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Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

ABSTRACT: Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestration, on its capacity to adhere to chondroitin sulfate A (CSA) present on the placental syncytium. We showed that phosphatase treatment of IEs impairs cytoadhesion to CSA. MS analysis of recombinant VAR2CSA phosphosites prior to and after phosphatase treatment, as well as of native VAR2CSA expressed on IEs, identified critical phosphoresidues associated with CSA binding. Site-directed mutagenesis on recombinant VAR2CSA of 3 phosphoresidues localised within the CSA-binding region confirmed in vitro their functional importance. Furthermore, using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), we generated a parasite line in which the phosphoresidue T934 is changed to alanine and showed that this mutation strongly impairs IEs cytoadhesion to CSA. Taken together, these results demonstrate that phosphorylation of the extracellular region of VAR2CSA plays a major role in IEs cytoadhesion to CSA and provide new molecular insights for strategies aiming to reduce the morbidity and mortality of PM.

SUBMITTER: Dorin-Semblat D

PROVIDER: S-EPMC6586358 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

Dorin-Semblat Dominique D Tétard Marilou M Claës Aurélie A Semblat Jean-Philippe JP Dechavanne Sébastien S Fourati Zaineb Z Hamelin Romain R Armand Florence F Matesic Graziella G Nunes-Silva Sofia S Srivastava Anand A Gangnard Stéphane S Lopez-Rubio Jose-Juan JJ Moniatte Marc M Doerig Christian C Scherf Artur A Gamain Benoît B

PLoS biology 20190610 6

Plasmodium falciparum is the main cause of disease and death from malaria. P. falciparum virulence resides in the ability of infected erythrocytes (IEs) to sequester in various tissues through the interaction between members of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesin family to various host receptors. Here, we investigated the effect of phosphorylation of variant surface antigen 2-CSA (VAR2CSA), a member of the PfEMP1 family associated to placental sequestrat ...[more]

PMID: 31181082

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Project description:Infection with Plasmodium falciparum during pregnancy is one of the major causes of malaria related morbidity and mortality in newborn and mothers. The complications of pregnancy-associated malaria result mainly from massive adhesion of Plasmodium falciparum-infected erythrocytes (IE) to chondroitin sulfate A (CSA) present in the placental intervillous blood spaces. Var2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is the predominant parasite ligand mediating CSA binding. However, experimental evidence suggests that other host receptors, such as hyaluronic acid (HA) and the neonatal Fc receptor, may also support placental binding. Here we used parasites in which var2csa was genetically disrupted to evaluate the contribution of these receptors to placental sequestration and to identify additional adhesion receptors that may be involved in pregnancy-associated malaria. By comparison to the wild-type parasites, the FCR3delta var2csa mutants could not be selected for HA adhesion, indicating that var2csa is not only essential for IE cytoadhesion to the placental receptor CSA, but also to HA. However, further studies using different pure sources of HA revealed that the previously observed binding results from CSA contamination in the bovine vitreous humor HA preparation. To identify CSA-independent placental interactions, FCR3delta var2csa mutant parasites were selected for adhesion to the human placental trophoblastic BeWo cell line. BeWo selected parasites revealed a multi-phenotypic adhesion population expressing multiple var genes. However, these parasites did not cytoadhere specifically to the syncytiotrophoblast lining of placental cryosections and were not recognized by sera from malaria-exposed women in a parity dependent manner, indicating that the surface molecules present on the surface of the BeWo selected population are not specifically expressed during the course of pregnancy-associated malaria. Taken together, these results demonstrate that the placental malaria associated phenotype can not be restored in FCR3delta var2csa mutant parasites and highlight the key role of var2CSA in pregnancy malaria pathogenesis and for vaccine development.

Project description:BACKGROUND: Pregnancy-associated malaria (PAM) is a serious consequence of Plasmodium falciparum-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA). Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies. METHODS: Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL). The human embryonic kidney 293 cell line (HEK293) was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The Escherichia coli expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-epsilon domains. RESULTS: Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-epsilon domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity. CONCLUSION: This is the first report showing inhibitory binding antibodies obtained through a var2CSA recombinant DBL domain immunization protocol. These results support the current strategies using var2CSA as immunogen in the aim of blocking placental sequestration of malaria parasites. This work is a step towards the development of a var2CSA based vaccine that will prevent pregnancy-associated malaria and improve pregnancy outcomes.

Project description:BACKGROUND: Protection against pregnancy associated malaria (PAM) is associated with high levels of anti-VAR2CSA antibodies. This protection is obtained by the parity dependent acquisition of anti-VAR2CSA antibodies. Distinct parity-associated molecular signatures have been identified in VAR2CSA domains. These two observations combined point to the importance of identifying VAR2CSA sequence variation, which facilitate parasitic evasion or subversion of host immune response. Highly conserved domains of VAR2CSA such as DBL5? are likely to contain conserved epitopes, and therefore do constitute attractive targets for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: VAR2CSA DBL5?-domain sequences obtained from cDNA of 40 placental isolates were analysed by a combination of experimental and in silico methods. Competition ELISA assays on two DBL5? variants, using plasma samples from women from two different areas and specific mice hyperimmune plasma, indicated that DBL5? possess conserved and cross-reactive B cell epitopes. Peptide ELISA identified conserved areas that are recognised by naturally acquired antibodies. Specific antibodies against these peptides labelled the native proteins on the surface of placental parasites. Despite high DBL5? sequence homology among parasite isolates, sequence analyses identified motifs in DBL5? that discriminate parasites according to donor's parity. Moreover, recombinant proteins of two VAR2CSA DBL5? variants displayed diverse recognition patterns by plasma from malaria-exposed women, and diverse proteoglycan binding abilities. CONCLUSIONS/SIGNIFICANCE: This study provides insights into conserved and exposed B cell epitopes in DBL5? that might be a focus for cross reactivity. The importance of sequence variation in VAR2CSA as a critical challenge for vaccine development is highlighted. VAR2CSA conformation seems to be essential to its functionality. Therefore, identification of sequence variation sites in distinct locations within VAR2CSA, affecting antigenicity and/or binding properties, is critical to the effort of developing an efficient VAR2CSA-based vaccine. Motifs associated with parasite segregation according to parity constitute one such site.

Dataset Information

Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

Publications

Phosphorylation of the VAR2CSA extracellular region is associated with enhanced adhesive properties to the placental receptor CSA.

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