A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites

Abstract

Background: The Plasmodium genome encodes for a number of 6-Cys proteins that contain a module of six cysteine residues forming three intramolecular disulphide bonds. These proteins have been well characterized at transmission as well as hepatic stages of the parasite life cycle. In the present study, a large complex of 6-Cys proteins: Pfs41, Pfs38 and Pfs12 and three other merozoite surface proteins: Glutamate-rich protein (GLURP), SERA5 and MSP-1 were identified on the Plasmodium falciparum merozoite surface.

Methods: Recombinant 6-cys proteins i.e. Pfs38, Pfs12, Pfs41 as well as PfMSP-1₆₅ were expressed and purified using Escherichia coli expression system and antibodies were raised against each of these proteins. These antibodies were used to immunoprecipitate the native proteins and their associated partners from parasite lysate. ELISA, Far western, surface plasmon resonance and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera.

Results: Immunoprecipitation of parasite-derived polypeptides, followed by LC-MS/MS analysis, identified a large Pfs38 complex comprising of 6-cys proteins: Pfs41, Pfs38, Pfs12 and other merozoite surface proteins: GLURP, SERA5 and MSP-1. The existence of such a complex was further corroborated by several protein-protein interaction tools, co-localization and co-sedimentation analysis. Pfs38 protein of Pfs38 complex binds to host red blood cells (RBCs) directly via glycophorin A as a receptor. Seroprevalence analysis showed that of the six antigens, prevalence varied from 40 to 99%, being generally highest for MSP-1₆₅ and GLURP proteins.

Conclusions: Together the data show the presence of a large Pfs38 protein-associated complex on the parasite surface which is involved in RBC binding. These results highlight the complex molecular interactions among the P. falciparum merozoite surface proteins and advocate the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface.

Keywords: 6-cys proteins; Glycophorin A; MSP-165; Plasmodium falciparum.

Fig. 1

Expression of 6-Cys domain proteins: Pfs41, Pfs38, Pfs12 and PfMSP-1₆₅. a Schematic showing the organization of the above-mentioned Plasmodium proteins. Arrows mark the regions that were expressed in Escherichia coli. b Coomassie stained SDS-PAGE and immunoblots showing the purified Pfs41, Pfs38, Pfs12 and PfMSP-1₆₅. Immunoblots were performed using mouse monoclonal anti-his antibody. c Recognition of native Pfs41, Pfs38 and Pfs12 proteins in parasite lysate from asexual blood stages using their respective antibodies generated against recombinant proteins

Fig. 2

Existence of a Pfs38 protein complex at Plasmodium asexual blood stages. A Far western analysis showing interactions of recombinant Pfs38 with Pfs12 (b), Pfs41(b), GLURPR2 (a), PfMSP-1₆₅(c), and SERA5 (d). Plasmodium PfMLH and PfMSP-1₁₉ (e) did not show interaction with Pfs38. B ELISA binding analysis confirming the interactions of Pfs38 with Pfs12 (a), Pfs41 (a), GLURPR2 (b), PfMSP-1₆₅ (c), and SERA5 (d) proteins, while Plasmodium PfMLH and PfMSP-1₁₉ did not show binding to Pfs38. Error bars represent mean ± SEM of duplicate measurements. C SPR analysis showing an interaction between Pfs38 and GLURPR2

Fig. 3

Expression and co-localization of proteins of Pfs38 complex on Plasmodium merozoites. Co-localization studies were performed by immunofluorescence assays. Merozoites were labelled with (a) mouse anti-Pfs38 and rabbit anti-GLURP or with (b) mouse anti-Pfs38 and rabbit anti-Pfs41 antibodies or with (c) mouse anti-Pfs38 and rabbit anti-Pfs12 antibodies or with (d) mouse anti-Pfs38 and rabbit anti-PfsMSP1₆₅ antibodies. Partial co-localization was observed between Pfs38 and other proteins of 6-Cys complex with co-localization coefficient of 0.75, 0.61, 0.82, and 0.83 for a–d, respectively. Td represent bright field images

Fig. 4

Pfs38 binds human erythrocytes via Glycophorin A and is required for invasion of red blood cells. a Recombinant Pfs38, Pfs12 and Pfs41 were incubated with uninfected human erythrocytes and bound proteins were eluted from the erythrocytes after centrifugation through oil. PvRII was used as a positive control for erythrocyte binding assay, while an internal protein ClpQ was used as a negative control for the assay; b Erythrocyte binding assay of recombinant Pfs38 with untreated (UN) and neuraminidase treated (NM) RBCs. c Glycophorin A is the erythrocyte receptor through which Pfs38 binds human erythrocytes. ELISA binding assay was performed to study the interaction between Glycophorin A and recombinant Pfs38. As a control, GLURPR2 and PfMLH protein were used. Error bars indicate mean ± SEM of duplicate measurements; d A model of Pfs38 protein complex based on protein–protein interactions and protein-erythrocyte interaction; e Invasion inhibition assay was performed on schizont stage using α-Pfs38 purified IgG at concentration of 2, 5 and 10 mg/mL and percent inhibition was calculated. Bars indicate mean ± SEM of duplicate measurements

Fig. 5

Naturally-acquired humoral IgG immune responses to proteins of Pfs38 complex. Human IgG antibodies against proteins of 6-Cys complex were detected by ELISA in sera from naturally infected patients from Liberia and India. Sera from Denmark were used as a negative control. The dotted line indicates positivity thresholds determined from the mean reactivities +2 SD of 28 sera samples from Danish non-immune volunteers. African and Indian sera with OD above the positive thresholds are considered seropositive for each of the antigen. Bars represent mean ± SEM for 28 sera samples