Limits...
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.

Show MeSH

Related in: MedlinePlus

In-silico, genomic and transcriptional analysis of thepvron5gene in thePlasmodium vivaxVCG-1 strain. (A) Schematic representation of the pvron5 gene (blue) localized in Pv_Sal1_chr05:629000..649500 (21 kb) and comparative analysis with P. falciparum: Pf3D7_08_v3:798000..818000 (20 kb) and P. knowlesi: Pk_strainH_chr05:663000..682000 (19 kb) and Pk_strainH_chr05:709,700..711,400 (1.7 kB). The transcription direction is shown for each gene and the identity and similarity values between ron5 and adjacent genes regarding the three Plasmodium species. Analysis was made concerning the information available in PlasmoDB [28] (B)pvron5 amplification from gDNA. Above, a representation of the pvron5 gene and the localization of the three sets of primers used for amplifying the gene are shown, along with the size of the expected products. ~3,404 bp (lane 1), ~1,815 bp (lane 2) and ~3,158 bp (lane 3) bands can be observed on the agarose gel, showing the weight of the expected products for pvron5. MWM: molecular weight marker. (C)pvron5 transcription in VCG-1 strain late schizonts. Lane 1: pvron5 positive RT-PCR. Lane 2: pvrhoph3 positive RT-PCR. Lane 3: pvron5 negative RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: In-silico, genomic and transcriptional analysis of thepvron5gene in thePlasmodium vivaxVCG-1 strain. (A) Schematic representation of the pvron5 gene (blue) localized in Pv_Sal1_chr05:629000..649500 (21 kb) and comparative analysis with P. falciparum: Pf3D7_08_v3:798000..818000 (20 kb) and P. knowlesi: Pk_strainH_chr05:663000..682000 (19 kb) and Pk_strainH_chr05:709,700..711,400 (1.7 kB). The transcription direction is shown for each gene and the identity and similarity values between ron5 and adjacent genes regarding the three Plasmodium species. Analysis was made concerning the information available in PlasmoDB [28] (B)pvron5 amplification from gDNA. Above, a representation of the pvron5 gene and the localization of the three sets of primers used for amplifying the gene are shown, along with the size of the expected products. ~3,404 bp (lane 1), ~1,815 bp (lane 2) and ~3,158 bp (lane 3) bands can be observed on the agarose gel, showing the weight of the expected products for pvron5. MWM: molecular weight marker. (C)pvron5 transcription in VCG-1 strain late schizonts. Lane 1: pvron5 positive RT-PCR. Lane 2: pvrhoph3 positive RT-PCR. Lane 3: pvron5 negative RT-PCR.

Mentions: The gene encoding the PvRON5 protein was identified in this study; this antigen is expressed in the schizont phase of the intra-erythrocyte cycle. An initial search was made in tBlastn regarding the pvron5 gene using the PfRON5 aa sequence (1,156 aa). The analysis revealed a high probability that the target gene would be localized in chromosome 5 (Pv_sal1_chr05) of the P. vivax genome between base pairs 635,954 to 642,743 (i.e., PfRON5 aa 105 to 1,156). The P. knowlesi RON5 aa sequence was used for searching for the start codon, as this parasite was phylogenetically closest to P. vivax. This analysis revealed that the pvron5 gene’s structural region began in position 634,671 and ended in chromosome 5 nucleotide 642,743; a gene (PVX_089530) having a putative function was found in this position (Figure 1A). Such analysis has been indispensable in re-annotating genes such as pvron4 [25] and pvrhoph3 [37] where incorrect annotations have been found regarding the start and termination of the gene as well as the number of exons.Figure 1


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)

In-silico, genomic and transcriptional analysis of thepvron5gene in thePlasmodium vivaxVCG-1 strain. (A) Schematic representation of the pvron5 gene (blue) localized in Pv_Sal1_chr05:629000..649500 (21 kb) and comparative analysis with P. falciparum: Pf3D7_08_v3:798000..818000 (20 kb) and P. knowlesi: Pk_strainH_chr05:663000..682000 (19 kb) and Pk_strainH_chr05:709,700..711,400 (1.7 kB). The transcription direction is shown for each gene and the identity and similarity values between ron5 and adjacent genes regarding the three Plasmodium species. Analysis was made concerning the information available in PlasmoDB [28] (B)pvron5 amplification from gDNA. Above, a representation of the pvron5 gene and the localization of the three sets of primers used for amplifying the gene are shown, along with the size of the expected products. ~3,404 bp (lane 1), ~1,815 bp (lane 2) and ~3,158 bp (lane 3) bands can be observed on the agarose gel, showing the weight of the expected products for pvron5. MWM: molecular weight marker. (C)pvron5 transcription in VCG-1 strain late schizonts. Lane 1: pvron5 positive RT-PCR. Lane 2: pvrhoph3 positive RT-PCR. Lane 3: pvron5 negative RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: In-silico, genomic and transcriptional analysis of thepvron5gene in thePlasmodium vivaxVCG-1 strain. (A) Schematic representation of the pvron5 gene (blue) localized in Pv_Sal1_chr05:629000..649500 (21 kb) and comparative analysis with P. falciparum: Pf3D7_08_v3:798000..818000 (20 kb) and P. knowlesi: Pk_strainH_chr05:663000..682000 (19 kb) and Pk_strainH_chr05:709,700..711,400 (1.7 kB). The transcription direction is shown for each gene and the identity and similarity values between ron5 and adjacent genes regarding the three Plasmodium species. Analysis was made concerning the information available in PlasmoDB [28] (B)pvron5 amplification from gDNA. Above, a representation of the pvron5 gene and the localization of the three sets of primers used for amplifying the gene are shown, along with the size of the expected products. ~3,404 bp (lane 1), ~1,815 bp (lane 2) and ~3,158 bp (lane 3) bands can be observed on the agarose gel, showing the weight of the expected products for pvron5. MWM: molecular weight marker. (C)pvron5 transcription in VCG-1 strain late schizonts. Lane 1: pvron5 positive RT-PCR. Lane 2: pvrhoph3 positive RT-PCR. Lane 3: pvron5 negative RT-PCR.
Mentions: The gene encoding the PvRON5 protein was identified in this study; this antigen is expressed in the schizont phase of the intra-erythrocyte cycle. An initial search was made in tBlastn regarding the pvron5 gene using the PfRON5 aa sequence (1,156 aa). The analysis revealed a high probability that the target gene would be localized in chromosome 5 (Pv_sal1_chr05) of the P. vivax genome between base pairs 635,954 to 642,743 (i.e., PfRON5 aa 105 to 1,156). The P. knowlesi RON5 aa sequence was used for searching for the start codon, as this parasite was phylogenetically closest to P. vivax. This analysis revealed that the pvron5 gene’s structural region began in position 634,671 and ended in chromosome 5 nucleotide 642,743; a gene (PVX_089530) having a putative function was found in this position (Figure 1A). Such analysis has been indispensable in re-annotating genes such as pvron4 [25] and pvrhoph3 [37] where incorrect annotations have been found regarding the start and termination of the gene as well as the number of exons.Figure 1

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