Functional analysis of band 3 protein in the process of Plasmodium vivax invasion

Little is known about the underlying biological mechanisms, and related molecular basis, involved in the invasion of reticulocytes by P. vivax. Indeed, research on P. vivax lags behind that on P. falciparum, partly due to the failure to achieve a continuous blood stage culture of P. vivax. Nevertheless, to date we have been able to set up a short-time culture (up to 5 days) and develop an efficient parasite-reticulocyte invasion test, which allows the study of the molecular mechanisms involved in the process of reticulocyte invasion. The only well described interaction so far is the one between the Duffy antigen (reticulocyte receptor) and the parasite Duffy binding protein (PvDBP). However, increasing reports on Duffy negative P. vivax patients in Sub-Saharan Africa and Brazil suggest that unrecognized parasite and reticulocytes molecules are involved in the process of invasion, either mediating invasion through a Duffy-independent pathway or building a parasite-host co-ligation in addition to PvDBP-Duffy.

Band 3, a transmembrane protein, is a member of macromolecular complex‟s found at the red blood cell (RBC) membrane in association with other proteins including Glycophorin C and the Duffy receptor. The erythrocyte polymorphism, Southeast Asian Ovalocytosis (SAO) caused by a 27-base pair deletion (SLC4A1Δ27) in the band 3 gene, has a distribution that is closely correlated with malaria prevalence [2]. Recently, three independent epidemiological studies conducted in Papua New Guinean children have shown that SAO-individuals have a significant 52% reduction in

P. vivax blood-stage reinfection [3]. The mechanism by which SAO may protect against P. vivax infection and disease is unknown. We hypothesize that SAO alters the ability of P. vivax to attach to and/or invade reticulocytes.

To test this hypothesis, we propose to characterize the expression and distribution of several RBC membrane proteins in SAO-reticulocytes compared to non-SAO-reticulocytes, and perform

ex vivo invasion assays with SAO reticulocytes to characterize the invasion mechanisms employed by P. vivax. Additionally, we will use whole genome sequence analysis to genetically characterize parasites able to invade SAO reticulocytes. Research will be divided in 3 work packages starting from understanding the structure of SAO cells, investigate the role of band 3 protein in P. vivax invasion, and analyze the genome and transcriptome of P. vivax isolates able to invade SAO cells. We expect results from this project to identify new host-parasite interactions needed for the invasion process. Results will be relevant to advance in the knowledge of P. vivax biology and develop new strategies to fight the most widespread malaria parasite, e.g. identifying novel drug targets. As such, the impact on society in countries where P. vivax is endemic will be substantial.