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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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rPvRON5 expression and antigenicity studies. (A) rPvRON5 expression and purification. Lane 1, rPvRON5 purified by affinity chromatography with Coomassie blue staining. A single ~33 kDa band was observed which coincided with the expected molecular weight. Lane 2, WB recognition of rPvRON5 using anti-polyhistidine monoclonal antibody. (B)P. vivax patients’ sera reactivity with rPvRON5. Top panel, recognition of recombinant protein by WB. H: reactivity by monoclonal anti-polyhistidine serum. Lanes 1 to 11, recognition of rPvRON5 by sera from P. vivax-infected patients. A single ~33 kDa band can be seen. Lanes 12 to 15, sera from healthy individuals. R: Polyclonal serum 2 reactivity with rPvRON5. Bottom panel, ELISA recognition of rPvRON5 by sera from P. vivax-infected patients (grey bars) and healthy individuals (black bars).
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Fig3: rPvRON5 expression and antigenicity studies. (A) rPvRON5 expression and purification. Lane 1, rPvRON5 purified by affinity chromatography with Coomassie blue staining. A single ~33 kDa band was observed which coincided with the expected molecular weight. Lane 2, WB recognition of rPvRON5 using anti-polyhistidine monoclonal antibody. (B)P. vivax patients’ sera reactivity with rPvRON5. Top panel, recognition of recombinant protein by WB. H: reactivity by monoclonal anti-polyhistidine serum. Lanes 1 to 11, recognition of rPvRON5 by sera from P. vivax-infected patients. A single ~33 kDa band can be seen. Lanes 12 to 15, sera from healthy individuals. R: Polyclonal serum 2 reactivity with rPvRON5. Bottom panel, ELISA recognition of rPvRON5 by sera from P. vivax-infected patients (grey bars) and healthy individuals (black bars).

Mentions: A ~33 kDa fragment was expressed in E. coli and purified by affinity chromatography based on the predicted topology for PvRON5 and findings regarding the function of the RON5 carboxy terminal region in T. gondii (Figure 3A). This recombinant protein was used for investigating (by ELISA and WB) antibodies’ natural response against PvRON5 in samples from P. vivax-infected patients. Preliminary results showed that the sera recognized a 33 kDa band with differing reactivity, which was correlated with ELISA assay data (Figure 3B). The differences so observed in recognition of rPvRON5 by sera from infected patients from different geographical regions in Colombia may have been due to the temporary acquisition of antibodies against the PvRON5 carboxyl-terminal region, as has been described for the RON6 carboxyl terminus in P. falciparum regarding sera from patients from Papua New Guinea and Vietnam [54]. Sera from healthy individuals did not recognize rPvRON5. It is worth emphasizing that reactivity was only evaluated regarding the protein’s carboxyl-terminal extreme. Future antigenicity studies could deal with evaluating the protein’s other regions.Figure 3


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)

rPvRON5 expression and antigenicity studies. (A) rPvRON5 expression and purification. Lane 1, rPvRON5 purified by affinity chromatography with Coomassie blue staining. A single ~33 kDa band was observed which coincided with the expected molecular weight. Lane 2, WB recognition of rPvRON5 using anti-polyhistidine monoclonal antibody. (B)P. vivax patients’ sera reactivity with rPvRON5. Top panel, recognition of recombinant protein by WB. H: reactivity by monoclonal anti-polyhistidine serum. Lanes 1 to 11, recognition of rPvRON5 by sera from P. vivax-infected patients. A single ~33 kDa band can be seen. Lanes 12 to 15, sera from healthy individuals. R: Polyclonal serum 2 reactivity with rPvRON5. Bottom panel, ELISA recognition of rPvRON5 by sera from P. vivax-infected patients (grey bars) and healthy individuals (black bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359499&req=5

Fig3: rPvRON5 expression and antigenicity studies. (A) rPvRON5 expression and purification. Lane 1, rPvRON5 purified by affinity chromatography with Coomassie blue staining. A single ~33 kDa band was observed which coincided with the expected molecular weight. Lane 2, WB recognition of rPvRON5 using anti-polyhistidine monoclonal antibody. (B)P. vivax patients’ sera reactivity with rPvRON5. Top panel, recognition of recombinant protein by WB. H: reactivity by monoclonal anti-polyhistidine serum. Lanes 1 to 11, recognition of rPvRON5 by sera from P. vivax-infected patients. A single ~33 kDa band can be seen. Lanes 12 to 15, sera from healthy individuals. R: Polyclonal serum 2 reactivity with rPvRON5. Bottom panel, ELISA recognition of rPvRON5 by sera from P. vivax-infected patients (grey bars) and healthy individuals (black bars).
Mentions: A ~33 kDa fragment was expressed in E. coli and purified by affinity chromatography based on the predicted topology for PvRON5 and findings regarding the function of the RON5 carboxy terminal region in T. gondii (Figure 3A). This recombinant protein was used for investigating (by ELISA and WB) antibodies’ natural response against PvRON5 in samples from P. vivax-infected patients. Preliminary results showed that the sera recognized a 33 kDa band with differing reactivity, which was correlated with ELISA assay data (Figure 3B). The differences so observed in recognition of rPvRON5 by sera from infected patients from different geographical regions in Colombia may have been due to the temporary acquisition of antibodies against the PvRON5 carboxyl-terminal region, as has been described for the RON6 carboxyl terminus in P. falciparum regarding sera from patients from Papua New Guinea and Vietnam [54]. Sera from healthy individuals did not recognize rPvRON5. It is worth emphasizing that reactivity was only evaluated regarding the protein’s carboxyl-terminal extreme. Future antigenicity studies could deal with evaluating the protein’s other regions.Figure 3

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