The malaria parasite Plasmodium falciparum in red blood cells selectively takes up serum proteins that affect host pathogenicity

Abstract

Background: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis.

Methods: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed.

Results: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca²⁺ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation.

Conclusions: Serum proteins were taken up by parasites under culture conditions with sufficient Ca²⁺ levels. This uptake phenomenon was associated with their pathogenicity.

Keywords: Blood coagulation; Calcium ion; Plasmodium falciparum; Serum protein uptake; Thrombin.

Fig. 1

Serum proteins were selectively taken up by Plasmodium falciparum. a Western blotting analysis of serum proteins. “Serum” and “Lysate” indicate plasma-derived serum (PDS) and isolated parasite lysate prepared from infected red blood cells (iRBCs) cultured in PDS-containing medium. Samples were prepared using the Percoll method. SERA5 was used as an internal control. Protein S vitamin K-dependent protein S, AHSG alpha-2-HS-glycoprotein, C4BPA C4b-binding protein alpha chain, SERA5 serine repeat antigen 5. The asterisk indicates a non-specific band. b Confocal microscopy images showing localization of prothrombin (green), EXP2/3C (red), and DNA (cyan). Samples were fixed using the paraformaldehyde/glutaraldehyde fixation method. Scale bar represents 5 µm. c Confocal microscopy images showing the localization of prothrombin (red) and DNA (cyan). Samples were fixed using the methanol fixation method. The scale bar represents 5 µm. All samples were prepared from 3D7 strain

Fig. 2

Uptake ability was dependent on the sample preparation method. a–c Western blotting analysis of human and parasite proteins. a Lysates prepared from iRBCs cultured in PDS- and naturally coagulated serum (NCS)-containing culture media. Samples were collected using the Percoll method and were treated with saponin. Band 3, β-actin, SERA5, and EXP2/3C were used as internal controls. b Protease protection assay in the RBC and PV lysates prepared from iRBCs cultured in PDS-containing culture medium. Samples were collected using the Percoll method and were treated with streptolysin O. “+” and “−” indicate treatment with or without proteinase K, respectively. Band 3, β-actin, SERA5, and EXP2/3C were used as internal controls. c (left panels) Serum proteins in the iRBC lysates cultured in PDS- and NCS-containing culture media. Samples were collected using the precipitation method and were treated with saponin. (right panels) Serum proteins in the original sera, PDS, and NCS. SERA5 was used as internal control. The asterisk indicates a non-specific band. d Confocal microscopy images showing the localization of protein S, prothrombin, vitronectin (red), and DNA (cyan) in iRBCs cultured in PDS- or NCS-containing medium. Samples were fixed using the methanol fixation method. As a control, rabbit control IgG was used instead of primary antibodies. The scale bar represents 5 µm. All samples were prepared from 3D7 strain

Fig. 3

The serum proteins were taken up under sufficient Ca²⁺ levels in the culture medium. a Concentration of Ca²⁺ in PDS and NCS. Data represents the mean ± standard deviation (SD) for eight serum samples prepared using each method. b Comparison of parasitaemia and morphology of parasites cultured in NCS-containing medium with and without 2 mM CaCl₂. (i) Parasitaemia. The ring stage-synchronized parasite culture (approximately 1% parasitaemia) was incubated with or without 2 mM CaCl₂ for 48 h. (ii) Light microscopy images of parasites. The scale bar represents 10 µm. c–e Western blotting analysis of serum proteins. Samples were prepared using the precipitation method and were treated with saponin. c Uptake of serum proteins after the addition of 2 mM CaCl₂ to NCS. d Uptake of serum proteins before (pre) and after (post) removal of Ca²⁺ from PDS by dialysis. e Uptake of serum proteins after removal of Ca²⁺ by the addition of 2.5 mM chelators (EDTA, EGTA, or sodium citrate). Approximately 5 IU/mL heparin, a non-chelating reagent, was added as a negative control. The asterisk indicates a non-specific band. All samples were prepared from 3D7 strain

Fig. 4

Assembly/disassembly of actin filaments was not required for the uptake of serum proteins. a Light microscopy images of parasites treated with actin-perturbing agents. The scale bar represents 5 µm. b M scattergrams showing the developmental stages of parasites after treatment. The horizontal and vertical axes represent DNA content and iRBC size, respectively. Colours indicate the following: red, ring-form; orange, trophozoite; purple, schizont; and blue, polychromatic red blood cell. These were assigned based on the default settings of the XN-30 analyser. c (i) Western blotting analysis of serum proteins. Samples were collected using the Percoll method and were treated with streptolysin O. “+” and “−” indicate treatment with or without proteinase K, respectively. (ii) Densitometry analysis of the observed bands in (i). Closed and open columns indicate treatment with or without proteinase K, respectively. a.u. arbitrary unit. Band 3 and SERA5 were used as internal controls. d Confocal microscopy images showing the localization of prothrombin (red) and DNA (cyan) after treatment. Samples were fixed using the methanol fixation method. Scale bar represents 5 µm. CytD cytochalasin D, MycB mycalolide B, Jas jasplakinolide. Each compound was added to the parasite culture at a final concentration of 50 µM. All experiments were performed in triplicate and representative data are shown

Fig. 5

Coagulation occurred after parasite egress from iRBCs. a–c (i) Western blotting analysis of coagulation. a “Med” and “Sup” indicate PDS- or NCS-containing medium and PDS- or NCS-cultured supernatant, respectively. b After the addition of protease inhibitors (PMSF phenylmethylsulfonyl fluoride, DE dabigatran etexilate). c “Lysate” indicates the isolated parasite lysate prepared using the precipitation method. The isolated parasite lysate prepared from iRBCs cultured in NCS-containing medium was added to plasma. (i) The absence of fibrinogen indicated coagulation, save for in the isolated parasite lysate (lanes 3 and 4). (ii) Visual confirmation of coagulation. Tubes, shown in lanes 3, 4, 5, and 6 in (i), containing 100 µL of the reaction mixture were rotated at a 90° angle onto their sides after completion of the coagulation reaction. All experiments were performed in triplicate and representative data are shown