title: PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen.
abstract: BACKGROUND
  Erythrocyte 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.
  
  
  RESULTS
  PfRON3 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.
  
  
  METHODS
  The 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.
  
  
  CONCLUSIONS
  This 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.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhao
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Chang
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Tu
authors.forename: Shengchao
authors.initials: S
authors.lastname: Yu
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wei
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhou
authors.forename: Huijun
authors.initials: H
authors.lastname: Lu
authors.affiliation: Key Laboratory of Zoonosis, Jilin University, Xi An Da Lu 5333, Changchun 5333, China. jiangning@jlu.edu.cn.
authors.forename: Ning
authors.initials: N
authors.lastname: Jiang
authors.forename: Qijun
authors.initials: Q
authors.lastname: Chen
mesh_terms: D000349:Africa; D000913:Antibodies, Protozoan / Q000097:blood*; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006801:Humans; D008018:Life Cycle Stages; D017780:Malaria Vaccines / Q000276:immunology / Q000302:isolation & purification; D016778:Malaria, Falciparum / Q000469:parasitology* / Q000517:prevention & control; D016253:Microscopy, Immunoelectron; D015388:Organelles / Q000737:chemistry*; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000913:Antibodies, Protozoan; D000951:Antigens, Neoplasm; D000953:Antigens, Protozoan; C066001:Duffy antigen binding protein, Plasmodium; D017780:Malaria Vaccines; D015800:Protozoan Proteins; C000598106:RON3 protein, Plasmodium falciparum; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins