Identification of a Novel RAMA/RON3 Rhoptry Protein Complex in Plasmodium falciparum Merozoites

Ito, Daisuke; Chen, Jun-Hu; Takashima, Eizo; Hasegawa, Tomoyuki; Otsuki, Hitoshi; Takeo, Satoru; Thongkukiatkul, Amporn; Han, Eun‐Taek; Tsuboi, Takafumi

doi:10.3389/fcimb.2020.605367

Cited by 4 publications

(3 citation statements)

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“…RON3 is localized at the body of the rhoptries, and not within the rhoptry neck as suggested by the name, and is discharged from the rhoptries into the PVM during invasion ( Ito et al., 2011 ; Geoghegan et al., 2021 ). RON3 interacts with RON2, RON4, and RAMA ( Ito et al., 2011 ; Ito et al., 2020 ). A global reverse genetics study indicates that the P. falciparum RON3 gene is essential for blood stage development ( Zhang et al., 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum

Ito

Kondo

Takashima

et al. 2023

Front. Cell. Infect. Microbiol.

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Plasmodium species cause malaria, and in the instance of Plasmodium falciparum is responsible for a societal burden of over 600,000 deaths annually. The symptoms and pathology of malaria are due to intraerythocytic parasites. Erythrocyte invasion is mediated by the parasite merozoite stage, and is accompanied by the formation of a parasitophorous vacuolar membrane (PVM), within which the parasite develops. The merozoite apical rhoptry organelle contains various proteins that contribute to erythrocyte attachment and invasion. RON3, a rhoptry bulb membrane protein, undergoes protein processing and is discharged into the PVM during invasion. RON3-deficient parasites fail to develop beyond the intraerythrocytic ring stage, and protein export into erythrocytes by the Plasmodium translocon of exported proteins (PTEX) apparatus is abrogated, as well as glucose uptake into parasites. It is known that truncated N- and C-terminal RON3 fragments are present in rhoptries, but it is unclear which RON3 fragments contribute to protein export by PTEX and glucose uptake through the PVM. To investigate and distinguish the roles of the RON3 C-terminal fragment at distinct developmental stages, we used a C-terminus tag for conditional and post-translational control. We demonstrated that RON3 is essential for blood-stage parasite survival, and knockdown of RON3 C-terminal fragment expression from the early schizont stage induces a defect in erythrocyte invasion and the subsequent development of ring stage parasites. Protein processing of full-length RON3 was partially inhibited in the schizont stage, and the RON3 C-terminal fragment was abolished in subsequent ring-stage parasites compared to the RON3 N-terminal fragment. Protein export and glucose uptake were abrogated specifically in the late ring stage. Plasmodial surface anion channel (PSAC) activity was partially retained, facilitating small molecule traffic across the erythrocyte membrane. The knockdown of the RON3 C-terminal fragment after erythrocyte invasion did not alter parasite growth. These data suggest that the RON3 C-terminal fragment participates in erythrocyte invasion and serves an essential role in the progression of ring-stage parasite growth by the establishment of the nutrient-permeable channel in the PVM, accompanying the transport of ring-stage parasite protein from the plasma membrane to the PVM.

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Section: Introductionmentioning

confidence: 99%

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum

Ito

Kondo

Takashima

et al. 2023

Front. Cell. Infect. Microbiol.

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“…coli cell-free expression platform, other approaches have been employed in the characterisation of protein targets for immunological assessment. The wheat germ cell-free expression system in particular has also proven to be an important platform in the advancement of biomarker discovery and malaria vaccine research [ 31 – 34 ]. This is not the focus of the current study.…”

Section: Introductionmentioning

confidence: 99%

Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray

et al. 2022

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The evaluation of protein antigens as putative serologic biomarkers of infection has increasingly shifted to high-throughput, multiplex approaches such as the protein microarray. In vitro transcription/translation (IVTT) systems–a similarly high-throughput protein expression method–are already widely utilised in the production of protein microarrays, though purified recombinant proteins derived from more traditional whole cell based expression systems also play an important role in biomarker characterisation. Here we have performed a side-by-side comparison of antigen-matched protein targets from an IVTT and purified recombinant system, on the same protein microarray. The magnitude and range of antibody responses to purified recombinants was found to be greater than that of IVTT proteins, and responses between targets from different expression systems did not clearly correlate. However, responses between amino acid sequence-matched targets from each expression system were more closely correlated. Despite the lack of a clear correlation between antigen-matched targets produced in each expression system, our data indicate that protein microarrays produced using either method can be used confidently, in a context dependent manner, though care should be taken when comparing data derived from contrasting approaches.

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“…Plasmodium falciparum rhoptry-associated protein 1 (PfRAP1) and P. falciparum high-molecular-weight rhoptry protein 3 (PfRhopH3) have been proved to be escorted to the rhoptry protein via an interaction with the PfRAMA [7]. Also, the identification of a novel PfRAMA/PfRON3 complex indicated the importance of PfRAMA in the invasion of merozoites [7,9]. These findings have led to several studies on PfRAMA immunogenicity.…”

Section: Introductionmentioning

confidence: 99%

Immunogenicity and antigenicity of a conserved fragment of the rhoptry-associated membrane antigen of Plasmodium vivax

Wang

Chen

et al. 2022

Parasites Vectors

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Background Plasmodium vivax rhoptry-associated membrane antigen (RAMA) is a glycophosphatidylinositol-anchored membrane protein currently under consideration as a malaria vaccine candidate. Immunoglobulin G (IgG) antibodies induced by P. vivax RAMA (PvRAMA) have been proved to persist over 12 months in the sera of people infected with P. vivax. It has also been shown that through stimulation of peripheral blood mononuclear cells with PvRAMA in vitro, the antigen can induce CD4+ T cells to produce interleukin-10. However, the genetic diversity of the RAMA gene in isolates of P. vivax (pvrama) and the immunogenicity of PvRAMA in animals remain unclear. Methods Genomic DNA was extracted from blood samples (n = 25) of patients in Jiangsu Province, China with imported infections of P. vivax from endemic countries in South and Southeast Asia. The extract genomic DNA was used as templates to amplify the P. vivax rama gene (pvrama) by PCR, and the PCR products were then sequenced and analyzed by the DnaSP, MEGA, and GeneDoc software packages. Recombinant PvRAMA (rPvRAMA) protein was expressed and purified, and then used to immunize mice. Levels of total IgG and different IgG subclasses of rPvRAMA-immunized mice were evaluated by enzyme-linked immunosorbent assay. Also, spleen cells of rPvRAMA-immunized mice were stimulated with rPvRAMA in vitro and levels of T cells were measured by flow cytometry. Results The average pairwise nucleotide diversity (π) of the pvrama gene was 0.00190, and the haplotype diversity (Hd) was 0.982. The C-terminal of PvRAMA showed lower haplotype diversity compared to the N-terminal and was completely conserved at amino acid sites related to erythrocyte binding. To further characterize immunogenicity of the C-terminal of PvRAMA, mice were immunized with rPvRAMA antigen. The rPvRAMA protein induced antibody responses, with the end-point titer ranging from 1:10,000 to 1:5,120,000. IgG1 was the predominant IgG subclass in rPvRAMA-immunized mice, followed by IgG2b. In addition, levels of CD4+ and CD8+ T cells in the rPvRAMA-stimulated group were significantly higher than those in the phosphate-buffered saline-stimulated group (normal control group). Conclusions The high conservation at specific amino acid sites and the high immunogenicity of the C-terminal of PvRAMA indicate the presence of conserved epitopes able to generate broadly reactive humoral and cellular immune responses. These findings support the potential of PvRAMA to serve as a vaccine candidate against P. vivax infection. Graphical Abstract

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Identification of a Novel RAMA/RON3 Rhoptry Protein Complex in Plasmodium falciparum Merozoites

Cited by 4 publications

References 44 publications

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum

Roles of the RON3 C-terminal fragment in erythrocyte invasion and blood-stage parasite proliferation in Plasmodium falciparum

Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray

Immunogenicity and antigenicity of a conserved fragment of the rhoptry-associated membrane antigen of Plasmodium vivax

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