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The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes.

Arévalo-Pinzón G, Bermúdez M, Curtidor H, Patarroyo MA - Malar. J. (2015)

Bottom Line: Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion.Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein.

View Article: PubMed Central - PubMed

Affiliation: Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50 # 26-20, Bogotá, Colombia. gabarpi@gmail.com.

ABSTRACT

Background: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes.

Methods: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.

Results: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes.

Conclusions: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.

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PvRON5 expression inPlasmodium vivaxschizonts. (A) Representation to scale of the P. vivax RON5 protein. The signal peptide and the two transmembrane domains predicted by bioinformatics tools and the localization and sequence of the linear B-cell epitope peptides selected for polyclonal antibody production are shown. Comparative analysis of amino acid sequences from different P. vivax strains revealed the deletion of a nine-residue-long region; black dotted lines show synonymous and non-synonymous changes. Aligning P. vivax, P. falciparum and P. knowlesi RON5 revealed nine conserved cysteines (red dotted lines). The recombinant protein (rPvRON5) produced in E. coli is shown in purple.(B)PvRON5 expression in VCG-1 strain schizont lysate. Lane 1, pre-immune serum 60; lane 2, immune serum 60; lane 3, immune serum 60 pre-incubated with peptides 36930 and 36927; lane 4, pre-immune serum 2; lane 5, immune serum 2 and lane 6, immune serum 2 pre-incubated with peptide 39274. (C)PvRON5 sub-cellular localization in P. vivax-infected RBCs in schizont stage. Green shows serum reactivity for PvRON5, having a dotted pattern similar to that observed for PvRON4 and PvRON2 (red). The arrows show the dotted pattern and the overlaying of the images (merging). DAPI (4′,6-diamidino-2-phenylindole) was used for staining the parasite nucleus.
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Fig2: PvRON5 expression inPlasmodium vivaxschizonts. (A) Representation to scale of the P. vivax RON5 protein. The signal peptide and the two transmembrane domains predicted by bioinformatics tools and the localization and sequence of the linear B-cell epitope peptides selected for polyclonal antibody production are shown. Comparative analysis of amino acid sequences from different P. vivax strains revealed the deletion of a nine-residue-long region; black dotted lines show synonymous and non-synonymous changes. Aligning P. vivax, P. falciparum and P. knowlesi RON5 revealed nine conserved cysteines (red dotted lines). The recombinant protein (rPvRON5) produced in E. coli is shown in purple.(B)PvRON5 expression in VCG-1 strain schizont lysate. Lane 1, pre-immune serum 60; lane 2, immune serum 60; lane 3, immune serum 60 pre-incubated with peptides 36930 and 36927; lane 4, pre-immune serum 2; lane 5, immune serum 2 and lane 6, immune serum 2 pre-incubated with peptide 39274. (C)PvRON5 sub-cellular localization in P. vivax-infected RBCs in schizont stage. Green shows serum reactivity for PvRON5, having a dotted pattern similar to that observed for PvRON4 and PvRON2 (red). The arrows show the dotted pattern and the overlaying of the images (merging). DAPI (4′,6-diamidino-2-phenylindole) was used for staining the parasite nucleus.

Mentions: The pvron5 cDNA sequence (VCG-1 strain) was deposited in the NCBI (GenBank accession number: KP026121) and was compared to strains distributed throughout different geographic regions, such as India, Brazil, Asia and Africa, taking the Sal-1 strain deposited in PlasmoDB as reference. Analysis at nucleotide level revealed a 27 nt deletion towards the 5′ extreme in the India VII strain, coinciding with the absence of nine amino acids between positions 100 to 108 (Figure 2A and Additional file 1). Six changes were also found, four of which were non-synonymous mutations in positions 544, 547, 730, and 929 at amino acid level (Figure 2A). Such amount of change in PvRON5 is low when compared to that present in other malarial antigens, such as MSP-1 and AMA-1 [45,46]. In fact, amino acids conserved physical-chemical properties in the first three changes mentioned (544, 547 and 730). However, it cannot be ruled out that such changes could have been associated with parasite evasion mechanisms. This is important when designing a completely effective anti-malaria vaccine as antigens having high genetic variability involve the expression of different alleles in different parasite strains, inducing allele-specific responses partly reducing vaccine efficacy [47,48].Figure 2


The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes.

Arévalo-Pinzón G, Bermúdez M, Curtidor H, Patarroyo MA - Malar. J. (2015)

PvRON5 expression inPlasmodium vivaxschizonts. (A) Representation to scale of the P. vivax RON5 protein. The signal peptide and the two transmembrane domains predicted by bioinformatics tools and the localization and sequence of the linear B-cell epitope peptides selected for polyclonal antibody production are shown. Comparative analysis of amino acid sequences from different P. vivax strains revealed the deletion of a nine-residue-long region; black dotted lines show synonymous and non-synonymous changes. Aligning P. vivax, P. falciparum and P. knowlesi RON5 revealed nine conserved cysteines (red dotted lines). The recombinant protein (rPvRON5) produced in E. coli is shown in purple.(B)PvRON5 expression in VCG-1 strain schizont lysate. Lane 1, pre-immune serum 60; lane 2, immune serum 60; lane 3, immune serum 60 pre-incubated with peptides 36930 and 36927; lane 4, pre-immune serum 2; lane 5, immune serum 2 and lane 6, immune serum 2 pre-incubated with peptide 39274. (C)PvRON5 sub-cellular localization in P. vivax-infected RBCs in schizont stage. Green shows serum reactivity for PvRON5, having a dotted pattern similar to that observed for PvRON4 and PvRON2 (red). The arrows show the dotted pattern and the overlaying of the images (merging). DAPI (4′,6-diamidino-2-phenylindole) was used for staining the parasite nucleus.
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Related In: Results  -  Collection

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Fig2: PvRON5 expression inPlasmodium vivaxschizonts. (A) Representation to scale of the P. vivax RON5 protein. The signal peptide and the two transmembrane domains predicted by bioinformatics tools and the localization and sequence of the linear B-cell epitope peptides selected for polyclonal antibody production are shown. Comparative analysis of amino acid sequences from different P. vivax strains revealed the deletion of a nine-residue-long region; black dotted lines show synonymous and non-synonymous changes. Aligning P. vivax, P. falciparum and P. knowlesi RON5 revealed nine conserved cysteines (red dotted lines). The recombinant protein (rPvRON5) produced in E. coli is shown in purple.(B)PvRON5 expression in VCG-1 strain schizont lysate. Lane 1, pre-immune serum 60; lane 2, immune serum 60; lane 3, immune serum 60 pre-incubated with peptides 36930 and 36927; lane 4, pre-immune serum 2; lane 5, immune serum 2 and lane 6, immune serum 2 pre-incubated with peptide 39274. (C)PvRON5 sub-cellular localization in P. vivax-infected RBCs in schizont stage. Green shows serum reactivity for PvRON5, having a dotted pattern similar to that observed for PvRON4 and PvRON2 (red). The arrows show the dotted pattern and the overlaying of the images (merging). DAPI (4′,6-diamidino-2-phenylindole) was used for staining the parasite nucleus.
Mentions: The pvron5 cDNA sequence (VCG-1 strain) was deposited in the NCBI (GenBank accession number: KP026121) and was compared to strains distributed throughout different geographic regions, such as India, Brazil, Asia and Africa, taking the Sal-1 strain deposited in PlasmoDB as reference. Analysis at nucleotide level revealed a 27 nt deletion towards the 5′ extreme in the India VII strain, coinciding with the absence of nine amino acids between positions 100 to 108 (Figure 2A and Additional file 1). Six changes were also found, four of which were non-synonymous mutations in positions 544, 547, 730, and 929 at amino acid level (Figure 2A). Such amount of change in PvRON5 is low when compared to that present in other malarial antigens, such as MSP-1 and AMA-1 [45,46]. In fact, amino acids conserved physical-chemical properties in the first three changes mentioned (544, 547 and 730). However, it cannot be ruled out that such changes could have been associated with parasite evasion mechanisms. This is important when designing a completely effective anti-malaria vaccine as antigens having high genetic variability involve the expression of different alleles in different parasite strains, inducing allele-specific responses partly reducing vaccine efficacy [47,48].Figure 2

Bottom Line: Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion.Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein.

View Article: PubMed Central - PubMed

Affiliation: Fundación Instituto de Inmunología de Colombia (FIDIC), Carrera 50 # 26-20, Bogotá, Colombia. gabarpi@gmail.com.

ABSTRACT

Background: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes.

Methods: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples.

Results: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes.

Conclusions: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.

Show MeSH
Related in: MedlinePlus