Acquisition of human plasminogen facilitates complement evasion by the malaria parasite <i>Plasmodium falciparum</i>

Abstract

We show that the intraerythrocytic stages of the malaria parasite Plasmodium falciparum bind plasminogen and mediate its conversion into plasmin to inactivate parasite-bound C3b. This complement evasion mechanism counteracts terminal complex formation and hence promotes parasite survival in human blood. The tropical disease malaria claims more than 400,000 victims every year. Most deaths are due to infections with Plasmodium falciparum, the causative agent of malignant malaria [1]. Affected people particularly suffer from high fever, anemia, hemoglobinuria, and neurological dysfunctions, caused by the red blood cell (RBC)-infecting stages of the unicellular parasite. To avoid destruction by human complement, the parasites acquire complement regulators like factor H, which bind to the surface of the infected RBC [2, 3]. In addition, the factor H-related protein FHR-1 is able to bind to infected RBCs, where it counteracts complement evasion [4]. Plasmodium falciparum is also able to acquire the plasma zymogen plasminogen (Plg) [5, 6]. Mediated by the urokinase-type and tissue-type plasminogen activators uPA and tPA, Plg can be converted into the serine protease plasmin (Plm), which is involved in fibrin degradation but can also process complement factors such as C3 and C5 [7, 8]. We here investigated the potential role of Plg acquisition during complement evasion by the P. falciparum blood stages. Schizonts cultivated with normal human serum were subjected to Western blotting, using a polyclonal Ab (pAb) against Plg, which detected a specific ∼92 kDa band, while a much fainter band was detected in noninfected RBCs (Fig. 1A). Immunoblotting with a mAb against the RBC-specific α1-spectrin and a pAb against the ER protein Pf39 of P. falciparum served as loading controls. ELISA and immunoblot quantification confirmed significantly increased binding of Plg to schizonts compared to noninfected RBCs (Fig. 1B; Supporting Information Fig. S1A). Subsequent immunolabeling demonstrated that Plg strongly binds to the surface of RBCs infected with rings, trophozoites, or schizonts, while Plg binding to noninfected RBCs is weak (Fig. 1C). An accumulation of Plg was frequently observed at the probable entry site of the invading merozoite. To study Plg-to-Plm conversion, RBCs infected with trophozoites or schizonts, devitalized paraformaldehyde-fixed schizonts and noninfected RBCs were incubated with Plg for 2 h in the presence or absence of tPA and lysates were immunoblotted with pAb Plg to detect Plg (92 kDa) and Plm (83 kDa). In the absence of tPA, Plg was partially processed into Plm by live trophozoites, while Plm levels increased in schizonts. Contrary, no Plm bands were detected in devitalized schizonts or noninfected RBCs (Fig. 1D and E). Addition of tPA resulted in complete Plg processing in all samples. Purified Plg and immunoblotting with mAb α1-spectrin and pAb Pf39 served as controls. Plm can inactivate C3b by different pathways; cleavage of the C3b α´-chain either leads to two fragments of 68 and 46 kDa or of 27 and 87 kDa (Fig. 1F; Supporting Information Fig. S1B), while further processing can result in additional fragments of approximately 40 kDa [7]. To study C3b processing, schizonts were incubated with Plg, tPA or both for 2 h, followed by incubation with C3b for 1 h. Lysates were immunoblotted with pAb C3 to detect C3b α′ (114 kDa) and C3b β (75 kDa) as well as additional C3b degradation products. In schizonts incubated with C3b alone, the C3b α′- and β-chains were detected. Pretreatment with Plg resulted in an additional band, representing C3b α′87 (87 kDa) (Fig. 1G). When Plg was added together with tPA, a strong C3b α′87 band was detected; in addition, a 40-kDa band was visible, indicating further processing of the α′-chain. Incubation with tPA alone had no effect on C3b inactivation. To study formation of the terminal complement complex (TCC), schizonts were incubated with normal human serum in the presence or absence of additional Plg. Supplementation resulted in decreased TCC band intensities (∼330 kDa) compared to parasite samples lacking additional Plg, when a mAb directed against the TCC was used for blotting (Supporting Information Fig. S1C). ELISA and immunoblot quantification confirmed significantly decreased TCC levels, when Plg and/or tPA was added to the parasite cultures (Fig. 2A, Supporting Information Fig. S1D). TCC formation was further investigated for cultures incubated with Plg-depleted plasma (Supporting Information Fig. S2A). Immunoblotting with mAb TCC demonstrated lower TCC levels in parasites incubated with Plg-depleted plasma, when this was supplemented with exogenous Plg (Supporting Information Fig. S2B). ELISA and immunoblot quantification confirmed a significant decrease of TCC levels, when Plg was added to the cultures (Fig. 2B, Fig. Supporting Information S2C). The TCC levels were even lower, when both Plg and tPA were present, while addition of tPA alone had no effect on TCC formation. In addition, a concentration-dependent decrease of TCC formation was observed by ELISA, when either Plg-depleted plasma or normal human serum was supplemented with Plg (Supporting Information Fig. S2D and E). Plg further affected parasite growth in vitro. When ring stage parasites were cultured in normal human serum for 96 h, the parasitemia increased in the presence of additional Plg with or without tPA (Fig. 2C, Supporting Information Fig. S2F). Heat-inactivated serum was used for control. Furthermore, the parasitemia significantly decreased when the parasites were cultivated in Plg-depleted plasma instead of plasma, while the addition of Plg and tPA could revert this effect (Fig. 2D). In summary, we show that Plg acquisition enhances the viability of the P. falciparum blood stages by promoting C3b inactivation and, in consequence, preventing TCC formation. Our data further suggest that a protease present on the surface of the infected RBC facilitates Plg-to-Plm conversion required for C3b inactivation. In accord with our findings, Plg internalization and processing by plasmodial proteases was reported [6]. Furthermore, acquisition of uPA by malaria parasites has previously been assigned to functions in RBC egress by merozoites, while Plm and uPA/tPA inhibitors impaired growth of blood stage parasites [9, 10]. Future studies need to investigate the detailed mechanism of Plg-mediated complement evasion by malaria parasites. This research was funded by grants PR905/12-1 to GP and SK46/4-1 to CS from the Deutsche Forschungsgemeinschaft. Open access funding enabled and organized by Projekt DEAL. The authors declare that there is no commercial or financial conflict of interest. The peer review history for this article is available at https://publons.com/publon/10.1002/eji.202048718. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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