Human plasma plasminogen internalization route in Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum clone 3D7 was cultured following the Trager and Jensen [32] method using standard HEPES-buffered RPMI 1640 medium (Gibco Life Technologies) supplemented with 0.5% AlbuMAX II (Life Technologies, UK). Parasite synchronization at ring stages was performed with 5% (w/v) D-sorbitol solution as described by Lambros and Vanderberg [33], and parasitaemia was determined by standard Giemsa staining and blood smear microscopy.

Purification of uninfected erythrocyte membranes was performed as described by Low et al. [34]. Briefly, the red blood cells (RBCs) were resuspended in three volumes of 0.9% w/v NaCl in 5 mM phosphate buffer at pH 8.0 and centrifuged at 500 g for 10 min (repeated 3 times). RBCs were lysed with cold 5 mM phosphate buffer, pH 8.0, containing 1 mM EDTA and a protease inhibitor cocktail (10 µM E-64, 1 mM PMSF, 1 µM pepstatin and 10 mM ortho-phenathrolin). The lysate was then centrifuged at 100,000g at 4 °C for 30 min in a Beckman L5-50B ultracentrifuge with a Ti 75 rotor. The supernatant (RBC cytosol fraction) was collected, and the membrane pellet was resuspended separately in the same buffer (RBC plasma membrane fraction).

The preparation of infected erythrocyte fractions was performed according to Hsiao and co-workers [35], with some modifications. Packed trophozoite-infected erythrocytes were resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM NaH₂PO₄) in the presence of the protease inhibitor cocktail cited above and lysed with 0.1% (w/v) saponin at room temperature for 10 min. The lysate was centrifuged at 16,000g for 1 min at room temperature. Isolated parasites were pelleted in the bottom, and the fragments of the erythrocyte membrane formed a visible band above the parasite pellet. The supernatant containing the iRBC cytosol fraction was collected in a new tube. The erythrocyte membrane layer was collected and placed in PBS with a protease inhibitor cocktail and centrifuged at 100,000g at 4 °C for 30 min. The resulting pellet was resuspended in the same buffer (iRBC plasma membrane fraction). Parasite pellets were disrupted by hypotonic lysis (1 mM HEPES, pH 7.2) on ice for 1 h in the presence of the protease inhibitor cocktail. To remove cell debris and plasma membranes, the lysate was centrifuged at 100,000g at 4 °C for 30 min, and the supernatant (parasite cytosol fraction) was collected. All fractions were stored at 80 °C until subjected to SDS-PAGE and Western blotting.

To further understand the mechanisms involved in plasminogen trafficking into infected RBCs, it was investigated the effect of inhibitors of different cellular processes on their uptake. Trophozoite-stage parasites at ~ 30% parasitaemia were incubated in PBS for 1 h, at 37 °C, with one the following compounds: 1 µM antimycin (Sigma-Aldrich, USA), 10 mM sodium azide, (Sigma-Aldrich, USA), 20 µM brefeldin (Sigma-Aldrich, USA), 1.3 µM cycloheximide (Sigma-Aldrich, USA), 10 µM FCCP + 10 mM 2-deoxy-d-glucose (Sigma-Aldrich, USA) or 50 µM genistein (Sigma-Aldrich, USA). After the treatment, samples were washed three times (300g, 2 min) with PBS and incubated with 4 µg/mL plasminogen (R&D Systems, USA) for 1 h at 37 °C. After incubation and washings, the parasite cytosol was purified as described in the fractionation section, and the effect of each treatment on plasminogen internalization was analysed by Western blotting to detect the presence of the human protein in the parasite.

Aliquots from each obtained cell fraction were resuspended in SDS-containing sample buffer and resolved on 10–12% Bis–Tris gels. The separated proteins were transferred onto polyvinylidene fluoride membranes and incubated in blocking buffer [PBS containing 0.05% (v/v) Tween 20 (PBS-T) with 5% (w/v) BSA] for 1 h. The primary antibodies were incubated overnight at 4 °C: anti-angiostatin (1:1000) (R&D Systems, USA), anti-Pfaldolase (1:1000) (Santa Cruz, USA), and anti-band3 (1:5000) (Sigma-Aldrich, USA) diluted in blocking buffer. After three washes with PBS-T, the membranes were incubated for 1 h in blocking buffer containing the secondary antibody anti-mouse IgG-HRP (1:1000). Membranes were washed with PBS-T and incubated with SuperSignal West Pico Chemiluminescent Substrate and imaged using the Odyssey® Fc Imaging System.

Flasks of asynchronous culture parasite (six negative control and six with plasminogen) with 30% parasitaemia collected from 3 consecutive days, were incubated with 4 µg/mL plasminogen (R&D Systems, USA) for 1 h at 37 °C. The control consisted of a protein-negative culture without the addition of human plasminogen. After three washes with PBS buffer to remove any plasminogen that hasn't been taken up, parasites were isolated with saponin as described above. After three washes with PBS (16,000g, 1 min), parasite pellets were suspended in Hanks’ Balanced Salt Solution (25 mM HEPES, 121 mM NaCl, 5 mM NaHCO₃, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 10 mM D-glucose, pH 7.4), and the chemical crosslinking of intracellular proteins was performed with 2 mM of the cell-permeable crosslinker DSS (Thermo Fisher Scientific Inc., USA) for 30 min at room temperature. Reactions were stopped by quenching DSS with 2 mM Tris–HCl (pH 7.4) for 15 min at room temperature.

Asynchronous parasite cultures were incubated with 4 µg/mL plasminogen (R&D Systems, USA) for 1 h at 37 °C. For immunoelectron microscopy of P. falciparum-infected erythrocytes, the previously described pre-embedding silver enhancement immunogold method was used with modifications [36]. The parasitized erythrocytes were fixed in 4% paraformaldehyde and 0.0075% glutaraldehyde dissolved in 0.1 M sodium phosphate buffer (pH 7.4) for 2 h and then washed three times (300g, 2 min) with PBS. Then, the cells were permeabilized in liquid nitrogen and incubated in a blocking buffer containing 0.005% saponin, 10% goat serum, 0.1% cold water fish gelatine, and 10% bovine serum albumin for 30 min and reacted overnight with mouse anti-angiostatin (1:250) in blocking buffer at 4 °C. Next, the cells were washed (300g, 2 min) in PBS containing 0.005% saponin and incubated with goat anti-mouse IgG conjugated with colloidal gold (1.4-nm diameter, Nanogold, Nanoprobes) (1:10) in blocking buffer for 2 h at room temperature. Cells were washed five times with PBS containing 0.005% saponin for 10 min, washed with PBS for 5 min, and fixed with 1% glutaraldehyde for 10 min. After washing, the gold particles were intensified using a silver enhancement kit (HQ silver, Nanoprobes) for 6 min at 20 °C in the dark. Cells were then centrifuged at 10,000g for 2 min and the pellets containing the P. falciparum-infected erythrocytes were incorporated to Agar 5% for further processing in epoxi resin. Next, the blocks were suspended and dehydrated with a graded ethanol, incubated in propylene oxide and embedded in Epon resin. Ulltrathin sections (70 nm) were doubly stained with uranyl acetate and lead citrate and observed at transmission electron microscopy at 80 kV (JEOL, EX II 1200, Japan). Images were acquired with a camera connected to microscope (GATAN Orius, USA).

Cellular fractionation of uninfected and infected RBCs was performed to investigate plasminogen localization in the different cell compartments, and the sub-cellular presence of the human protein was demonstrated by Western blotting analysis. Uninfected erythrocytes possess plasminogen associated with the plasma membrane; however, the protein had not been detected in significant amounts in the RBC cytoplasm (Fig. 1). In infected erythrocytes, part of the plasminogen added to the culture was located in the cytosol of P. falciparum, and part remained associated with the iRBC plasma membrane. Interestingly, plasminogen was not detected in the erythrocyte cytosol (Fig. 1). These results suggested that human plasminogen from extracellular medium can access the parasite cytosol inside erythrocytes.

The IEM micrographs are able to define the subcellular localization of plasminogen. In uninfected RBCs, the gold particles were founded consistently associated with the plasma membrane surface, while labelling was not detected in the cell cytoplasm (Fig. 2a). In infected RBCs, electron micrographs showed intense plasminogen staining located in the parasite cytoplasm and in the iRBC plasma membrane. In addition, the protein was also observed in the parasitophorous vacuolar space. Furthermore, the ultrastructural analysis revealed another interesting feature of the distribution pattern: when plasminogen is present in the iRBC cytoplasm, it is associated with the exomembrane system lumen, such as the tubovesicular network (TVN) and Maurer’s clefts (MCs) (Figs. 2b, c, e), revealing the protein trafficking route. The observed plasminogen staining pattern in these structures is comparable to other published IEMs [18]. Interestingly, plasminogen signals were not detected on the FV containing residual hemozoin (Fig. 2d) or in endocytic vesicles containing haemoglobin (Fig. 2c).

Immunoprecipitation followed by mass spectrometry analysis were used to investigate global plasminogen interactions that may assist this protein import into infected erythrocytes. Among all proteins identified (Additional file 1: Table S1), P. falciparum proteins that were unique (unique peptides negative = 0) to, or ≥ threefold enriched (unique peptides positive ≥ 3) in plasminogen immunoprecipitation were selected compared to a plasminogen negative control (Table 1). Furthermore, the enriched proteins with Relative Values above 3 were selected. With this analysis, it was possible to evaluate relevant interacting partners that may be involved in plasminogen transport in P. falciparum-infected erythrocytes, as P. falciparum Maurer’s cleft 2 transmembrane (PF3D7_1039700), Pf113 (PF3D7_1420700), a recently described PTEX-associated protein [37], vacuolar protein sorting-associated proteins (PF3D7_1250300, PF3D7_1110500). The study also identified the cyto-adherence linked asexual protein 3.1 (PF3D7_0302500) and the cyto-adherence linked asexual protein 3.2 (PF3D7_0302200), that together with RhopH2 and RhopH3 contribute to the nutrient uptake via PSAC (the plasmodial surface anion channel).

Some regulators of vesicular trafficking were also identified, including Ras-related protein Rab-11A (PF3D7_1320600), Ras-related protein Rab-2 (PF3D7_1231100), Coatomer subunits (PF3D7_1429800, PF3D7_1134800) and Dynamin-like protein (PF3D7_1145400). Furthermore, several proteins that belong to the parasite's chaperonin system were detected, among them, the T-complex protein 1 sub-units (PF3D7_0608700, PF3D7_1132200), Heat shock protein 60 (PF3D7_1015600), Heat shock protein 110 (PF3D7_0708800), Heat shock protein 90 (PF3D7_1118200), 60 kDa chaperonin (PF3D7_1232100), Hsp70/Hsp90 organizing protein (PF3D7_1434300), Heat shock protein DNAJ homologue Pfj4 (PF3D7_1211400). It should be mentioned other binding partners identified that are involved in erythrocyte invasion, as Rhoptry neck proteins (PF3D7_0905400, PF3D7_0817700, PF3D7_1116000, PF3D7_1452000) and Glideosome-associated connector (PF3D7_1361800). Regarding protein degradation, some sub-units of the parasite’s Proteasome complex (PF3D7_0727400, PF3D7_0807500) were also identified.

The effect of several pharmacological agents that may modulate different cellular processes on the import of plasminogen was investigated. As shown in Fig. 3a, the treatment of infected erythrocytes with antimycin (oxidative phosphorylation inhibitor [38, 39]), sodium azide (oxidative phosphorylation inhibitor [26]), brefeldin (protein transport inhibitor [26, 40]), cycloheximide (protein synthesis inhibitor [41]), FCCP (uncoupler of oxidative phosphorylation in mitochondria [38]), 2-deoxy-d-glucose (glycolysis inhibitor [38]) and genistein (protein tyrosine kinase inhibitor [42]), did not affect the uptake of plasminogen by the parasite. The soluble parasite protein Pfaldolase was used as a loading control. The same samples used were probed with anti-band3, an integral erythrocyte membrane protein, to confirm that samples constituted only the parasite cytosol fraction and were not contaminated with host cell membrane (Fig. 3b).