# Functional characterization of the Plasmodium falciparum invasion ligand bindome

> **NIH NIH R21** · HARVARD UNIVERSITY D/B/A HARVARD SCHOOL OF PUBLIC HEALTH · 2024 · $238,060

## Abstract

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 ...

## Key facts

- **NIH application ID:** 10790197
- **Project number:** 1R21AI180609-01
- **Recipient organization:** HARVARD UNIVERSITY D/B/A HARVARD SCHOOL OF PUBLIC HEALTH
- **Principal Investigator:** Usheer Kanjee
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $238,060
- **Award type:** 1
- **Project period:** 2024-05-16 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10790197

## Citation

> US National Institutes of Health, RePORTER application 10790197, Functional characterization of the Plasmodium falciparum invasion ligand bindome (1R21AI180609-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10790197. Licensed CC0.

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