion protein used to generate the serum. However, only the full-length protein (and neither of the fragments) possessed protease activity. We were able to purify enough of the 29-kDa fragment for use as a competitor in a radioisotopic assay of T. cruzi myoblast infection (Fig. 6B). Myoblast cultures were preincubated with fractions containing the 29-kDa TcGP63 fragment and were compared to myoblast cultures from which the fragment had been removed by anti-TcGP63 immunodepletion. Radiolabeled trypomastigotes were then allowed to infect these myoblast cultures. At the indicated times, infected monolayers were washed to remove unbound parasites and processed for scintillation counting. Control infections of myoblasts preincubated with buffer alone demonstrated slowly increasing association of parasites with myoblasts, becoming maximal by 60 min, similar to what was found for the immunodepleted control (not shown). Addition of the 29-kDa fragment diminished the association of parasites with myoblasts by as much as 50% over a 2-h period; this inhibition was abolished when the fragment was depleted with anti-TcGP63.

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FIG. 6. TcGP63 or the C-terminal fragment can reduce T. cruzi infection of myoblasts. (A) SDS-PAGE of purified TcGP63 and its proteolytic fragments. A schematic of the full-length TcGP63 protein and its putative site of cleavage (arrow) for liberation of the indicated fragments are shown. The antibody symbol shows the area of reactivity of the antiserum generated against the GST-TcGP63 fusion protein. The protease activity of each fraction was determined by zymogram analysis with casein and gelatin as indicated; only full-length TcGP63 possessed proteolytic activity. (B) Radiolabeled T. cruzi trypomastigotes were used to infect myoblasts which had been preincubated at 37°C with fractions containing the purified 29-kDa TcGP63 fragment (29 kDa). Controls for each were fractions of the 29-kDa fragment depleted by anti-TcGP63 immunoprecipitation (IP) (29-kDa TcGP63).

Second, TCTs were preincubated with either preimmune serum, anti-FCaBP, or Ab-1 and Ab-2; then, after extensive washing, these parasites were used to infect cardiac myoblasts pregrown on coverslips. At increasing times postinfection, the coverslips were washed to remove unbound parasites, fixed with methanol, and stained with DAPI to visualize host cell and parasite DNA. The percentage of host cells that had been infected was determined microscopically by counting the number of infected and uninfected myoblasts and determining the number of parasites within each infected cell (Fig. 7). Examination of host myoblasts infected with parasites preincubated with controls (preimmune serum or anti-FCaBP) or anti-TcGP63 revealed a clear difference in the percentages of infected host cells. The average numbers of parasites per infected host cell were not statistically different, averaging 5 to 10 (not shown). At 90 min, the percentage of host cells infected with parasites preincubated with either TcGP63 antiserum was significantly lower than that for the preimmune control. This indicated that both TcGP63 proteins contribute to host cell infection. Addition of more antiserum to the reaction mixtures did not result in further decrease in infection (data not shown). In reactions in which both antibodies were used together to block infection, there were more-significant decreases in cell association than in those with either antiserum alone.

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FIG. 7. Antisera against different TcGP63 proteins inhibit parasite infection of host myoblasts. TCTs were preincubated with either preimmune serum (PI), anti-FCaBP (FCaBP), or TcGP63-specific antisera (Ab-1 or -2 or both) on ice for 30 min at a dilution of 1:200. After being washed, the trypomastigotes were used to infect rat heart myoblasts at a multiplicity of infection of 5:1. Coverslips were rinsed of free parasites at the indicated times postinfection, fixed in methanol, stained with DAPI, and analyzed by fluorescence microscopy. Ten to 20 high-power fields were assessed for percentages of infected cells and average numbers of parasites per infected cell. The numbers in the latter group ranged from 1 to 5 and were not significantly different among preparations. The experiments were done in triplicate, and the results were consistent among three independent experiments. Mean values ± standard deviations are shown.

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In this report, we describe the identification of the unique 61-kDa GP63 homologue of T. cruzi expressed on the surfaces of epimastigotes, amastigotes, and TCTs. The expression of this TcGP63 protein at these stages suggests its functional importance at multiple stages in the parasite life cycle. The TcGP63 isoform expressed in metacyclic trypomastigotes is smaller, lacks the glycosylation, and is located intracellularly. The reactivity of this GP63 isoform with polyclonal antiserum specific for LaGP63 indicates that this isoform possesses epitopes in common with the Leishmania protein that are not present in the TcGP63 proteins expressed at the other life cycle stages. The TcGP63 protein used to generate the antiserum in this study has significant homology to 7 of the 29 TcGP63 proteins identified in the T. cruzi proteome and has, at most, only 46% overall amino acid identity with the corresponding regions of some leishmanial GP63 proteins, with the most significant regions of homology (up to 68%) being within the first 60 residues of the TcGP63 sequence. Epitopes in these areas may account for the cross-reactivity of LaGP63 antiserum to TcGP63 proteins. Whether the inability of the metacyclic isoform to traffic to the surface is based on its lack of glycosylation is an interesting possibility. However, LaGP63 is found on the surface even when glycosylation is eliminated (21). The intracellular nature of TcGP63 in metacyclic trypomastigotes may, in fact, be important for their infectivity. Assuming that the protein is a protease, its substrate may be cytoplasmic and not extracellular. Alternatively, the protease may be sequestered until the parasite comes in contact with or invades its host cell, at which time it may be modified and/or transported to the surface. TcGP63 is irregularly distributed on the surfaces of epimastigotes, which might reflect the concentration of this protein in particular membrane domains. This may be functionally related inasmuch as TcGP63 may be localized with other cell surface molecules in the membrane.

In their report, Cuevas et al. (9) found TcGP63 in all life stages as a 75- to 78-kDa, GPI-linked protein. We have verified these results and found that there are additional proteins recognized by this antiserum in different parasite strains. Interestingly, the recombinant TcGP63 protein used to generate our antiserum also contains this sequence, yet each antiserum clearly identifies unique subsets of TcGP63 proteins, suggesting that the recombinant protein contains an immunodominant epitope specific for that TcGP63 protein that is not present in other TcGP63 proteins.

In Leishmania, the GP63 protein is linked to the cell membrane by a GPI anchor (31) and is associated with lipid rafts (11). We found that the Triton X-100-insoluble and -soluble fractions of Leishmania GP63 are distributed equally at 4°C and that this protein is detergent soluble at 37°C. In stark contrast, both TcGP63 proteins in this study are completely soluble in detergent at both 4°C and 37°C. This strongly suggests that T. cruzi GP63, unlike Leishmania GP63, is not associated with lipid rafts. This may indirectly reflect the lack of a GPI anchor on our TcGP63 protein. This is supported by the lack of biosynthetic incorporation of palmitate into our T. cruzi TcGP63 protein and the inability of TcGP63 to partition into Triton X-114 (data not shown). The TcGP63 protein reactive with Ab-1, however, possesses a GPI anchor yet is not contained in detergent-insoluble pellets, indicating that despite being anchored via a GPI anchor, it is not associated with lipid rafts. The size of the 61-kDa TcGP63 protein corresponds closely with the predicted sizes of the TcGP63 proteins reported by Grandgenett et al., the C termini of which are predicted to be somewhat longer and slightly less hydrophobic than that of Leishmania GP63 (14). Thus, the 61-kDa TcGP63 protein is likely to be a transmembrane protein.

We investigated whether TcGP63 proteins recognized by either antiserum are involved in infection of host myoblasts and found significant reduction of infection of myoblasts when trypomastigotes were pretreated with either TcGP63 antiserum. Antibody treatment of parasites did not alter their viability inasmuch as they appeared intact and highly motile during the course of the assay. Preincubation of parasites with twofold-higher concentrations of immune or preimmune serum did not alter the relative reduction in infection, discounting the possibility that nonspecific blocking of parasite proteins accounts for the reduction. Since using both antisera together had a greater effect on infection than using either antiserum alone, this suggests that they independently contribute to infection, possibly by different mechanisms. Our TcGP63 protein was reactive to the C-terminal portion of the protein, which suggests that this portion of the molecule contains epitopes which are important for host cell recognition. This notion was further supported by competition experiments using the 29-kDa TcGP63 fragment. Preincubation of myoblasts with this fragment led to a reduction in parasite association of approximately 50%. These results suggest that a domain(s) within the 29-kDa C-terminal fragment possesses a binding site(s) for the surfaces of host cells. An additional domain(s) within and/or the protease activity of the full-length molecule may serve to disrupt or modify this interaction in some way.

In Leishmania, GP63 is thought to interact with the fibronectin receptor via an RGD-like motif (28, 32). In addition, GP63 can proteolyze surface-bound complement, generating neoepitopes for receptor binding (2). The former mechanism is not likely for T. cruzi, since the known genes encoding T. cruzi GP63 do not possess RGD-like motifs (14). Other T. cruzi GP63 isoforms, not yet identified, may possess similar recognition RGD- or non-RGD-like sequences for binding intracellular adhesion molecule receptors. Interactions with other portions of TcGP63 or other parasite molecules may facilitate further parasite binding or internalization. Autoproteolytic processing of TcGP63 or proteolysis of other parasite or host molecules may indeed be necessary for efficient parasite binding or invasion. It is known that modification of some parasite molecules is important for various steps in infection (3). This is noted to be the case with oligopeptidase B, a serine protease, which is thought to process an unknown protein necessary for calcium-dependent signaling of host cells (5). Finally, an array of other functions for TcGP63 proteins can also be envisioned. The protein may help mediate complement resistance, in conjunction with other factors (24, 25), at some stages of T. cruzi (33). A trypomastigote-specific GP63 isoform might protect this stage from complement-mediated lysis, as is the case for GP63 of Leishmania (2). Our results pave the way for a more detailed investigation of the functions of TcGP63 proteins in host parasitism by T. cruzi.

 
This work was supported in part by grants from the American Heart Association (a Physician-Scientist Postdoctoral Fellowship and a Beginning Grant-in-Aid) and the OSU (Davis-Bremer Research Award) to B.S.M. and the National Institutes of Health to D.M.E.

We thank Daniel Sanchez for the anti-TcGP63 anti-peptide antiserum, K.-P. Chang (University of the Health Sciences) for the LaGP63- and P36-specific sera, Juan Leon for clones of the Brazil strain of T. cruzi, and Kegiang Wang and William O'Connell for excellent technical assistance.

 
* Corresponding author. Mailing address: Division of Infectious Diseases and Center for Microbial Interface Biology, The Ohio State University, Biomedical Research Tower, Rm. 1012, 460 W. 12th Ave., Columbus, OH 43210-1240. Phone: (614) 292-3226. Fax: (614) 292-9616. E-mail: brad.mcgwire{at}osumc.edu

Published ahead of print on 9 March 2009.

Editor: W. A. Petri, Jr.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, May 2009, p. 2193-2200, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01542-08
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