Project Grant R21AI180609

FUNCTIONAL CHARACTERIZATION OF THE PLASMODIUM FALCIPARUM INVASION LIGAND BINDOME - MALARIA REMAINS A MAJOR GLOBAL INFECTIOUS DISEASE THAT INFECTS HUNDREDS OF MILLIONS OF PEOPLE AND IS RESPONSIBLE FOR HUNDREDS OF THOUSANDS OF DEATHS ANNUALLY. TWO MAJOR SPECIES OF PARASITE, PLASMODIUM FALCIPARUM AND P. VIVAX ARE RESPONSIBLE FOR THE MAJORITY OF CLINICAL CASES. DURING THE COURSE OF A MALARIA INFECTION, PARASITES UNDERGO REPEATED CYCLES OF INVASION AND REPLICATION IN RED BLOOD CELLS (RBC) WHICH LEADS TO ALL SYMPTOM OF THE DISEASE. AN ESSENTIAL STEP IN THIS PROCESS IS PARASITE INVASION: A MATURE SCHIZONT WILL BURST RELEASING 16 - 20 MEROZOITES WHICH WILL BIND TO AND INVADE A NEW HOST RBC IN A PROCESS THAT TAKES LESS THAN A MINUTE. DURING THIS TIME, THE MEROZOITE DEPLOYS AN ARRAY OF INVASION LIGAND PROTEINS THAT FACILITATE THE DIFFERENT STEPS OF THE INVASION PROCESS. OVER 70 CANDIDATE P. FALCIPARUM INVASION LIGAND PROTEINS HAVE BEEN IDENTIFIED AND ARE THOUGHT TO BIND TO COGNATE HOST RECEPTOR PROTEINS ON THE SURFACE OF THE RBC. HOWEVER, TO DATE LESS THAN A THIRD OF THE CANDIDATE INVASION LIGAND PROTEINS HAVE BEEN DEMONSTRATED TO BIND TO RBCS AND EVEN FEWER HOST RECEPTORS HAVE BEEN IDENTIFIED. COMPREHENSIVE IDENTIFICATION OF A GLOBAL P. FALCIPARUM INVASION LIGAND BINDOME IS A MAJOR GAP IN THE FIELD AND WOULD GREATLY FACILITATE PRIORITIZING TARGETS FOR A BLOOD STAGE MALARIA VACCINE. HERE WE PROPOSE A NEW APPROACH: BIOTINYLATED SUPERNATANT ERYTHROCYTE BINDING ASSAY PROTEOMICS (BSEP) IN ORDER TO GLOBALLY IDENTIFY THE P. FALCIPARUM BINDOME. IN BSEP, PURIFIED SCHIZONT STAGE PARASITES ARE ALLOWED TO EGRESS IN PROTEIN FREE MEDIA TO GENERATE A SUPERNATANT ENRICHED IN INVASION LIGAND PROTEINS. THESE INVASION LIGAND PROTEINS ARE BIOTINYLATED, INCUBATED WITH RBCS WHICH ARE THEN SPUN THROUGH MINERAL OIL. BOUND PROTEINS ARE ELUTED VIA HIGH SALT TREATMENT, PURIFIED VIA STREPTAVIDIN BEADS AND SUBJECTED TO QUANTITATIVE TANDEM MASS-TAG BASED PROTEOMICS. IN THIS PROPOSAL WE AIM TO SYSTEMATICALLY DEVELOP THE BSEP PROTOCOL BY TESTING FOR THE OPTIMAL WAY TO GENERATE AND BIOTINYLATE PARASITE SUPERNATANTS, AND TO DETERMINE BINDING SPECIFICITY BY USING RBC BINDING SATURATION AND SUPERNATANT DEPLETION ASSAYS (WITH ANTIBODIES AGAINST KNOWN INVASION LIGANDS AS CONTROLS). WE WILL ALSO USE BSEP TO IDENTIFY INVASION LIGANDS BINDING TO CANDIDATE HOST RECEPTORS BY COMPARING THE BINDOMES BETWEEN WT AND CANDIDATE HOST RECEPTOR KNOCKOUTS GENERATED IN AN IMMORTALIZED ERYTHROID CELL LINE AND ALREADY AVAILABLE IN THE LAB. WE WILL VALIDATE THIS APPROACH USING THE WELL-KNOWN INTERACTION BETWEEN PFEBA175 INVASION LIGAND AND HOST GLYCOPHORIN A (GYPA) USING A GYPA KNOCKOUT. WE WILL USE A SIMILAR APPROACH WITH OUR CANDIDATE HOST RECEPTOR GLYCOPHORIN B (GYPB). GYPB IS THOUGHT TO INTERACT WITH PFEBL-1 INVASION LIGAND; HOWEVER, GYPB KNOCKDOWN RBCS SHOW A STRONG INVASION DEFECT WITH PARASITE STRAINS THAT HAVE EITHER WILD TYPE OR EBL-1 DELETIONS, THUS SUGGESTING THE PRESENCE OF AN ALTERNATIVE INVASION. WE WILL VALIDATE CANDIDATE INVASION LIGANDS BY RECOMBINANT EXPRESSION FOLLOWED BY FLOW CYTOMETRY BINDING ASSAYS WITH WT OR GYPB CELLS. ONCE ESTABLISHED, WE BELIEVE THAT THE BSEP APPROACH WILL RAPIDLY ENHANCE OUR UNDERSTANDING OF INVASION LIGAND/HOST RECEPTOR INTERACTIONS AND WOULD BE HIGHLY GENERALIZABLE TO OTHER MALARIA PARASITE SPECIES, INCLUDING P. VIVAX, ABOUT WHICH EVEN LESS IS KNOWN.