Structure of Rhoptry Neck Protein 2 is essential for the interaction in vitro with Apical Membrane Antigen 1 in Plasmodium vivax

Abstract

BackgroundIn several Apicomplexa, the formation of moving junctions (MJs) at the interface between the external membranes of the invading parasite and the host cell is essential for the process of parasite invasion. In Plasmodium falciparum and Toxoplasma gondii, the MJ is composed of the Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck Proteins (RONs) complex; specifically, AMA1 interacts with RON2 during host cell invasion.MethodsRecombinant proteins based on Plasmodium vivax RON2 (A2033-P2100) and its synthetic peptide fragments, one cyclic and one linear, based on PvRON2 (D2035-T2074) were generated and used to evaluate the interaction with P. vivax AMA1 (PvAMA1) by the far western blot, surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) methods. The structural studies of peptides were performed by circular dichroism, and the structural analysis of the complex of PvAMA1 with peptides based on PvRON2 (D2035-T2074) was conducted with small-angle X-ray scattering (SAXS).ResultsSurface plasmon resonance (KD = 23.91 ± 2.078 μmol/L) and ITC (K = 3 × 105 mol/L) studies conclusively showed an interaction between the cyclic peptide based on PvRON2 and PvAMA1-His6. In contrast, the linear peptide and recombinant PvRON2 (GST fusion protein) did not show an interaction with PvAMA1. However, the interaction among recombinant proteins PvRON2.2 and PvAMA1-His6 was possible to show by far western blot.ConclusionsThe results show that the PvRON2 structure, particularly the S–S bond between C2051 and C2063, is determinant for the existence of the interaction between PvAMA1 and PvRON2.Electronic supplementary materialThe online version of this article (10.1186/s12936-019-2649-6) contains supplementary material, which is available to authorized users.

RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B-cell epitopes that induce antibodies that block merozoite invasion

“…Instead, three hydrophobic domains were predicted from amino acids 1093–1112, 1143–1160 and 1214–1232. These transmembrane domains are used in other species as ligands for AMA-1 (Richard et al ., 2010; Srinivasan et al ., 2013; Bermúdez et al ., 2018; Salgado-Mejias et al ., 2019), suggesting that, although the function of this protein is also conserved in this species, the topology of B. bigemina RON2 could be not the same as that of B. bovis RON2. More functional studies are necessary to test this hypothesis.…”

“…To date, RON2 has not been identified in B. bigemina , and the present work represents the first report of the identification, transcription and expression of this protein in B. bigemina . The moving junction (MJ) is the irreversible interaction between AMA-1 and RON2 in Apicomplexa parasites, and both proteins have an important role in parasite invasion of erythrocytes (Richard et al ., 2010; Srinivasan et al ., 2013; Bermúdez et al ., 2018; Salgado-Mejias et al ., 2019). AMA-1 and RON2 were initially characterized in Toxoplasma and Plasmodium (Curtidor et al ., 2011); where RON2 is integrated into the RBC membrane and there it is used as an AMA-1 ligand on the cell surface (Silvie et al ., 2004; Shen and Sibley, 2012).…”

“…This complex is discharged towards the RBC, and RON2 is integrated into the RBC membrane where it acts as an AMA-1 ligand on the parasite surface (Silvie et al ., 2004; Shen and Sibley, 2012). Blocking this interaction halts merozoite invasion, suggesting that RON2 may be a target for vaccine development (Srinivasan et al ., 2013, 2014; Zhang et al ., 2015; Bittencourt et al ., 2018; Salgado-Mejias et al ., 2019). Although RON2 has been described in Babesia divergens, B. microti and B. bovis (Ord et al ., 2016; Hidalgo-Ruiz et al ., 2018), to date, there is no evidence of RON2 in other species of Babesia , such as B. bigemina, where the presence of AMA-1 has been reported (Torina et al ., 2010).…”

“…important role in parasite invasion of erythrocytes (Richard et al, 2010;Srinivasan et al, 2013;Bermúdez et al, 2018;Salgado-Mejias et al, 2019). AMA-1 and RON2 were initially characterized in Toxoplasma and Plasmodium (Curtidor et al, 2011); where RON2 is integrated into the RBC membrane and there it is used as an AMA-1 ligand on the cell surface (Silvie et al, 2004;Shen and Sibley, 2012).…”

Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli

“…A correctly folded AMA1 can be obtained from insect cell or yeast cell expression [ 6 , 12 , 13 ]. However, to eliminate the aberrant glycosylation in the protein expressed from eukaryotic cells, the AMA1 protein was expressed in bacteria by several groups [ [4] , [14] , [15] , [16] ].…”