Deciphering Membrane Protein Complexes in Plasmodium falciparum Gametocytes via Integrative Structural Systems Biology

Malaria is caused by protozoan parasites of the genus Plasmodium that proliferate asexually in human erythrocytes. During each replication cycle, a small fraction of the parasites differentiates into gametocytes. These sexual gametocytes are the only stages of the parasite that can infect the mosquito vector and transmit malaria. Their progression and maturation depend on a profound remodeling of the erythrocyte. This is achieved by the export of parasite proteins into the erythrocyte, leading to structural and mechanistic changes in the cytoskeleton and membrane of the host cell, crucial for gametocyte development. Since gametocytes are not susceptible to most antimalarials, they pose a major obstacle to current malaria intervention strategies. At the same time, they are a bottleneck within the parasite life cycle, making them an excellent target for future transmission-blocking interventions, which are critical in the context of malaria eradication efforts. Despite this, our current understanding of the composition and interactions within the gametocyte-specific proteome is limited, particularly with respect to membrane proteins and membrane-associated multiprotein complexes. To address this knowledge gap, we employed cross-linking mass spectrometry to detect residue-level proximities between proteins in the gametocyte membrane proteome. We then used a size-exclusion-based cofractionation mass spectrometry dataset as an orthogonal source of support for candidate protein associations. Furthermore, we modeled representative complexes using AlphaFold and AF3x, capable of using cross-links as restraints. By integrating these data types, we prioritized known and previously undescribed host–pathogen and pathogen–pathogen membrane-associated complexes distributed among the cellular compartments of the parasite and the erythrocyte host cell. The results of this work advance the molecular understanding of gametocyte biology and provide a valuable resource to inform future studies that have the potential to serve as a springboard for research aimed at transmission-blocking interventions.