PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen

Abstract

BackgroundErythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes.ResultsPfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes.MethodsThe expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies.ConclusionThis study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-490) contains supplementary material, which is available to authorized users.

Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

“…We focussed validation experiments on the rhoptry protein PfRON3 (Plasmodb ID: PFL2505c / PF3D7_1252100, in view of its major role in invasion [ 24 , 32 ]; this protein is listed in the high-probability interactors ( S2 Table ). We also selected two potential interactors that appeared in the full list of proteins co-purifying with PfCK1 ( S1 Table ) but did not make the threshold value on the volcano plot ( S2 Table ): (i) PfCK2α (Plasmodb ID: PF11_0096 / PF3D7_1108400), as we had previously shown in a reciprocal experiment that PfCK1 co-precipitates with a PfCK2 regulatory subunit (PfCK2β) [ 25 ].…”

“…Co-localisation immunofluorescence experiments indicate partial overlap of PfCK1 with PfRON3 in schizonts, but the proteins segregate into distinct, close but non-overlapping dots in segmenters ( Fig 6A ). Since RON3 is localised in the rhoptry bulb [ 32 ], the non-overlapping localisation with PfCK1 suggests that the kinase is located in a nearby but distinct secretory organelle, the microneme. Indeed, in isolated merozoites, PfCK1 co-localises exactly with the micronemal protein AMA1 ( Fig 6B ; see Discussion ).…”

“…The cellular distribution of PfCK1 appears to be dynamic, with a pool of enzyme being exported to the host erythrocyte periphery at the early stage of infection, and subsequently detectable mostly within the parasite in late trophozoites and schizonts. A punctate pattern emerges in segmenters, with one PfCK1 dot in each developing merozoite, adjacent to the rhoptry marker RON3 [ 32 ] ( Fig 6A ). In line with this observation, PfCK1 colocalises with AMA-1 ( Fig 6B ), a protein of micronemes that is released at the very early stages of invasion [ 38 , 39 ].…”

The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

“…SERA5 and SERA6 have been shown to be required for egress from the RBC, which would be after the PfGRP170-GFP-DDD parasites die (Collins, Hackett, Atid, Tan, & Blackman, 2017;Ruecker et al, 2012;Thomas et al, 2018). RON3 has been shown to been suggested to be a protein important for RBC invasion, which implies this protein interaction is also not why PfGRP170-GFP-DDD parasites are dying (Zhao et al, 2014). CLAG9, another identified protein, has been proposed to play a role in cytoadherence to CD36 and remodelling the host RBC after invasion by a merozoite (Ling et al, 2004;Trenholme et al, 2000).…”

The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

“…SERA5 and SERA6 have been shown to be required for egress from the RBC, which would be after the PfGRP170-GFP-DDD parasites die 69–71 . RON3 has been shown to been suggested to be a protein important for RBC invasion, which implies this protein interaction is also not why PfGRP170-GFP-DDD parasites are dying 72 . CLAG9, another identified protein, has been proposed to play a role in cytoadherence to CD36 and remodeling the host RBC after invasion by a merozoite 73,74 .…”