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Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro.

ABSTRACT: Plasmodium vivax is the most widely distributed human malaria parasite. Despite its importance, both clinical research and basic research have been hampered by lack of a convenient in vitro culture system, in part due to the parasite's infection preference of reticulocytes rather than mature erythrocytes. The use of reticulocyte-producing hematopoietic stem cell culture has been proposed for the maintenance of the parasite, but good numbers of reticulocytes and P. vivax parasites sufficient for practical use in research have been difficult to produce from this system. Here, we report an improved method of hematopoietic stem cell culture for P. vivax infection, which requires less time and produces higher or equivalent percentage of reticulocytes than previously reported systems. Reticulocytes were cultured from cryopreserved erythroblasts that were frozen after 8day-cultivation of purified CD34+ cells from human umbilical cord blood. This method of production allowed the recovery of reticulocytes in a shorter time than with continuous stem cell culture. We obtained a relatively high percentage of peak reticulocyte production by using co-cultivation with a mouse stromal cell line. Using P. vivax mature stage parasites obtained from infected Aotus monkeys, we observed substantial numbers (up to 0.8% of the total number of the cells) of newly invaded reticulocytes 24h after initial cultivation. The addition of fresh reticulocytes after 48h culture, however, did not result in significant increase of second cycle reticulocyte invasion. Assays of invasion inhibition with specific antibodies were successful with this system, demonstrating potential for study of biological processes as well as the conditions necessary for long-term maintenance of P. vivax in vitro.

SUBMITTER: Furuya T

PROVIDER: S-EPMC3943572 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro.

Furuya Tetsuya T Sá Juliana M JM Chitnis Chetan E CE Wellems Thomas E TE Stedman Timothy T TT

Parasitology international 20131201 2

Plasmodium vivax is the most widely distributed human malaria parasite. Despite its importance, both clinical research and basic research have been hampered by lack of a convenient in vitro culture system, in part due to the parasite's infection preference of reticulocytes rather than mature erythrocytes. The use of reticulocyte-producing hematopoietic stem cell culture has been proposed for the maintenance of the parasite, but good numbers of reticulocytes and P. vivax parasites sufficient for ...[more]

PMID: 24291603

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Project description:BackgroundDifferent 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.MethodsTaking 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.ResultsThe 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.ConclusionsThe 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.

Project description:Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71(high)) Duffy-positive (Fy(+)) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes. For decades, P. vivax infections in humans have been defined by a unique requirement for the interaction between the Duffy binding protein ligand of the parasite and the Duffy blood group antigen receptor (DARC). Recent reports of P. vivax infections in Duffy-negative individuals challenge this paradigm and suggest an alternate pathway of infection, potentially using the recently discovered EBP2. However, we demonstrate that EBP2 host cell specificity is more restricted than DBP binding and that EBP2 binds preferentially to Duffy-positive, young reticulocytes. This finding indicates that this DBP paralog does mediate a Duffy-independent pathway of infection.

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Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro.

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Reticulocytes from cryopreserved erythroblasts support Plasmodium vivax infection in vitro.

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