mAb CZP-315.D9: An Antirecombinant Cruzipain Monoclonal Antibody That Specifically Labels the Reservosomes of Trypanosoma cruzi Epimastigotes

Reservosomes are large round vesicles located at the posterior end of epimastigote forms of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. They are the specific end organelles of the endocytosis pathway of T. cruzi, and they play key roles in nutrient uptake and cell differentiation. These lysosome-like organelles accumulate ingested macromolecules and contain large amounts of a major cysteine proteinase (cruzipain or GP57/51 protein). Aim of this study was to produce a monoclonal antibody (mAb) against a recombinant T. cruzi cruzipain (TcCruzipain) that specifically labels the reservosomes. BALB/c mice were immunized with purified recombinant TcCruzipain to obtain the mAb. After fusion of isolated splenocytes with myeloma cells and screening, a mAb was obtained by limiting dilution and characterized by capture ELISA. We report here the production of a kappa-positive monoclonal IgG antibody (mAb CZP-315.D9) that recognizes recombinant TcCruzipain. This mAb binds preferentially to a protein with a molecular weight of about 50 kDa on western blots and specifically labels reservosomes by immunofluorescence and transmission electron microscopy. The monoclonal CZP-315.D9 constitutes a potentially powerful marker for use in studies on the function of reservosomes of T. cruzi.


Introduction
The kinetoplastid protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, which affects about eight million people in the 18 countries in which it is endemic, mostly in Latin America [1,2]. This parasite has a complex life cycle, with two developmental stages in the insect host (replicative epimastigotes and infective metacyclic trypomastigotes) and two stages in mammalian hosts (replicative intracellular amastigotes and infective bloodstream trypomastigotes). Macromolecule endocytosis plays an important role in this flagellate protozoan, allowing survival in the very different environments it colonizes. The endocytosis pathway has been elucidated mainly in epimastigote forms: molecules enter the cells via the flagellar pocket and cytostome, both located in the anterior region of the cell and accumulate in the reservosomes, the end compartments of the endocytosis pathway [3][4][5][6].
Reservosomes are large round vesicles located at the posterior end of T. cruzi epimastigotes [7]. The lack of molecular markers for cytoplasmic compartments in this parasite makes it difficult to clarify all the functions of reservosomes, which have characteristics typical of prelysosomes, lysosomes, and recycling compartments [8]. Subcellular localization [9] and proteomics [10] experiments have shown reservosomes to contain large amounts of a cysteine proteinase, known as cruzipain [11] or GP57/51 [12]. The native GP57/51 has been isolated from epimastigotes and used to generate a monoclonal antibody (mAb) [13]. Subcellular localization experiments demonstrated the presence of this protein in vesicles of the endosomal/lysosomal system and close to the flagellar pocket [12,14]. At about the same time, the native cysteine proteinase (cruzipain) was isolated and characterized [11,15]. A monospecific rabbit polyclonal antibody against this protein labeled reservosomes, the membrane lining the cell body and flagellum, the inside of the flagellar pocket, and even the cytostome [16]. Thus, no antibody directed against cruzipain has yet been reported to label reservosomes specifically, despite the accumulation of the enzyme in this organelle.
We report here the characterization of a mouse monoclonal antibody (mAb CZP-315.D9) against recombinant T. cruzi cruzipain (TcCruzipain) that specifically recognizes reservosomes. This mAb has potential as a powerful molecular marker for studies on the function of this organelle.

Ethics Statement.
Experiments involving animals were approved by the Ethics Committee of Fiocruz (Protocol P-47/12-3 with license number LW-15/13).

Construction and Purification of Recombinant
TcCruzipain Protein. The whole gene encoding T. cruzi cruzipain (TcCruzipain, 1404 bp, gene ID Tc00 .1047053507603.260) was used to design primers (Forward: 5 -ATGTCTGGCTGGGCTCGTGCGCTG-3 and Reverse: 5 -TCAGAGGCGACGATGACGGCTGTGGGTA-3 ) with recombination sites (attBs) for use on the Gateway cloning platform (Life Technologies-Invitrogen, USA). Escherichia coli strain C43+ was used for recombinant protein production (TcCruzipain + pDEST17 vector expressing a histidine tag), which was induced by incubating the cell culture for 7 h with 1 mM IPTG. The production of the recombinant protein (50 kDa TcCruzipain + 6 kDa histidine tag) was confirmed by western blotting with a probe directed against the histidine tag, and the recombinant protein was purified from the polyacrylamide gel by elution. . Escherichia coli strain C43+ was used for recombinant protein production (TcCruzipain protein domains + pDEST17 vector expressing a histidine tag), which was induced by incubating the cell culture for 4 h with 1 mM IPTG. Production of recombinant proteins was confirmed by western blot with a probe directed against the histidine tag.
2.6. Monoclonal Antibody Production. Three male BALB/c mice (30-45-days old) received four intraperitoneal doses of 20 g TcCruzipain + Alu-Gel-S and a last intravenous (without Alu-Gel-S) injection, separated by intervals of one week. The animals were checked before immunization for antibody cross reactivity with protein extracts of T. cruzi epimastigotes (preimmune serum) by western blot assay.
The spleen of a TcCruzipain-reactive mouse was used in a cell fusion protocol [20]. Spleen cells were obtained by filtration, centrifugation, and washing and were fused with Ag8XP3653 myeloma cells (generously supplied by Dr. Carlos R. Zanetti, from Laboratório de Imunologia Aplicada, Universidade Federal de Santa Catarina, Brazil) in the presence of 50% polyethylene glycol (PEG). After fusion, the cells were resuspended at a density of 2.5 × 10 6 cells/mL in RPMI medium supplemented with 20% FCS and 100 L of this suspension was added to each well of a 96-well plate. The cells were allowed to grow for 24 h at 37 ∘ C, under an atmosphere containing 5% CO 2 , and 100 L of HAT medium was then added to the cell culture. The medium was replaced every 48 h. Hybrid cells were selected over a period of 14 days, and the medium was then replaced with HT medium for an additional four days. Hybrid cells were selected and propagated in RPMI medium containing 20% FCS. Positive hybridomas were selected by indirect ELISA, western blotting, and indirect immunofluorescence (see below).
The most stable hybridoma in cryosurvival assays [20] was cloned by limiting dilution. The SBA Clonotyping-HRP System (Southern Biotech, Birmingham, USA), based on capture ELISA, was used to identify mAb isotype, in accordance with the manufacturer's instructions. Positive hybridomas and clones were cryopreserved at the Laboratório de Biologia Celular (ICC/FIOCRUZ-PR).

ELISA.
For indirect ELISA, recombinant TcCruzipain (0.15 g/well) was adsorbed onto 96-well immunoplates (Nunc, Roskilde, Denmark) by incubation overnight at 4 ∘ C with sensitizing buffer (0.05 M sodium carbonate and sodium bicarbonate, pH 9.6). The plates were then blocked by incubation for 1 h with 5% nonfat milk powder in PBS supplemented with 0.01% Tween 20 (PBS-T). The hybridoma supernatants were added to the immunoplates and incubated for 1 h at 37 ∘ C. The plates were washed five times with PBS-T and incubated for 1 h at 37 ∘ C with HRP-conjugated goat anti-mouse IgG (1 : 4,000). The plates were then washed five times with PBS-T and immunoreactivity was visualized with the SureBlue TMB Substrate, with optical density (OD) being read at 450 nm in an EL800 ELISA reader (BioTek, Winooski, VT, USA). Only OD values higher than 0.300 were considered positive.
To verify the specificity of mAb CZP-315.D9, whole protein extracts of E. coli (15 g protein/lane) and purified recombinant TcCruzipain (2 g protein/lane) were fractionated by SDS-PAGE in 10% polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with either the antirecombinant TcCruzipain polyclonal serum (diluted 1 : 1000 in blocking buffer) or the mAb CZP-315.D9 (diluted 1 : 100 in blocking buffer). The membrane was washed three times in 0.05% Tween-20/PBS and then incubated for 1 h with AP-conjugated rabbit anti-mouse IgG (diluted 1 : 10,000). The membrane was then washed three times with 0.05% Tween-20/PBS and the reactive bands were visualized with BCIP-NBT solution, as described by the manufacturer.
For analysis of cruzipain domain labeling, protein extracts of E. coli vector (with cruzipain domains) were fractionated by SDS-PAGE (15 g protein/lane) in 10% polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with the antirecombinant TcCruzipain polyclonal serum (diluted 1 : 1000) or with the mAb CZP-315.D9 (diluted 1 : 100). The experiment then continued was described above. We further analyzed colocalization of native cruzipain in the Golgi apparatus in transfected epimastigotes expressing the T. cruzi Golgi marker TcHIP/AC [24]. Three-day-old culture transfected epimastigotes were washed twice in PBS, fixed for 30 min with 4% paraformaldehyde, and incubated for one hour at 37 ∘ C with anti-TcCruzipain mouse polyclonal serum (1 : 500) and rabbit anti-protein A antibody (1 : 40,000). The samples were washed three times in PBS and incubated, in the same conditions, with the secondary antibodies: goat anti-mouse antibody coupled to AlexaFluor 488 and goat anti-rabbit antibody coupled to AlexaFluor 594 (both diluted 1 : 600). Fluorescence microscopy was then carried out as described above.  After washing in this buffer, the grids were incubated for 1 h with a rabbit anti-mouse antibody coupled to 10 nm gold particles diluted at 1 : 20 in incubation buffer. After washing in buffer and distilled water, the grids were stained for 45 min with 5% uranyl acetate and for 5 min with lead citrate and observed in a JEOL 1200EXII transmission electron microscope operated at 80 kV.

Production, Characterization, and Specificity of the Anti-
TcCruzipain Monoclonal Antibody. The T. cruzi cruzipain gene was amplified, cloned (as confirmed by sequencing), and expressed in E. coli, producing a 56 kDa recombinant protein (50 kDa of TcCruzipain sequence + 6 kDa of his-tag) that was purified and used to immunize BALB/c mice. The mouse with the most responsive and specific anti-TcCruzipain serum (as determined by western blotting and subcellular localization by indirect immunofluorescence) was chosen for fusion of splenocytes with myeloma cells. Seven positive hybridomas were detected by indirect ELISA. The most stable hybridoma (CZP-315) was used to obtain clones by limiting dilution. An IgG1 isotype (OD value: 1.105) and kappa-positive (OD value: 0.459) monoclonal antibody (mAb CZP-315.D9) was obtained after selection by indirect ELISA, western blotting, and indirect immunofluorescence assays (Table 1). A western blotting assay was performed to compare the reactivity of the anti-TcCruzipain polyclonal serum and the mAb CZP-315.D9 to E. coli protein extracts and to purified recombinant TcCruzipain. The anti-TcCruzipain serum recognized three protein bands between 80 and 110 kDa in E. coli (Figure 1(a), Ec lane 1) and several protein bands with the recombinant TcCruzipain (Figure 1(a), Czp lane 1), but with higher reactivity to a protein band between 50 and 60 kDa, compatible with TcCruzipain (50 kDa TcCruzipain + 6 kDa histidine tag). On the other hand, the mAb CZP-315.D9 recognized no protein bands in E. coli (Figure 1(a), Ec lane 2) but recognized the protein band between 50 and 60 kDa in the purified TcCruzipain fraction (Figure 1(a), Czp lane 2). Furthermore, both polyclonal and monoclonal antibodies recognized three protein bands below 50 kDa in the TcCruzipain fraction.
We further assessed the specificity of the CZP-315.D9 mAb against whole-epimastigote extracts by western blotting. Both anti-TcCruzipain polyclonal serum and CZP-315 hybridoma supernatant recognized two protein bands between 50 and 60 kDa, whereas the CZP-315.D9 mAb recognized mainly the protein band with about 50 kDa. The preimmune serum did not recognize any proteins. Actin (42 kDa), used for normalization, was detected with a polyclonal anti-TcActin mouse serum (Figure 1(b)).
Western blot assay was performed to determine which cruzipain domain (pre-pro domain, catalytic domain or Cterminal extension) was recognized by the polyclonal serum and by mAb CZP-315.D9. While the polyclonal serum recognized all protein domains and crossreacted with E. coli (protein bands below 50 kDa, Figure 1(c)), the mAb CZP-315.D9 did not, or weakly, recognize the pre-pro domain (Figure 1(c)).

Localization of Cruzipain in Reservosomes and
Colocalization with Ingested Transferrin. T. cruzi epimastigotes were incubated with preimmune serum, anti-TcCruzipain polyclonal serum, CZP-315 hybridoma supernatant, or mAb CZP-315.D9. As expected, no labeling was observed with the preimmune serum (Figure 2(a)). The anti-TcCruzipain polyclonal serum labeled several round spots at the posterior end of the parasites (reservosomes) and a single spot at the anterior end of the cells, lateral to the kinetoplast (Figure 2(b)), corresponding to the Golgi complex (see below). The CZP-315 An endocytosis assay was performed to validate the mAb CZP-315.D9. Alexa 633-conjugated transferrin was internalized and directed to the reservosomes, where it colocalized with cruzipain labeling (Figures 2(i)-2(l)).
We further assessed the immunolocalization of cruzipain by transmission electron microscopy (TEM). After incubating mAb CZP-315.D9 with ultrathin sections of epimastigote forms, gold labeling was found specifically in reservosomes ( Figure 3). Weaker labeling was found in reservosomes from cells embedded with Lowicryl resin, which appeared electronlucent (Figures 3(a)-3(c)). More intense labeling was found in reservosomes from cells embedded with Lowicryl MonoStep resin (Figures 3(d) and 3(e)), which appeared more electrondense.

Discussion
Reservosomes are large round vesicles at the posterior end of T. cruzi epimastigote forms, in which the macromolecules taken up by the parasites accumulate [3]. Reservosomes are thus specific end organelles of the endocytosis pathway of this protozoan and can be used as exclusive markers/targets for these parasites. Proteomics analyses have shown that reservosomes contain several lysosomal enzymes [10], including a major cysteine proteinase known as cruzipain [11] or GP57/51 [12]. However, the antibodies against cruzipain currently available do not specifically target the reservosomes [11,12]. We, therefore, aimed to produce a monoclonal antibody (mAb) against recombinant cruzipain (TcCruzipain) that specifically labeled reservosomes.
Indirect immunofluorescence assays to detect cruzipain in T. cruzi epimastigotes showed that (a) following incubation with a polyclonal serum against TcCruzipain, labeling was restricted to the reservosomes and in a single spot lateral to the kinetoplast and (b) following incubation with hybridoma supernatant and the mAb against TcCruzipain, labeling was restricted to the reservosomes. Immunolocalization of cruzipain by transmission electron microscopy showed gold labeling specifically in reservosomes. More intense labeling in electrondense reservosomes could be due to sample preservation in different resins (Lowicryl K4M and Lowicryl K4M MonoStep). Previous antibodies against cruzipain have labeled reservosomes, the membrane lining the cell body and flagellum, the inside of the flagellar pocket, and even the cytostome [9,12,14,16]. Our monoclonal antibody, therefore, appears to be a suitable tool for the specific labeling of reservosomes.
TcHIP is a marker of the Golgi apparatus of T. cruzi [24]. Incubation of TcHIP/AC-transfected epimastigotes with both anti-protein-A tag and anti-TcCruzipain polyclonal sera revealed colocalization of protein A and TcCruzipain in a single spot at the anterior end of the cells, close to the kinetoplast, in a region corresponding to the Golgi complex. Cruzipain is a glycoprotein that is edited in the Golgi complex  and then directed to the endosomal/lysosomal system via the trans-Golgi network [25,26]. Our polyclonal serum, therefore, also recognized immature cruzipain in transit through the Golgi complex, whereas the CZP-315 hybridoma and CZP-315.D9 mAb recognized the mature cruzipain in the reservosomes.
In western blot assays with whole extracts of T. cruzi epimastigote forms, both the anti-TcCruzipain serum and the CZP-315 hybridoma supernatant recognized two protein bands between 50 and 60 kDa, whereas the CZP-315.D9 mAb reacted strongly with a protein band at about 50 kDa. Cruzipain is produced as a 57 kDa protein, from which 6 kDa is cleaved to generate the mature cysteine protease, which thus has a molecular weight of 51 kDa (GP57/51) [12]. These data thus indicate that our CZP-315.D9 mAb recognizes mainly the mature enzyme in the reservosomes. Western blot assay to compare recognition of the anti-TcCruzipain polyclonal serum and the mAb CZP-315.D9 to purified recombinant TcCruzipain showed that the monoclonal recognized mainly a protein band between 50 and 60 kDa (50 kDa TcCruzipain + 6 kDa histidine tag), thus confirming the higher specificity of this mAb, as compared to a polyclonal antiserum. Both polyclonal and monoclonal antibodies recognized three protein bands below 50 kDa in a TcCruzipain fraction, probably due to proteolysis.
Cruzipain has three protein domains: pre-pro, catalytic, and C-terminal extension [27]. Our polyclonal serum recognized all protein domains by western blot analysis. On the other hand, mAb CZP-315.D9 recognized the catalytic domain and the C-terminal extension but did not, or weakly, recognize the pre-pro domain. This double binding can be dependent on conformational epitopes. No labeling with