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Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

ABSTRACT: The pathological consequences of malaria infection are the result of parasite replication within red blood cells (RBCs). Invasion into RBCs is mediated by a large repertoire of parasite proteins that are distributed on the parasite surface and within specialised apical secretory organelles. As invasion is an essential step in the parasite life-cycle, targeting invasion-related molecules provides an avenue for therapeutic intervention. We have used genome and transcriptome data available for Plasmodium falciparum to identify proteins likely to be involved in RBC invasion. Of these candidates, we selected a protein which we have dubbed PfRON6 for detailed characterisation. PfRON6 contains a novel cysteine-rich domain that is conserved in other Apicomplexan parasites. We show that PfRON6 is localised in the rhoptry neck of merozoites and is transferred to the newly formed parasitophorous vacuole during invasion. Transfection experiments indicate that the gene which encodes PfRON6 is refractory to integration that disrupts the coding sequence, suggesting its absence is incompatible with the parasite life-cycle. Further, the cysteine-rich domain appears to be functionally important as it cannot be truncated. Taken together, these data identify PfRON6 as a novel and potentially important component of the Plasmodium invasion machinery.

SUBMITTER: Proellocks NI

PROVIDER: S-EPMC4766589 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

Proellocks Nicholas I NI Kats Lev M LM Sheffield David A DA Hanssen Eric E Black Casilda G CG Waller Karena L KL Coppel Ross L RL

International journal for parasitology 20081127 6

The pathological consequences of malaria infection are the result of parasite replication within red blood cells (RBCs). Invasion into RBCs is mediated by a large repertoire of parasite proteins that are distributed on the parasite surface and within specialised apical secretory organelles. As invasion is an essential step in the parasite life-cycle, targeting invasion-related molecules provides an avenue for therapeutic intervention. We have used genome and transcriptome data available for Plas ...[more]

PMID: 19073187

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Project description:BACKGROUND: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites' apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. METHODS: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. RESULTS: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a "smokescreen". PvRON4 contains a predicted signal sequence, a coiled-coil ?-helical motif, two tandem repeats and six conserved cysteines towards the carboxy-terminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2. CONCLUSIONS: Genomic, transcriptional and expression data reported for PvRON4, as well as its primary structure characteristics suggest that this protein participates in reticulocyte invasion, as has been shown for its homologue PfRON4.

Project description:Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5.IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it-and the other parasite proteins it interacts with-promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

Project description:BACKGROUND: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. METHODS: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. RESULTS AND DISCUSSION: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil ?-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host's immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. CONCLUSIONS: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2.

Dataset Information

Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

Publications

Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

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